Antidiabetic potential of volavetki sea fish protein hydrolysates in high lipid diet and streptozotocin-induced diabetes rats

Antidiabetic potential of volavetki sea fish protein hydrolysates in high lipid diet and streptozotocin-induced diabetes rats | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Antidiabetic potential of volavetki sea fish protein hydrolysates in high lipid diet and streptozotocin-induced diabetes rats Supriya Bhowmick, Shayan Panda, Madhumita Pal, Sanjay Das, Meghamala Mandal, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7469537/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 19 Jan, 2026 Read the published version in The Journal of Basic and Applied Zoology → Version 1 posted You are reading this latest preprint version Abstract Background In the present study, the antidiabetic properties of Panna microdon sea fish protein hydrolysates (FPH) in high lipid diet and streptozotocin-induced type 2 diabetes mellitus (T2DM) rats were investigated through the mechanistic way using glucagon like peptide-1 (GLP-1) agonistic and dipeptidyl peptidase-4 (DPP-4) inhibitory activities. There were eight groups of 5 rats each: control, vehicle control, and six T2DM groups out of these one T2DM control, four T2DM groups supplemented with FPH at the doses of 12.5, 25, 50, and 75 mg/ kg b.w./day respectively, and one T2DM group treated with gliptin at the dose of 12 mg/kg b.w./day for 28 days. Results After 28 days of experiment the T2DM group presented a significant increase in fasting blood glucose (FBG) and glycated hemoglobin (HbA1c) levels in plasma, hepatic glucose-6 phosphatase activities in liver, level of lipid peroxidation, pancreatic DPP-4 activities as well as a significant decrease in the levels of plasma insulin, c-peptides, GLP-1, and antioxidant activities of superoxide dismutase, catalase, glutathione, and glucokinase activity in pancreas compared to control group. However, the daily supplementation of FPH for 28 days improved plasma insulin, c-peptides, GLP-1 levels and as well as significantly decrease plasma FBG and HbA1c levels, DPP-4 activities, lipid peroxidation and hepatic glucose-6 phosphatase activities compared to T2DM group. FPH supplementation reduced pancreatic ROS generation and pancreatic necrosis. Conclusion Results have been presumed that antidiabetes activities were shown by various potential peptides in FPH identified by nLC‒MS/MS analysis. FPH DPP-4 GLP-1 T2DM Panna microdon and FBG Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Background Low or no insulin emission from pancreatic β-cells cause hyperglycaemia, called type 1 diabetes mellitus (T1DM) (Zhao et al., 2012 ) Partly insulin production from pancreatic β-cells and /or insulin refusal to accept by skeletal muscle and fat cells causes elevation of blood glucose levels which is known as type 2 diabetes mellitus (T2DM) (Fig. 1 ) (Wan et al., 2023 ). T1DM is stemmed by the collapse of the pancreas to synthesize insulin due to autoimmune desolation of pancreatic β-cells linked with human leukocyte antigen (HLA) gene defect (Zhao et al., 2012 ; Kilari et al., 2020 ). T2DM is promoted by excessive food intake, a high lipid diet, oxidative and mental stress, obesity, lack of physical activity and other many causes. Amidst, T2DM is comprehensively prevalent above 90% of all cases (Nasri et al., 2015 ). T2DM patients have been raising the intensity of hyperglycaemia as a consequence of glucagon like peptide-1 (GLP-1) degradation. GLP-1 is a peptide hormone secreted from intestinal L cells in response to food intake (Nasri et al., 2015 ; Iwase et al., 2015 ). GLP-1 binds with GLP-1 receptors present in pancreatic β-cells and exerts insulinotropic action by withdrawn the effect of glucagon. Dipeptidyl peptidase-4 (DPP-4) rapidly degrades GLP-1 and inhibits the insulin secretion from pancreatic β-cells in a T2DM patients (Fig. 1 .) (Iwase et al., 2015 ; Rivero-Pino et al., 2020 ). International Diabetes Federation (IDF) reported in 2021 that 1 in 10 adults (20–79 years) has been suffering with diabetes which will be projected 1 in 7 adults for the year 2030 (Ashraf et al., 2021 ; IDF, 2021). DPP-4 inhibitors like gliptin have been successfully used to treat and manage T2DM. However, this medicine has gastrointestinal side effects, weight gain, bone loss, headaches, upper respiratory tract infections, and other side effects that restrict their use (Yun et al., 2021; Harnedy et al., 2017 ). The scientific community was inspired to look for risk-free complementary therapy of diabetes that may be exploited to treat or prevent T2DM without having any negative side effects. The therapy and prevention of T2DM both heavily rely on dietary components (Wan et al., 2023 ; Kilari et al., 2020 ). Proteins, peptides, fatty acids, and polyphenols are examples of food-derived bioactive or therapeutic components that have gained considerable attention recently because to their high safety and wide range of biological capabilities, including anti-oxidant, anti-inflammatory, anti-hypertensive, anti-cancer, and immunomodulatory actions (Zhou et al., 2021 ; Bunsroem et al., 2022 ). Sea fish protein hydrolysates such as, tuna fish protein hydrolysates (Kaleshkumar et al., 2019 ), sardine fish protein hydrolysates (Nasri et al., 2015 ), Atlantic salmon fish protein hydrolysates (Harnedy et al., 2017 ) are significant sources of useful bioactive substances such proteins, peptides, and fatty acids that have been shown to have anti-oxidant, anti-inflammatory, anti-hypertensive, anti-cancer, and immunomodulatory characteristics (Harnedy et al., 2017 ; Wang et al., 2023 ). As well as, a mediterranean diet that includes a moderate amount of fish hydrolysate has revealed the benefits for the prevention T2DM (Wang et al., 2023 ). Numerous systematic reviews and meta-analyses have found a link between higher fish/fish product consumption and a decreased incidence or risk of T2DM (Das et al., 2024 ; Pyne et al., 2023 ). In our earlier cross-sectional study, it has been revealed that sea fish eaters showed lower the incidence of T2DM when compared to fresh water fish eaters (Pyne et al., 2021 ). We have also experimentally established the potential effects of sea fish oil for the management of T2DM (Pyne et al., 2023 ). The present study was aimed to scout out the antidiabetic sea fish protein hydrolysates from volavetki ( Panna microdon) in high lipid diet along with streptozotocin induced T2DM rats. Results Identification of bioactive peptides and amino acid sequences in FPH by nLC‒MS/MS analysis Thirty-five unique peptides were analysed in FPH by nLC-MS/MS method which were given in supplementary file no.1. Tentative identification of peptides present in FPH were histone H2B, 2-iminobutanoate/2-iminopropanoate deaminase, histone H3, hemoglobin alpha chain, alpha1 hemoglobin, hemoglobin subunit beta, hemoglobin beta chain, methylmalonyl-CoA epimerase, O-acetyl-ADP-ribose deacetylase MACROD2, peptidyl-prolyl cis-trans isomerase, THAP domain-containing protein 4, receptor expression-enhancing protein, natural killer cell enhancer factor, myosin light chain 5, serine protease 57, ES1 protein, mitochondrial, carbonic anhydrase, homer protein-like protein 2, cytosolic 5'-nucleotidase 1B, keratin, type I cytoskeletal 50 kDa GK50, keratin, type II cytoskeletal 8, protein disulfide-isomerase, putative aminopeptidase NPEPL1, transmembrane protease serine 9, drebrin Developmentally-regulated brain protein, A-kinase anchor protein 8-like, junction plakoglobin, aconitate hydratase, mitochondrial, glycogen debranching enzyme, uncharacterized protein, nidogen-2, ryanodine receptor 3, treslin TopBP1-interacting checkpoint and replication regulator, gelsolin, nebulin. Effects of FPH on FBG and HbA1c levels Figure 2 (A & B). shows the results of glucose metabolism monitored in the control, vehicle control, and experimental rats on day 0, 7, 14, 21 and 28 of the experiment. Overall, no significant (p > 0.05) changes in FBG and HbA1c were measured in day 0 of the experiment of all groups of rats. A continuous increase in FBG level was observed in the T2DM rats from the day 3, which continued throughout the experimental period. On the other hand, there was no significant changes in HbA1c level between day 0 to day 21, at 28 days showed the significant changes of HbA1c level in T2DM rats. Administration of STZ led to a significant increase in FBG and HbA1c levels compared with those of the control and vehicle control rats. However, administered with various doses of FPH, FBG levels began to diminish from day 14 of the experiment. A significant reduction in FBG and HbA1c levels was found in the T2DM + FPH 50 (50 mg/kg of b.w.) rats compared with those in the T2DM rats (P < 0.001) at the end of the experiment. The mean FBG and HbA1c levels in the T2DM + FPH 50 rats were progressively declined at the end of the final experimental day. These results indicated that T2DM + FPH 50 has the ability to reduce FBG and HbA1c levels, implying its anti-diabetic effects. Effects of FPH on the levels on insulin, c-peptide, GLP-1 in plasma Plasma insulin and c-peptides levels were significantly (P < 0.05) lower in the STZ-induced T2DM rats when compared with the control rats. The supplementation of FPH at different doses (12.5, 25, 50, and 75 mg/kg b. w./day) increased the plasma insulin and c-peptides levels compared to T2DM rats. The effect was more pronounced at the dose of FPH 50 supplemented rats than in the other doses supplemented rats shown in Fig. 3 (A&B). Effects of FPH on DPP-4, glucokinase activities in pancreas tissue and glucose-6-phosphatase activity in liver tissue T2DM rats unveiled significantly (P < 0.05) increased pancreatic DPP-4 activity as compared to the control as well as vehicle control rats. The supplementation of FPH at different doses significantly (P < 0.001) decreased the pancreatic DPP-4 activity as opposed to T2DM rats Fig. 4 (A). The glucokinase activity in pancreas tissue was significantly (P < 0.05) lower in T2DM rats compared with the control rats. The supplementation of FPH at different doses significantly (P < 0.001) increased the glucokinase activities compared to T2DM rats Fig. 4 (B). The glucose 6-phosphatase activity of liver tissue was increased in T2DM rats as compared to control rats. A significant (P < 0.001) reduction in glucose 6- phosphatase activity was observed in the FPH supplemented rats as compared to T2DM rats Fig. 4 (C). Effects of FPH on oxidative stress markers Effects of FPH in pancreatic intracellular ROS generation and apoptosis Effects of FPH in pancreatic nuclear integrity and apoptosis in DAPI stanning Effects of FPH in cell apoptosis The apoptotic cell death results obtained by flow cytometer were shown in Table 1 and Fig. 8 The results show scattered plot, which was created fluorescence colour to generate through FITC and PI staining. It shows early cell apoptosis followed by late apoptosis due to induction of STZ in T2DM rats which coincidentally converts into necrotic cell death. Supplementation of FPH at the dose of 50 mg/kg b. w. shown the lowest apoptosis. Table 1 Effect of FPH on late apoptosis cells, necrotic cells, live cells, and early apoptosis cells of HLD and STZ induced T2DM rats in different groups. Data are expressed as mean ± SEM (n = 5). * P < 0.05, and ** P < 0.01 when compared with T2DM group. Groups Live cells Early apoptotic cell Apoptotic cell Necrotic cell Control 23.922 ± 0.135* 1.25 ± 0.135** 43.806 ± 0.085** 34.148 ± 0.294** Vehicle control 17.258 ± 0.220** 54.312 ± 0.307* 4.05 ± 0.366** 20.694 ± 0.386** T2DM 4.514 ± 0.304 76.214 ± 0.195 5.844 ± 0.262 15.806 ± 0.281 T2DM + FPH 12.5 5.048 ± 0.276** 75.846 ± 0.251* 7.722 ± 0.294** 14.998 ± 0.366** T2DM + FPH 25 11.07 ± 0.367** 67.134 ± 0.324** 2.052 ± 0.168* 20.742 ± 0.312** T2DM + FPH 50 22.742 ± 0.290* 44.11 ± 0.234* 1.124 ± 0.135* 36.128 ± .353* T2DM + FPH 75 12.442 ± 0.309* 72.64 ± 0.291* 3.166 ± 0.201* 14.042 ± 0.099** T2DM + OHA 13.48 ± 0.268** 63.992 ± 0.384* 15.026 ± 0.156** 9.814 ± 0.264* Histological observations The pancreas histological examination was performed using haematoxylin and eosin staining, and the results are presented in Fig. 9 Control rats exhibited normal architecture of pancreatic sections, with evenly organized islet of β-cells. Sections from the T2DM rats revealed damaged β-cells with irregular shape and reduced numbers, infiltration of lymphocytes, necrosis, and damaged globules. The pancreatic sections prepared from the FPH-supplemented rats showed necrotic changes with reduced β-cells and partially deranged structure. FPH 50 rats demonstrated preservation of pancreatic islets, with an increase in their number and size along with attenuation of the histological changes from severe to mild. Discussion In the present study, focused on antidiabetic effects of FPH in HLD feeding and low dose STZ-induced T2DM rat model has been investigated. Our results demonstrated that Panna microdon FPH has immensely bring down the blood glucose level endlessly 28 days of treatment and retrieved the rats from diabetes and reclaimed back to normal. T2DM is an incurable disorder, and it necessitates routine check-up, proper management by a patient to control the disease's progression and stop complications from emerging. There are many commercial medicines are available in the market that can be used to treat these complications (Wan et al., 2023 ). However, more recently, researchers focused in different approach by using marine organisms, their bioactive compounds against the occurrence of certain diseases. The nLC-MS/MS investigation recorded the presence of various bioactive compounds in Panna microdon FPH. Unspecified amalgamation was reported for displaying antidiabetic property. Histone H2B, a positively charged nuclear protein, plays a crucial role in the compaction and organization of chromosomal DNA (Ali et al., 2020 ). The enzyme 2-iminobutanoate/2-iminopropanoate deaminase catalyses the hydrolytic deamination of enamine and imine intermediates, which are transiently formed during cellular metabolic processes (Harnedy et al., 2017 ). Histone H3 is a key epigenetic regulator that modulates gene expression by influencing chromatin structure and transcription factor accessibility (Muscle Blaze Biozyme Iso-Zero, 2022 ). Methylmalonyl-CoA epimerase catalyses a crucial step in the metabolic degradation of branched-chain amino acids and odd-chain fatty acids, facilitating their conversion to succinyl-CoA for entry into the citric acid cycle (Kilari et al., 2020 ). MACROD2, an O-acetyl-ADP-ribose deacetylase, is a crucial enzyme in cellular nucleotide metabolism, regulating the turnover of O-acetyl-ADP-ribose and modulating intracellular signalling pathways, thereby influencing various cellular regulatory processes (Graham et al., 2018 ). Peptidyl-prolyl cis-trans isomerases (PPIases) facilitate protein folding and refolding by catalysing the interconversion of cis and trans conformations at peptide bonds, thereby modulating protein structure and function (Bianchetti et al., 2011 ). THAP domain-containing protein 4 exhibits multifunctional enzymatic activities, including hemi binding, protein-protein interaction, and peroxynitrite isomerase functions, underscoring its potential role in various cellular processes (Fan et al., 2022 ). Receptor expression-enhancing protein modulates the function and homeostasis of the endoplasmic reticulum (ER), playing a crucial role in regulating cellular processes, including protein synthesis, folding, and transport, thereby impacting overall cell function and survival (Masuda et al., 2016 ). ES1 protein regulates transcription, DNA synthesis, and cellular transformation (Dodgson et al., 1980 ). Mitochondrial carbonic anhydrase is essential for the expression of carbonic anhydrase V in pancreatic β-cells, which in turn enhances insulin secretion, highlighting its critical role in glucose metabolism and insulin regulation (Shiraishi-Yamaguchi et al., 2007). Homer protein-like 2 (HPL2) modulates intracellular signal transduction and vesicular trafficking, playing a crucial role in regulating cellular communication and protein transport (Ipata et al., 2013). Cytosolic 5'-nucleotidase 1B (cN1B) exerts inhibitory effects on basal lipid oxidation and glucose transport in skeletal muscle, thereby regulating metabolic flux and energy homeostasis (Raszewski et al., 2024). Ryanodine receptor 3 (RyR3) plays a crucial role in regulating the resting calcium ion concentration ([Ca2+] rest) in skeletal muscle cells, thereby modulating muscle tone and excitability (Bucki et al., 2008 ). Gelsolin exerts anti-inflammatory effects in T2DM by decreasing microparticle-driven inflammation and suppressing NLRP3 inflammasome activation, leading to improved disease outcomes (Bae et al., 2018 ). Nebulin plays a crucial role in maintaining the structural and functional integrity of skeletal muscle, ensuring proper contractile function and regulating actomyosin interactions (Huang et al., 2010). According to Li-Chan et al., the antidiabetic properties of Salmon protein hydrolysate (Li-Chan et al., 2012 ). Wan et al. also revealed the antidiabetic effects of protein hydrolysates of Trachinotus ovatus and their derived peptides, which are promising natural ingredients with the potential to be used for the treatment or prevention of diabetes (Wan et al., 2023 ). These peptides together play and important role in cellular as well as membrane signalling pathway through activating Ca2 + channel- gene expression of transmembrane receptor protein- activating intracellular kinase which may contribute antidiabetic activity. The inhibitory effect of FPH on digestive enzymes prompted an investigation into its potential antidiabetic properties in a T2DM rat model induced by a combination of HLD and STZ. Previous studies have shown that low-dose STZ administration induces diabetes in animals, but this model lacks the insulin resistance characteristic of T2DM. To better mimic T2DM pathogenesis, we employed a combination of HLD and STZ, where HLD induces low-dose STZ along with insulin aversion causes pancreatic β-cell destruction, recapitulating the human disease (Bucki et al., 2008 ). For progression of T2DM, HLD for 180 days was required, followed by a single intraperitoneal injection of STZ at 40 mg/kg concentration was needed. According to earlier reports by Pyne et al. (Pyne et al., 2023 ) and Das et al. (Das et al., 2024 ), the optimal paradigm for the development of diabetes involves use of HLD for the period of 24 weeks and STZ injection at 40 mg/kg was one of the best tools. One of the important symptoms of diabetes is hyperglycaemia. Our research revealed that the blood glucose level of the T2DM group were predominantly raised throughout the study. Blood glucose was successfully regulated and T2DM was kept within normal ranges with the supplementation of FPH 50. Interestingly, the level of plasma insulin remained constant throughout the study. By raising blood glucose levels by HLD and STZ, it was seen that all of the induced group rats had developed T2DM. Our findings were vindicated by an earlier investigation by Huang and Wu (Huang et al., 2010). According to his reports, the peptide that is isolated from the liver of sharks can assist blood glucose regulation. As well as Zhu et al., (Zhu et al., 2010 ) have also observed the same that the HLD and STZ-induced diabetic rats the blood glucose levels were lowered by an oligopeptide from the skin of marine Salmon. Furthermore, they reported that there was no discernible alteration in the insulin level during the experimental period. In T2DM rat, plasma insulin and c-peptide levels decreased but these diabetic sensors significantly increased by FPH supplemented group indicated to T2DM which managed by probably therapeutic or anti-T2DM peptides present in FPH. Several studies demonstrated that consumption of various sea fish protein hydrolysates can effectively enhance the postprandial insulin response and reduce postprandial blood glucose levels (Wan et al., 2023 ; Graham et al., 2018 ). The results of our study clarified that supplementation with FPH ameliorated FBG and HbA1c levels, reduced glucose-6-phosphatase activity, GLP-1 levels, insulin secretion, and DPP-4 inhibition in diabetic rats. Mainly involved is the antidiabetic action in intestinal cells release GLP-1, while pancreatic tissue inhibits DPP-4. Identification of the pathways that FPH supplementation targets for both combinatorial and synergistic effects is being attempted. By utilizing DPP-4 inhibitors and GLP-1 agonists as new mechanistic targets, this may lead to the discovery of unique therapeutic advantages. Many synthetic medications, including metformin, glimepiride, voglibose, pioglitazone, and sitagliptin are used today to treat T2DM. Nevertheless, these medications have a number of adverse effects, such as gastrointestinal, renal, and urinary tract infections (Wang et al., 2023 ; Anang et al., 2019 ). With understanding the existing circumstances, our investigation was expanded by the antidiabetic effects of FPH at different doses were investigated against rats with T2DM caused by STZ and HLD. Furthermore, we carried out a mechanistic analysis of the synergistic effects of DPP-4, GLP-1, glucose 6-phosphate, and glucokinase using STZ-induced in diabetic rat model. When 50 mg/kg body weight FPH was given to diabetic rats, the rats' fasting plasma glucose levels were dramatically lowered, and insulin production was increased, hence enhancing the antihyperglycemic benefits of the supplement (Ashraf et al., 2021 ; Das et al., 2024 ). Several mechanisms have been investigated to explain the antihyperglycemic effects of FPH, such as the inhibition of DPP-4 and the elevation of plasma GLP-1 levels. Reducing FBG and increasing insulin production are two important effects of DPP-4 inhibition. Increased DPP-4 activity leads to the breakdown of GLP-1, a protein that increases insulin production and inhibits the release of glucagon. Pancreatic β cells exhibit high levels of expression for this enzyme, especially when oxidative stress occurs. Being a serine protease, DPP-4 breaks the peptide link that unites proline and alanine in GLP-1 (Kaleshkumar et al., 2019 ; Pyne et al., 2021 ) The study reveals that different doses of FPH mainly 50 mg/kg body weight, considerably lowers the amount of DPP-4 in pancreatic tissue as compared to other doses. Lipid peroxidation and antioxidant enzyme activity in pancreatic tissue were analysed to assess oxidative stress. Free radicals are created in order to cause T2DM in rats by a high-fat, low-dose STZ regime (Kaleshkumar et al., 2019 ; Das et al., 2024 ). In the diabetic population, an extend the duration of hyperglycaemic state results in an elevated oxidative stress state. Our body produces antioxidants to combat oxidative stress, which is caused by free radicals. Some of the antioxidants which is produced in our bodies are- SOD, CAT, and GSH (Bhowmick et al., 2023 ). In this work, we chronicled how the beginning of diabetes distressed the oxidative stress marker. A crucial function of the antioxidant enzyme SOD is to protecting cells from oxidative damage. Oxidative stress causes the superoxide radical to transfigured to hydrogen peroxide, which is subsequently bring down prior to water molecules (Das et al., 2024 ). GSH lowers peroxidative stress by averting free radicals from oxidizing through lipid peroxidation. As fatty acid disassembles releases MDA, our bodies exhibit higher levels of lipid peroxidation, which leads to increased oxidative stress, and higher MDA congregation (Bhowmick et al., 2023 ). According to our findings, the rats' oxidative stress from the disease's progression has decreased. Rats receiving various dosages of FPH escaped from death. Huang and Wu have documented the function of shark fish liver in lowering oxidative stress during the incident of diabetes (Huang et al., 2010) AO and Et-Br fluorescent staining enables the detection of morphological changes associated with apoptosis, allowing for the discrimination between normal cells, early and late apoptotic cells, and necrotic cells. AO penetrates cells with intact membranes, including normal and early apoptotic cells, emitting green fluorescence in untreated pancreatic cells. In contrast, Et-Br only enters cells with compromised membranes, such as late apoptotic and dead cells, producing orange-red fluorescence upon binding to condensed DNA fragments or apoptotic bodies. Notably, dual AO/Et-Br staining can detect mild DNA damage. Our results show that rats fed 50 mg/kg FPH exhibit similar characteristics to control rats and vehicle control rats, suggesting a potential protective effect of FPH (Dutta et al., 2023 ; Muscle Blaze Biozyme Iso-Zero, 2023). Moreover, DAPI staining revealed that pancreatic cells from T2DM rats treated with FPH displayed characteristic apoptotic features, including chromatin condensation and nuclear fragmentation, indicative of cellular apoptosis (Dutta et al., 2023 ). Histopathological examination of the pancreas further substantiated this hypothesis, revealing that FPH protected against the detrimental effects of STZ-induced T2DM. Notably, FPH displayed the comparable mode of action to that of the vehicle control and control groups, specifically, enhancing insulin secretion and promoting pancreatic function. This hypothesis has been further confirmed by the basic morphological changes in apoptotic cells and identifies the distinction between normal cells, early and late apoptotic cells, and necrotic cells. The apoptotic cell death results obtained by flow cytometer were shown in Table 1 and Fig. 8 The results show scattered plot, which was created fluorescence colour to generate through FITC and PI staining. It shows early cell apoptosis followed by late apoptosis due to induction of STZ in T2DM rats which coincidentally converts into necrotic cell death. Supplementation of FPH at the dose of 50 mg/kg b. w. shown the lowest apoptosis. Supposition of this study that many therapeutic peptides present in FPH and they play important role to manage ROS and elevation of GLP-1 level with impediment of DPP-4 action in pancreas, thereby, lower the hyperglycaemia (Fig. 10 ). Conclusion The emergence of the population with diabetes has been climbing day by day. Hence, the exploration for an acceptable food gleaned integrant is in great demand. Fish protein hydrolysates (FPH) of volavetki ( Panna microdon ) plays a key role in controlling and the management of type 2 diabetes mellitus (T2DM) through inhibiting pancreatic DPP-4 activity and hepatic glucose 6- phosphatase activity also enhancing pancreatic glucokinase activity with increasing the action of GLP-1. Various peptides in FPH presupposed to up-down regulation in cellular enzymes which approbates anti-diabetic activity. Principally, the FPH exemplified their potential to improve glycemia and ought to be contemplated in the admonishment of T2DM. Methods Chemicals and instruments Major biochemical parameter was measured using diagnostic kit of glycosylated haemoglobin (HbA1c) purchased from agappe diagnostics LTD, Hills, Ernakulam, Kerala, India. Fasting blood glucose (FBG) performed from tail vein of rats by using blood glucose monitoring system with strips (Apollo pharmacy, APG01). We assayed enzyme-linked immunosorbent assay (ELISA) kits like insulin, and c-peptides purchased from Weldon biotech Pvt. Ltd, Patparganj, Mayur Vihar, Delhi, India, glucose 6-phosphatase, glucagon like peptide-1 (GLP-1), and DPP-4, purchased from wuxidonglin sci & tech development Co. Ltd, Wuxi, Jiangsu, India. Anti-diabetic drug and Streptozotocin (STZ) were come by sisco research laboratories Pvt. Ltd, Taloja, Maharashtra, India. We brought the Protease enzyme (LOT 0000562173) from HIMEDIA laboratories Pvt. Ltd., Nashik, India. All other chemicals and enzyme like calcium chloride (Cacl 2 ), magnesium chloride (MgCl 2 ), hank’s balanced salt solution (HBSS), phosphate buffer saline (PBS) solution, potassium dihydrogen phosphate (KH 2 PO 4 ), potassium chloride (KCL), D- glucose, sodium chloride (NaCl), magnesium sulphate (MgSo 4 ), sodium hydrogen phosphate (Na 2 HPO 4 ), and bicarbonate of soda (NaHco 3 ) were picked from SRL, India, and MERCK, India, HiMedia Laboratories Pvt. Ltd. Mumbai, India, and Crest Biosystems Goa, India and protease enzyme was purchased from Caltech life science, Bhubaneswar, Odisha, India. Anti-oxidant enzyme parameters like glutathione (GSH), superoxide dismutase (SOD), catalase (CAT) was determined by absorbance UV-VIS double beam Spectrophotometer (systronics, India). Protein hydrolysate from volavetki ( Panna microdon ) was performed using tissue homogenizer (Eltek), pH matter (Digital), Incubator with shaker (YONA R ), cold centrifuge (Eltek), freeze dryer (ICC-INSTIND). And other instruments were used for various examination like fluorescence inverted microscope (Magnus), flow cytometer (Beckman coulter), semiautoanalizer (MISPA VIVA) and fluorescence microscope (Olympus, Model No. BX43F). Digital weight balance (accuracy-0.1mg) Adhair Dutta and Sons was used for weighted all materials. Preparation of volavetki fish protein hydrolysates (FPH) Sea fish volavetki ( Panna microdon , Bleeker, 1949) sea fish was freshly purchased from the local market of Digha mohona (21°38′18″N 87°30′35″E), coastal area which is located along the Bay of Bengal, Purba Medinipur, West Bengal, India. The samples were packed in ice container. The sample were identified by Assistant professor, Department of Zoology, Belda College and a specimen was deposited at department of Zoology Belda College, Belda, Paschim Medinipur, West Bengal, India. FPH was prepared by step by step, muscles were separated from volavetki sea fish then rinsed with cold distilled water and make fine chopped. Next, 1 kg of fish muscle were mixed in 10L HBSS buffer keeping on ice container and completed the homogenization process. Then, enzymatic hydrolysis was conducted in protease at an enzyme-to-substrate ratio (wt/wt) of 1:100 at 37°C (pH 7.4) under constant stirring in incubator cum shaker (CareLab) for 30 minutes. Next, the samples were centrifuged at 5,000 × g for 10 min at 4°C, and the supernatants were collected and freeze-dried (Harnedy et al., 2017 ; Bunsroem et al., 2022 ; Asokan et al., 2019 ). Identification of peptides and amino acid sequences by nano liquid chromatography mass spectrometry (nLC‒MS/MS) Finding the bioactive substances that are contained in the FPH by nLC‒MS/MS. In briefly, 50 mM iodoacetamide alkylated with 5 mM and tris 2- carboxyethyl phosphine (TCEP) then breakdown with Trypsin (1:50, Trypsin/lysate ratio) at 37°C for 16 hours. A speed vacuum was used to dry the digests after they had been cleaned with C18 silica cartridge for elimination of any salt. After dying the pellet was re-suspended with buffer A, concentrated at 0.1% formic acid and 5% acetonitrile. Thermo QE and nano LC 1200 were used for the experiments. On a Thermo Fisher Scientific C18 column measuring 50 cm and 3.0µm Easy-spray, one unit was loaded. Peptides were injected for MS analysis after being eluted using a 0–40% gradient of buffer B (80% acetonitrile, 0.1% formic acid) at a flow rate of 300 nl/min. The LC gradients ran for 60 minutes. At 70k resolution, MS1 spectra were obtained using the Orbitrap. Dynamic exclusion was used to exclude all charge states for a given precursor for 10 seconds. At 17500 resolutions MS2 spectra were obtained (Harnedy et al., 2017 ; Elam et al., 2021 ; Asokan et al., 2019 ). Selection of animal’s care, and handling Healthy adult male Wistar strain albino rats weighing about 150 ± 20 g were purchased from Saha Enterprise, 386/2, Nilachal, Birati, Kolkata-700051, Registration number: 1828/PO/BT/S/15/CPCSEA. Experimental process was approved by Institutional animal ethics committee (IAEC-CPCSEA) registration number of 1905/PO/Re/S/16/CCSEA for Raja Narendra Lal Khan Women’s College (Autonomous). The rats were housed inside a well-ventilated room in metal wire cages with free access to food and water, at 25 ± 1°C, humidity 60 ± 5%- and 12-hours dark-light cycle and throughout the experimental period. We strictly followed the scientific procedure using experimental animal as per CPCSEA (Approval No. 01/IAEC (1)/S/RNLKWC/2023) and ARRIVE guideline. T2DM induction Animals were acclimatized on high lipid diet (HLD) diet (Pyne et al., 2023 ), which included the following ingredients: cholesterol 0.5g, corn starch 40g, mineral mixture 0.5g, casein 20g, milk powder, sucrose 10g, bengal gram flour 15g, vitamin premix 0.5g, coconut oil 13ml, and salt 0.5g for 180 days. After acclimatization, for all rats other than the vehicle control rats and control rats, a single intraperitoneal injection of freshly made STZ (0.5 mL of pH 4.5, 0.01 M citrate buffer; 40 mg/kg b.w.) was used to induce T2DM. Vehicle control rats were injected with 0.5 mL of 0.01 M citrate buffer (pH 4.5). After 72 hours of injection, animals exhibiting the blood glucose levels greater than 250 mg/dL were considered as T2DM and sorted into 8 groups of 5 animals per group. Experimental design Randomly allocated total 40 rats and divided into 8 groups (n = 5) as follows: Group 1: Control rats fed HLD. Group 2: Vehicle control rats fed HLD and a single intraperitoneal injection of 0.5 mL of 0.01 M citrate buffer. Group 3: T2DM rats fed HLD without any supplementation. Group 4: T2DM rats fed HLD and FPH (12.5mg/kg of b.w.) supplementation for 28 days. Group 5: T2DM rats fed HLD and FPH (25mg/kg of b.w.) supplementation for 28 days. Group 6: T2DM rats fed HLD and FPH (50mg/kg of b.w.) supplementation for 28 days. Group 7: T2DM rats fed HLD and FPH (75mg/kg of b.w.) supplementation for 28 days. Group 8: T2DM rats fed HLD and standard anti-T2DM oral medicine, namely, gliptin dose of 12mg/kg of b.w. for 28 days through oral gavage After completion of the experimental period, the rats were decapitated to avoid stress. Blood was collected from all rats, and centrifuged at 5000 × g for 10 min to separate the plasma. Then plasma and the organs i.e., pancreas and liver were stored at − 20°C for further analysis. Measurement of FBG and HbA1c levels FBG and HbA1c from whole blood was measured 5 times to confirm sustained hyperglycemia during the experimental period at 0, 7, and 14, 21, 28 days from tail vein of rats, using a glucometer and FBG level was expressed as mg/dL. The standard kit method was utilised to assay HbA1c. Briefly mixing 10 µL of whole blood with 0.5 mL of haemolyzed reagent and allow at room temperature for 10 minutes to ensure complete lysis. Next, 300 µL latex and 10 µL hemolysate sample were combined, and the mixture was incubated at 37 ◦ C for 5 minutes. Next, 100 µL anti-human HbA1c mouse monoclonal antibody was added, and the mixture was again incubated at 37 ◦ C for 5 minutes. The output was then measured using a semiautoanalyser at 360 nm wavelengths. The expression for HbA1c was g% (Pyne et al., 2023 ). Estimation of plasma insulin, c-peptide, and GLP-1 The plasma insulin and c-peptide were estimated by an ELISA plate reader with standard kit method. In brief, 50 µL of plasma was combined with 100 µL of insulin and c-peptides enzyme reagent to each well and allow for 120 mins at 25 ◦ C -27 ◦ C. Then discard the contents and added 350 µL, 0.01 M of PBS buffer (pH 7.2) with deionized water and washed for two times. Then added 100 µL tetramethylbenzidine substrate and incubated for 15 mins at room temperature and finally added 50 µL hydrogen peroxide (H2O2) in buffer (0.01M PBS buffer, pH 7.4 containing deionized water) and read the absorbance at 450 nm in ELISA plate reader within 30 mins. Unit of c-peptide and insulin were expressed as ng/mL of plasma (Das et al., 2024 ; Pyne et al., 2023 ). GLP-1 was estimated by an ELISA plate reader with standard kit method. In short, 50 µL of plasma was combined with 50 µL of biotin labelled GLP-1 to each well and allow for 60 mins at 37 ◦ C. Then discard the contents and added 300 µL, 0.01 M of PBS buffer (pH 7.2) containing deionized water and washed three times. Then added 100 µL substrate and incubated for 60 mins at 37 ◦ C. Finally added 50 µL hydrogen peroxide (H2O2) in buffer (0.01M PBS buffer, pH 7.4 containing deionized water) and read the absorbance at 450nm in ELISA plate reader within 30 mins. GLP-1 was expressed as ng/mL of plasma (Das et al., 2024 ). Pancreas and liver tissue preparation for determination of in vivo antioxidant activities Homogenates of pancreas and liver tissues were prepared using a 150 mM, pH 7.4 phosphate buffer solution and then used to measure the activities of enzymes (Das et al., 2024 ; Das et al., 2016 ;). Determination of DPP-4 and glucokinase activity in pancreas and glucose 6- phosphatase activity in liver Activities of DPP-4 and glucokinase in pancreatic tissue, and activities of glucose 6-phosphatase in liver were estimated by using ELISA kit method. Briefly, 20 µL of pancreatic tissue sample, 30 µL of 0.02 M Tris-HCl buffer (pH 8.0), 0.10 M NaCl and 1 mM EDTA were mixed with 50 µL of DPP-4 standard and incubated at 37 ◦ C for 60 minutes. Then discard the contents and 300 µL of 0.02 M Tris-HCl buffer (pH 8.0) was added and washed for three times. Then 100 µL substrate of 200 µM H-Gly-Pro-AMC was added and incubated at 37 ◦ C for 60 minutes. Finally, we measured the absorbance at 450nm in ELISA plate reader within 30 mins. DPP-4 was expressed as pg/g of tissue (Pyne et al., 2023 ). For determination of glucokinase activity- 20 µL of pancreatic tissue sample, 30 µL of 0.02 M Tris-HCl buffer (pH 8.0), 0.10 M NaCl and 1 mM EDTA were combined with 50 µL of glucokinase standard and incubated at 37 ◦ C for 60 minutes. Then discard the contents and 300 µL of 0.02 M Tris-HCl buffer (pH 8.0) was added and washed for three times. Then added 100 µL substrate (200 µM of H-Gly-Pro-AMC) and incubated for 60 mins at 37 ◦ C. Absorbance was measured at 360nm and 460 nm wavelengths respectively in ELISA plate reader within 30 min. Glucokinase activity was expressed as ng/µg (Das et al., 2024 ; Pyne et al., 2023 ). For activities of glucose 6- phosphatase in brief, 20 µL of hepatic tissue sample, 30 µL of 0.02 M Tris-HCl buffer (pH 8.0), 0.10 M NaCl and 1 mM EDTA were combined with 50 µL of glucose 6- phosphatase standard and incubated at 37 ◦ C for 60 minutes. Then discard the contents and 300 µL of 0.02 M Tris-HCl buffer (pH 8.0) was added and washed for three times. Then 100 µL of 200 µM H-Gly-Pro-AMC substrate was added and incubated for 60 minutes at 37 ◦ C. Then measured the absorbance at 360nm and 460 nm wavelengths respectively in ELISA plate reader within 30 mins. Glucose 6- phosphatase activity was demonstrated as U/g of tissue (Das et al., 2024 ; Pyne et al., 2023 ). Determination of antioxidant enzyme activities and oxidative stress marker At first pancreatic tissues were homogenised with 100 mM ice-cold Tris-cacodylate buffer with 50 mg/mL of tissue concentration and centrifuged at 4ºC for 20 minutes at 10,000 rpm. Utilising a UV-VIS double beam spectrophotometer set at 240 nm wavelength. 50% reduction of pyrogallol's auto-oxidation was demonstrated by one unit of SOD enzyme activity. The activity of SOD was defined just as Unit/mg of tissue/min (Giri et al., 2019 ; Bhowmick et al., 2023 ). 50 mg per each mL of pancreatic tissues were homogenised with 0.05 M Tris-HCl (pH 7.0) buffer. Separate centrifugations of these homogenates were conducted at 4°C for 10 minutes with 4,000 rpm. CAT was determined in tissue supernatant using the protocol developed in our laboratory, and measured the absorbance at 240nm using a UV-VIS double beam spectrophotometer. The CAT activity was demonstrated as mmol of H2O2 consumption/mg of tissue/ min (Giri et al., 2019 ; Bhowmick et al., 2023 ). Our standard laboratory procedure was used to determine the MDA level. At 535nm of spectrophotometer the measurement was carried out including moral values are intimate as nmol/dL of pancreatic tissue (Pyne et al., 2023 ). Quantification of GSH was done by pancreatic tissue homogenate of 0.2 M sodium phosphate (pH 8.0) buffer and combined with dithio-bis-nitrobenzoic acid and measured using a UV-VIS double beam spectrophotometer set to 405 nm according to the standard method. The levels of GSH were demonstrated as Units/mg protein (Das et al., 2016 ). Apoptotic morphological changes by acridine orange (AO) and ethidium bromide (AO/ EtBr) staining detection Two amazing DNA-binding dyes called AO and Et-Br were used to detect apoptotic and necrotic cells. Pancreatic cells were washed with 50 mM of cold PBS, pH 7.4. Then AO and Et-Br mixture were used for staining. The staining procedure was taking place for 5 minutes at 25°C. After that, the cells were washed for three times with PBS, then under a fluorescence microscope it was examined at 40x magnifications (Dutta et al., 2023 ). Determination of nuclear integrity and apoptosis by 4’,6-diamidino-2-phenylindole (DAPI) stanning For preparation of DAPI staining, cold PBS buffer was used for cells washed, 2.5% glutaraldehyde was used for cells fixing for 15 minutes, permeabilized with 0. 1% Triton X-100, and then stained with 1 mg/mL DAPI for 5 minutes at 37°C. After that, the cells were washed again with PBS and examined under fluorescence microscopy at 40x (Dutta et al., 2023 ). Pancreatic cell apoptosis assay by flow cytometry To assess the mechanism of pancreas tissue cell death, the apoptosis and necrosis assay were measured by flow cytometer using Annexin A5 - FITC apoptosis detection kits. Briefly, cells were placed in cultured solution (DNase100 mg in HBSS) then centrifuged at 4˚ C for 5 minutes at 5,000 rpm. Next, afloat was collected and add 200µl binding buffer for cell suspension. Before analysis, 5µL of propidium iodide and 1µL of annexin A5-FITC was mixed and incubate at room temperature in dark place for 15 minutes. After that 400µL 1x ice cold binding buffer was added. With the help of Flow Cytometer, the quantity of apoptosis and necrosis cells were analysed (Dutta et al., 2023 ). Histological preparation The pancreas specimen was fixed in 5% formaldehyde solution and surrounded in paraffin. In the histopathological analysis, 3 sections were evaluated from each rat and 3 fields were evaluated from each section. Sections were cut at 5µm thick with the microtome machine consequently deparaffined with xylene. Every piece of the rat pancreas paraffin block was sectioned (5µm) prior to staining with haematoxylin and eosin staining. Then slides were observed by using invert microscope with digital camera (Das et al., 2024 ; Pyne et al., 2023 ). Statistical analysis Data are presented as mean ± standard error of means obtained from 5 rats per group to evaluate variances between the groups. All assays were tested in 3 replicates by one way ANOVA test the statistical analysis was performed by originpro 6.1 software, following the Bonferroni's modification for determination of statistical significance (P < 0.05, P < 0.01, P < 0.001) among all groups compared to diabetic group (Das et al., 2024 ). Abbreviations T1DM Type 1 diabetes mellitus T2DM Type 2 diabetes mellitus GLP-1 Glucagon like peptide-1 DPP4 Dipeptidyl peptidase-4 IFD International Diabetes Federation HLD High lipid diet FPH Fish protein hydrolysates STZ Streptozotocin HbA1c Glycosylated haemoglobin FBG Fasting blood glucose GSH Glutathione SOD Superoxide dismutase CAT Catalase nLC-MS/MS Nano liquid mass spectrometric DAPI 4’,6-diamidino-2-phenylindole AO/ Et‑Br Acridine orange and ethidium bromide Declarations Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Dr. Koushik Das reports financial support was provided by Belda College. Koushik Das reports a relationship with Belda College that includes: employment. No If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Ethics approval and consent to participate Study was designed on Wister stain albino rat model which was approved by the Institutional Animal Ethics Committee (Approval No. 01/IAEC (1)/S/RNLKWC/2023) dated 15.06.2023 of Raja Narendra Lal Khan Women’s College (Autonomous). Consent for publication Not applicable. Animal Ethics declaration All experimental procedures involving animals were conducted in accordance with the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) and the ARRIVE guidelines. The study protocol was approved by the Institutional Animal Ethics Committee (Approval No. 01/IAEC (1)/S/RNLKWC/2023) dated 15.06.2023 of Raja Narendra Lal Khan Women’s College (Autonomous). Availability of data and materials Raw data were generated by the Raja Narendra Lal Khan Women’s College (Autonomous) and data supporting the finding of the study are available from the corresponding author upon reasonable request. Competing interests The authors declare that no conflict of interest. Funding No fund Authors' contributions Supriya Bhowmick: Writing – original draft, Methodology, Data analysis Shayan Panda: Methodology, Data analysis, Investigation, Writing – original draft Madhumita Pal: Methodology, Investigation, Writing – original draft Sanjay Das: Methodology, Investigation Meghamala Mandal: Writing – review & editing Shrabanti Pyne: Writing – review & editing Deblina Giri: Methodology, Investigation Koushik Das: Conceptualization, Writing – review & editing Acknowledgment The authors thank the DST-FIST (Memo no. SR/FST/College-2017/03 (C), 29 th August 2018) for equipment and infrastructure facility for Raja Narendra Lal Khan Women’s College (Autonomous). 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19:32:54","extension":"html","order_by":60,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":181007,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7469537/v1/ab1e6e14527a36adbc1fb900.html"},{"id":92208678,"identity":"9e4e3b3d-4100-4396-a14b-35a0398faf70","added_by":"auto","created_at":"2025-09-25 19:32:52","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":153561,"visible":true,"origin":"","legend":"\u003cp\u003ePathophysiological main mechanism of type 2 diabetes mellitus.\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7469537/v1/1f4877ed547f6917a4cf0e63.jpg"},{"id":92208679,"identity":"145bf332-3766-45a5-b82f-38d52f74dd6e","added_by":"auto","created_at":"2025-09-25 19:32:52","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":43729,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of different doses of FPH on blood glucose (mg/dl) (A) and HbA1c (mg/dL) (B) level in STZ- induced T2DM rats. Data were expressed as mean ± SE (n=5). \u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e*\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e P \u0026lt; 0.05, \u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e**\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e P \u0026lt; 0.01, and \u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e***\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eP \u0026lt; 0.001 when compared with diabetic group.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7469537/v1/390dfcc9558c3756c9da1b8c.jpg"},{"id":92208680,"identity":"639bcbfb-5f9c-4daf-99cc-26e74720eebf","added_by":"auto","created_at":"2025-09-25 19:32:52","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":72162,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of FPH on plasma insulin (ng/mL) (A), C-peptide (ng/mL) (B), and GLP-1 (ng/mL) (C) levels in STZ- induced T2DM rats. Data are expressed as mean ± SE (n=5). \u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e*\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e P \u0026lt; 0.05, \u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e**\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e P \u0026lt; 0.01, and \u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e***\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eP \u0026lt; 0.001 when compared with diabetic group.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7469537/v1/02c792450da39ea1126525b8.jpg"},{"id":92209357,"identity":"73daaa44-730f-43aa-839c-958a285ea054","added_by":"auto","created_at":"2025-09-25 19:48:53","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":66314,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of FPH on DPP-4 (pg/mL) (A) and glucokinase (ng/µg) (B) activities in pancreas tissue and Glucose-6-phosphatase (U/g of tissue) (C) activity of liver tissue in STZ- induced T2DM rats. Data are expressed as mean ± SE (n=5). \u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e*\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e P \u0026lt; 0.05, \u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e**\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e P \u0026lt; 0.01, and \u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e***\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eP \u0026lt; 0.001 when compared with diabetic group.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7469537/v1/99b5724f6a9c749f01b360b8.jpg"},{"id":92209175,"identity":"344f3b47-7dbe-4bd5-82bd-351244a8763f","added_by":"auto","created_at":"2025-09-25 19:40:52","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":85839,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of FPH on antioxidant enzyme activities like SOD (Unit/mg of tissue/min) (A), CAT (m mol of H2O2 consumption/mg of tissue/min) (B), and GSH (Units/mg of protein) (C) and lipid peroxidation (nmol/mg of tissue/min) (D) activity in STZ- induced T2DM rats. Data are expressed as mean ± SE (n=5). \u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e*\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e P \u0026lt; 0.05, \u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e**\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e P \u0026lt; 0.01, and \u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e***\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eP \u0026lt; 0.001 when compared with diabetic group.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Picture5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7469537/v1/d2cd1673b34d11a427fb3dd4.jpg"},{"id":92208688,"identity":"8477d12d-b631-43a7-a445-6c0b7120785c","added_by":"auto","created_at":"2025-09-25 19:32:53","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":261447,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDetection of apoptosis by acridine orange and ethidium bromide (AO-EtBr) staining in pancreas were done at 40x magnification in fluorescence microscope (Olympus) of different treatment groups control, vehicle control, T2DM, T2DM + FPH 12.5mg/ day, T2DM + FPH 25 mg/ day, T2DM + FPH 50 mg/ day, T2DM + FPH 75 mg/ day, and T2DM + OHA.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Picture6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7469537/v1/216b3f4133547a2049afae6f.jpg"},{"id":92209358,"identity":"db497851-3d2d-4a61-b6a2-1c7803835c1f","added_by":"auto","created_at":"2025-09-25 19:48:53","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":153282,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDetection of intracellular reactive oxygen species (ROS) measurement by DCFH2 staining in pancreas were done at 40x magnification in fluorescence microscope (Olympus) of different treatment groups control, vehicle control, T2DM, T2DM + FPH 12.5mg/ day, T2DM + FPH 25 mg/ day, T2DM + FPH 50 mg/ day, T2DM + FPH 75 mg/ day, and T2DM + OHA.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Picture7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7469537/v1/1ed253ca86398abcd4da6b7c.jpg"},{"id":92208694,"identity":"ce95eb95-7d87-4f48-9d41-329d4d52e421","added_by":"auto","created_at":"2025-09-25 19:32:53","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":334932,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFlow cytometry illustrating represents the effects of FPH of late apoptosis cells (Q1-UL), necrotic cells (Q1-UR), live cells (Q1-LL), and early apoptosis cells (Q1-LR) on HLD and STZ induced T2DM rats in different groups; control, vehicle control, T2DM, T2DM + FPH 12.5mg/ day, T2DM + FPH 25 mg/ day, T2DM + FPH 50 mg/ day, T2DM + FPH 75 mg/ day, and T2DM + OHA.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Picture8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7469537/v1/3c65ea96f7aacfa7e413fcdc.jpg"},{"id":92209179,"identity":"da082def-b47d-4bce-9e4a-7f4c49a01286","added_by":"auto","created_at":"2025-09-25 19:40:53","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":173689,"visible":true,"origin":"","legend":"\u003cp\u003eMicrophotographs of histological changes in pancreas at 40x magnification of different treatment groups control, vehicle control, T2DM, T2DM + FPH 12.5mg/ day, T2DM + FPH 25 mg/ day, T2DM + FPH 50 mg/ day, T2DM + FPH 75 mg/ day, and T2DM + OHA. Here, each square box denotes the area (μm2) of the islets of Langerhans of pancreas. Where control 292.76 μm2, vehicle control 322.31 μm2, T2DM 31.84 μm2, T2DM+FPH12.5 59.54 μm2, T2DM+FPH25 55.67 μm2, T2DM+FPH50 264.77 μm2, T2DM+FPH75 257.84 μm2.61, and T2DM+OHA 86.76 μm2. Scales bar = 10μm. Magnification= 10x.\u003c/p\u003e","description":"","filename":"Picture9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7469537/v1/62a41bb0d8b3ccdbfe1238ec.jpg"},{"id":92273662,"identity":"e57da31c-dc76-45c9-96c4-206d1c21baaa","added_by":"auto","created_at":"2025-09-26 15:14:13","extension":"jpg","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":63921,"visible":true,"origin":"","legend":"\u003cp\u003eA notion of antidiabetes role of FPH via DPP-4 and GLP-1 mechanistic pathway.\u003c/p\u003e","description":"","filename":"Picture10.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7469537/v1/dbb547293e130243caa7e8b8.jpg"},{"id":101151723,"identity":"bded15a0-dad1-4551-85d0-98e928b09604","added_by":"auto","created_at":"2026-01-26 16:03:05","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3521777,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7469537/v1/63e3a986-b1e9-49c0-bc8b-fc89244b42a9.pdf"},{"id":92209178,"identity":"fc587395-ce02-4022-b49b-0e18d071cb8f","added_by":"auto","created_at":"2025-09-25 19:40:53","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":36332,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFile.docx","url":"https://assets-eu.researchsquare.com/files/rs-7469537/v1/1f012dedff7fec2cac3054f2.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Antidiabetic potential of volavetki sea fish protein hydrolysates in high lipid diet and streptozotocin-induced diabetes rats","fulltext":[{"header":"Background","content":"\u003cp\u003e\u003c/p\u003e\u003cp\u003eLow or no insulin emission from pancreatic β-cells cause hyperglycaemia, called type 1 diabetes mellitus (T1DM) (Zhao et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) Partly insulin production from pancreatic β-cells and /or insulin refusal to accept by skeletal muscle and fat cells causes elevation of blood glucose levels which is known as type 2 diabetes mellitus (T2DM) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) (Wan et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). T1DM is stemmed by the collapse of the pancreas to synthesize insulin due to autoimmune desolation of pancreatic β-cells linked with human leukocyte antigen (HLA) gene defect (Zhao et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Kilari et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). T2DM is promoted by excessive food intake, a high lipid diet, oxidative and mental stress, obesity, lack of physical activity and other many causes. Amidst, T2DM is comprehensively prevalent above 90% of all cases (Nasri et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). T2DM patients have been raising the intensity of hyperglycaemia as a consequence of glucagon like peptide-1 (GLP-1) degradation. GLP-1 is a peptide hormone secreted from intestinal L cells in response to food intake (Nasri et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Iwase et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). GLP-1 binds with GLP-1 receptors present in pancreatic β-cells and exerts insulinotropic action by withdrawn the effect of glucagon. Dipeptidyl peptidase-4 (DPP-4) rapidly degrades GLP-1 and inhibits the insulin secretion from pancreatic β-cells in a T2DM patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.) (Iwase et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Rivero-Pino et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eInternational Diabetes Federation (IDF) reported in 2021 that 1 in 10 adults (20\u0026ndash;79 years) has been suffering with diabetes which will be projected 1 in 7 adults for the year 2030 (Ashraf et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; IDF, 2021). DPP-4 inhibitors like gliptin have been successfully used to treat and manage T2DM. However, this medicine has gastrointestinal side effects, weight gain, bone loss, headaches, upper respiratory tract infections, and other side effects that restrict their use (Yun et al., 2021; Harnedy et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The scientific community was inspired to look for risk-free complementary therapy of diabetes that may be exploited to treat or prevent T2DM without having any negative side effects. The therapy and prevention of T2DM both heavily rely on dietary components (Wan et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Kilari et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Proteins, peptides, fatty acids, and polyphenols are examples of food-derived bioactive or therapeutic components that have gained considerable attention recently because to their high safety and wide range of biological capabilities, including anti-oxidant, anti-inflammatory, anti-hypertensive, anti-cancer, and immunomodulatory actions (Zhou et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Bunsroem et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Sea fish protein hydrolysates such as, tuna fish protein hydrolysates (Kaleshkumar et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), sardine fish protein hydrolysates (Nasri et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), Atlantic salmon fish protein hydrolysates (Harnedy et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) are significant sources of useful bioactive substances such proteins, peptides, and fatty acids that have been shown to have anti-oxidant, anti-inflammatory, anti-hypertensive, anti-cancer, and immunomodulatory characteristics (Harnedy et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). As well as, a mediterranean diet that includes a moderate amount of fish hydrolysate has revealed the benefits for the prevention T2DM (Wang et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Numerous systematic reviews and meta-analyses have found a link between higher fish/fish product consumption and a decreased incidence or risk of T2DM (Das et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Pyne et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In our earlier cross-sectional study, it has been revealed that sea fish eaters showed lower the incidence of T2DM when compared to fresh water fish eaters (Pyne et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). We have also experimentally established the potential effects of sea fish oil for the management of T2DM (Pyne et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The present study was aimed to scout out the antidiabetic sea fish protein hydrolysates from volavetki \u003cb\u003e(\u003c/b\u003e\u003cem\u003ePanna microdon)\u003c/em\u003e in high lipid diet along with streptozotocin induced T2DM rats.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eIdentification of bioactive peptides and amino acid sequences in FPH by nLC‒MS/MS analysis\u003c/h2\u003e\u003cp\u003eThirty-five unique peptides were analysed in FPH by nLC-MS/MS method which were given in supplementary file no.1. Tentative identification of peptides present in FPH were histone H2B, 2-iminobutanoate/2-iminopropanoate deaminase, histone H3, hemoglobin alpha chain, alpha1 hemoglobin, hemoglobin subunit beta, hemoglobin beta chain, methylmalonyl-CoA epimerase, O-acetyl-ADP-ribose deacetylase MACROD2, peptidyl-prolyl cis-trans isomerase, THAP domain-containing protein 4, receptor expression-enhancing protein, natural killer cell enhancer factor, myosin light chain 5, serine protease 57, ES1 protein, mitochondrial, carbonic anhydrase, homer protein-like protein 2, cytosolic 5'-nucleotidase 1B, keratin, type I cytoskeletal 50 kDa GK50, keratin, type II cytoskeletal 8, protein disulfide-isomerase, putative aminopeptidase NPEPL1, transmembrane protease serine 9, drebrin Developmentally-regulated brain protein, A-kinase anchor protein 8-like, junction plakoglobin, aconitate hydratase, mitochondrial, glycogen debranching enzyme, uncharacterized protein, nidogen-2, ryanodine receptor 3, treslin TopBP1-interacting checkpoint and replication regulator, gelsolin, nebulin.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eEffects of FPH on FBG and HbA1c levels\u003c/h3\u003e\n\u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e (A \u0026amp; B). shows the results of glucose metabolism monitored in the control, vehicle control, and experimental rats on day 0, 7, 14, 21 and 28 of the experiment. Overall, no significant (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) changes in FBG and HbA1c were measured in day 0 of the experiment of all groups of rats. A continuous increase in FBG level was observed in the T2DM rats from the day 3, which continued throughout the experimental period. On the other hand, there was no significant changes in HbA1c level between day 0 to day 21, at 28 days showed the significant changes of HbA1c level in T2DM rats. Administration of STZ led to a significant increase in FBG and HbA1c levels compared with those of the control and vehicle control rats. However, administered with various doses of FPH, FBG levels began to diminish from day 14 of the experiment. A significant reduction in FBG and HbA1c levels was found in the T2DM\u0026thinsp;+\u0026thinsp;FPH 50 (50 mg/kg of b.w.) rats compared with those in the T2DM rats (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) at the end of the experiment. The mean FBG and HbA1c levels in the T2DM\u0026thinsp;+\u0026thinsp;FPH 50 rats were progressively declined at the end of the final experimental day. These results indicated that T2DM\u0026thinsp;+\u0026thinsp;FPH 50 has the ability to reduce FBG and HbA1c levels, implying its anti-diabetic effects.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eEffects of FPH on the levels on insulin, c-peptide, GLP-1 in plasma\u003c/h3\u003e\n\u003cp\u003ePlasma insulin and c-peptides levels were significantly (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) lower in the STZ-induced T2DM rats when compared with the control rats. The supplementation of FPH at different doses (12.5, 25, 50, and 75 mg/kg b. w./day) increased the plasma insulin and c-peptides levels compared to T2DM rats. The effect was more pronounced at the dose of FPH 50 supplemented rats than in the other doses supplemented rats shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003e (A\u0026amp;B).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eEffects of FPH on DPP-4, glucokinase activities in pancreas tissue and glucose-6-phosphatase activity in liver tissue\u003c/b\u003e\u003c/p\u003e\u003cp\u003eT2DM rats unveiled significantly (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) increased pancreatic DPP-4 activity as compared to the control as well as vehicle control rats. The supplementation of FPH at different doses significantly (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) decreased the pancreatic DPP-4 activity as opposed to T2DM rats Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003e (A).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe glucokinase activity in pancreas tissue was significantly (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) lower in T2DM rats compared with the control rats. The supplementation of FPH at different doses significantly (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) increased the glucokinase activities compared to T2DM rats Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003e (B).\u003c/p\u003e\u003cp\u003eThe glucose 6-phosphatase activity of liver tissue was increased in T2DM rats as compared to control rats. A significant (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) reduction in glucose 6- phosphatase activity was observed in the FPH supplemented rats as compared to T2DM rats Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003e (C).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eEffects of FPH on oxidative stress markers\u003c/h3\u003e\n\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eEffects of FPH in pancreatic intracellular ROS generation and apoptosis\u003c/h3\u003e\n\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eEffects of FPH in pancreatic nuclear integrity and apoptosis in DAPI stanning\u003c/h2\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eEffects of FPH in cell apoptosis\u003c/h3\u003e\n\u003cp\u003eThe apoptotic cell death results obtained by flow cytometer were shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e8\u003c/span\u003e The results show scattered plot, which was created fluorescence colour to generate through FITC and PI staining. It shows early cell apoptosis followed by late apoptosis due to induction of STZ in T2DM rats which coincidentally converts into necrotic cell death. Supplementation of FPH at the dose of 50 mg/kg b. w. shown the lowest apoptosis.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eEffect of FPH on late apoptosis cells, necrotic cells, live cells, and early apoptosis cells of HLD and STZ induced T2DM rats in different groups. Data are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM (n\u0026thinsp;=\u0026thinsp;5). \u003csup\u003e*\u003c/sup\u003e P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, and \u003csup\u003e**\u003c/sup\u003e P\u0026thinsp;\u0026lt;\u0026thinsp;0.01 when compared with T2DM group.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGroups\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLive cells\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eEarly apoptotic cell\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eApoptotic cell\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNecrotic cell\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eControl\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e23.922\u0026thinsp;\u0026plusmn;\u0026thinsp;0.135*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e1.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.135**\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e43.806\u0026thinsp;\u0026plusmn;\u0026thinsp;0.085**\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e34.148\u0026thinsp;\u0026plusmn;\u0026thinsp;0.294**\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eVehicle control\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e17.258\u0026thinsp;\u0026plusmn;\u0026thinsp;0.220**\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e54.312\u0026thinsp;\u0026plusmn;\u0026thinsp;0.307*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e4.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.366**\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e20.694\u0026thinsp;\u0026plusmn;\u0026thinsp;0.386**\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eT2DM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e4.514\u0026thinsp;\u0026plusmn;\u0026thinsp;0.304\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e76.214\u0026thinsp;\u0026plusmn;\u0026thinsp;0.195\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e5.844\u0026thinsp;\u0026plusmn;\u0026thinsp;0.262\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e15.806\u0026thinsp;\u0026plusmn;\u0026thinsp;0.281\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eT2DM\u0026thinsp;+\u0026thinsp;FPH 12.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e5.048\u0026thinsp;\u0026plusmn;\u0026thinsp;0.276**\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e75.846\u0026thinsp;\u0026plusmn;\u0026thinsp;0.251*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e7.722\u0026thinsp;\u0026plusmn;\u0026thinsp;0.294**\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e14.998\u0026thinsp;\u0026plusmn;\u0026thinsp;0.366**\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eT2DM\u0026thinsp;+\u0026thinsp;FPH 25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e11.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.367**\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e67.134\u0026thinsp;\u0026plusmn;\u0026thinsp;0.324**\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e2.052\u0026thinsp;\u0026plusmn;\u0026thinsp;0.168*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e20.742\u0026thinsp;\u0026plusmn;\u0026thinsp;0.312**\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eT2DM\u0026thinsp;+\u0026thinsp;FPH 50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e22.742\u0026thinsp;\u0026plusmn;\u0026thinsp;0.290*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e44.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.234*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e1.124\u0026thinsp;\u0026plusmn;\u0026thinsp;0.135*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e36.128\u0026thinsp;\u0026plusmn;\u0026thinsp;.353*\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eT2DM\u0026thinsp;+\u0026thinsp;FPH 75\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e12.442\u0026thinsp;\u0026plusmn;\u0026thinsp;0.309*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e72.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.291*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e3.166\u0026thinsp;\u0026plusmn;\u0026thinsp;0.201*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e14.042\u0026thinsp;\u0026plusmn;\u0026thinsp;0.099**\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eT2DM\u0026thinsp;+\u0026thinsp;OHA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e13.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.268**\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e63.992\u0026thinsp;\u0026plusmn;\u0026thinsp;0.384*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e15.026\u0026thinsp;\u0026plusmn;\u0026thinsp;0.156**\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e9.814\u0026thinsp;\u0026plusmn;\u0026thinsp;0.264*\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eHistological observations\u003c/h3\u003e\n\u003cp\u003eThe pancreas histological examination was performed using haematoxylin and eosin staining, and the results are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e9\u003c/span\u003e Control rats exhibited normal architecture of pancreatic sections, with evenly organized islet of β-cells. Sections from the T2DM rats revealed damaged β-cells with irregular shape and reduced numbers, infiltration of lymphocytes, necrosis, and damaged globules. The pancreatic sections prepared from the FPH-supplemented rats showed necrotic changes with reduced β-cells and partially deranged structure. FPH 50 rats demonstrated preservation of pancreatic islets, with an increase in their number and size along with attenuation of the histological changes from severe to mild.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn the present study, focused on antidiabetic effects of FPH in HLD feeding and low dose STZ-induced T2DM rat model has been investigated. Our results demonstrated that \u003cem\u003ePanna microdon\u003c/em\u003e FPH has immensely bring down the blood glucose level endlessly 28 days of treatment and retrieved the rats from diabetes and reclaimed back to normal. T2DM is an incurable disorder, and it necessitates routine check-up, proper management by a patient to control the disease's progression and stop complications from emerging. There are many commercial medicines are available in the market that can be used to treat these complications (Wan et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). However, more recently, researchers focused in different approach by using marine organisms, their bioactive compounds against the occurrence of certain diseases.\u003c/p\u003e\u003cp\u003eThe nLC-MS/MS investigation recorded the presence of various bioactive compounds in \u003cem\u003ePanna microdon\u003c/em\u003e FPH. Unspecified amalgamation was reported for displaying antidiabetic property. Histone H2B, a positively charged nuclear protein, plays a crucial role in the compaction and organization of chromosomal DNA (Ali et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The enzyme 2-iminobutanoate/2-iminopropanoate deaminase catalyses the hydrolytic deamination of enamine and imine intermediates, which are transiently formed during cellular metabolic processes (Harnedy et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Histone H3 is a key epigenetic regulator that modulates gene expression by influencing chromatin structure and transcription factor accessibility (Muscle Blaze Biozyme Iso-Zero, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Methylmalonyl-CoA epimerase catalyses a crucial step in the metabolic degradation of branched-chain amino acids and odd-chain fatty acids, facilitating their conversion to succinyl-CoA for entry into the citric acid cycle (Kilari et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). MACROD2, an O-acetyl-ADP-ribose deacetylase, is a crucial enzyme in cellular nucleotide metabolism, regulating the turnover of O-acetyl-ADP-ribose and modulating intracellular signalling pathways, thereby influencing various cellular regulatory processes (Graham et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Peptidyl-prolyl cis-trans isomerases (PPIases) facilitate protein folding and refolding by catalysing the interconversion of cis and trans conformations at peptide bonds, thereby modulating protein structure and function (Bianchetti et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). THAP domain-containing protein 4 exhibits multifunctional enzymatic activities, including hemi binding, protein-protein interaction, and peroxynitrite isomerase functions, underscoring its potential role in various cellular processes (Fan et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Receptor expression-enhancing protein modulates the function and homeostasis of the endoplasmic reticulum (ER), playing a crucial role in regulating cellular processes, including protein synthesis, folding, and transport, thereby impacting overall cell function and survival (Masuda et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). ES1 protein regulates transcription, DNA synthesis, and cellular transformation (Dodgson et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1980\u003c/span\u003e). Mitochondrial carbonic anhydrase is essential for the expression of carbonic anhydrase V in pancreatic β-cells, which in turn enhances insulin secretion, highlighting its critical role in glucose metabolism and insulin regulation (Shiraishi-Yamaguchi et al., 2007). Homer protein-like 2 (HPL2) modulates intracellular signal transduction and vesicular trafficking, playing a crucial role in regulating cellular communication and protein transport (Ipata et al., 2013). Cytosolic 5'-nucleotidase 1B (cN1B) exerts inhibitory effects on basal lipid oxidation and glucose transport in skeletal muscle, thereby regulating metabolic flux and energy homeostasis (Raszewski et al., 2024). Ryanodine receptor 3 (RyR3) plays a crucial role in regulating the resting calcium ion concentration ([Ca2+] rest) in skeletal muscle cells, thereby modulating muscle tone and excitability (Bucki et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Gelsolin exerts anti-inflammatory effects in T2DM by decreasing microparticle-driven inflammation and suppressing NLRP3 inflammasome activation, leading to improved disease outcomes (Bae et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Nebulin plays a crucial role in maintaining the structural and functional integrity of skeletal muscle, ensuring proper contractile function and regulating actomyosin interactions (Huang et al., 2010). According to Li-Chan et al., the antidiabetic properties of Salmon protein hydrolysate (Li-Chan et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Wan et al. also revealed the antidiabetic effects of protein hydrolysates of \u003cem\u003eTrachinotus ovatus\u003c/em\u003e and their derived peptides, which are promising natural ingredients with the potential to be used for the treatment or prevention of diabetes (Wan et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). These peptides together play and important role in cellular as well as membrane signalling pathway through activating Ca2\u0026thinsp;+\u0026thinsp;channel- gene expression of transmembrane receptor protein- activating intracellular kinase which may contribute antidiabetic activity. The inhibitory effect of FPH on digestive enzymes prompted an investigation into its potential antidiabetic properties in a T2DM rat model induced by a combination of HLD and STZ. Previous studies have shown that low-dose STZ administration induces diabetes in animals, but this model lacks the insulin resistance characteristic of T2DM. To better mimic T2DM pathogenesis, we employed a combination of HLD and STZ, where HLD induces low-dose STZ along with insulin aversion causes pancreatic β-cell destruction, recapitulating the human disease (Bucki et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). For progression of T2DM, HLD for 180 days was required, followed by a single intraperitoneal injection of STZ at 40 mg/kg concentration was needed. According to earlier reports by Pyne et al. (Pyne et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) and Das et al. (Das et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), the optimal paradigm for the development of diabetes involves use of HLD for the period of 24 weeks and STZ injection at 40 mg/kg was one of the best tools.\u003c/p\u003e\u003cp\u003eOne of the important symptoms of diabetes is hyperglycaemia. Our research revealed that the blood glucose level of the T2DM group were predominantly raised throughout the study. Blood glucose was successfully regulated and T2DM was kept within normal ranges with the supplementation of FPH 50. Interestingly, the level of plasma insulin remained constant throughout the study. By raising blood glucose levels by HLD and STZ, it was seen that all of the induced group rats had developed T2DM.\u003c/p\u003e\u003cp\u003eOur findings were vindicated by an earlier investigation by Huang and Wu (Huang et al., 2010). According to his reports, the peptide that is isolated from the liver of sharks can assist blood glucose regulation. As well as Zhu et al., (Zhu et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) have also observed the same that the HLD and STZ-induced diabetic rats the blood glucose levels were lowered by an oligopeptide from the skin of marine Salmon. Furthermore, they reported that there was no discernible alteration in the insulin level during the experimental period.\u003c/p\u003e\u003cp\u003eIn T2DM rat, plasma insulin and c-peptide levels decreased but these diabetic sensors significantly increased by FPH supplemented group indicated to T2DM which managed by probably therapeutic or anti-T2DM peptides present in FPH. Several studies demonstrated that consumption of various sea fish protein hydrolysates can effectively enhance the postprandial insulin response and reduce postprandial blood glucose levels (Wan et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Graham et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The results of our study clarified that supplementation with FPH ameliorated FBG and HbA1c levels, reduced glucose-6-phosphatase activity, GLP-1 levels, insulin secretion, and DPP-4 inhibition in diabetic rats. Mainly involved is the antidiabetic action in intestinal cells release GLP-1, while pancreatic tissue inhibits DPP-4.\u003c/p\u003e\u003cp\u003eIdentification of the pathways that FPH supplementation targets for both combinatorial and synergistic effects is being attempted. By utilizing DPP-4 inhibitors and GLP-1 agonists as new mechanistic targets, this may lead to the discovery of unique therapeutic advantages. Many synthetic medications, including metformin, glimepiride, voglibose, pioglitazone, and sitagliptin are used today to treat T2DM. Nevertheless, these medications have a number of adverse effects, such as gastrointestinal, renal, and urinary tract infections (Wang et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Anang et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eWith understanding the existing circumstances, our investigation was expanded by the antidiabetic effects of FPH at different doses were investigated against rats with T2DM caused by STZ and HLD. Furthermore, we carried out a mechanistic analysis of the synergistic effects of DPP-4, GLP-1, glucose 6-phosphate, and glucokinase using STZ-induced in diabetic rat model. When 50 mg/kg body weight FPH was given to diabetic rats, the rats' fasting plasma glucose levels were dramatically lowered, and insulin production was increased, hence enhancing the antihyperglycemic benefits of the supplement (Ashraf et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Das et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eSeveral mechanisms have been investigated to explain the antihyperglycemic effects of FPH, such as the inhibition of DPP-4 and the elevation of plasma GLP-1 levels. Reducing FBG and increasing insulin production are two important effects of DPP-4 inhibition. Increased DPP-4 activity leads to the breakdown of GLP-1, a protein that increases insulin production and inhibits the release of glucagon. Pancreatic β cells exhibit high levels of expression for this enzyme, especially when oxidative stress occurs. Being a serine protease, DPP-4 breaks the peptide link that unites proline and alanine in GLP-1 (Kaleshkumar et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Pyne et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) The study reveals that different doses of FPH mainly 50 mg/kg body weight, considerably lowers the amount of DPP-4 in pancreatic tissue as compared to other doses. Lipid peroxidation and antioxidant enzyme activity in pancreatic tissue were analysed to assess oxidative stress. Free radicals are created in order to cause T2DM in rats by a high-fat, low-dose STZ regime (Kaleshkumar et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Das et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn the diabetic population, an extend the duration of hyperglycaemic state results in an elevated oxidative stress state. Our body produces antioxidants to combat oxidative stress, which is caused by free radicals. Some of the antioxidants which is produced in our bodies are- SOD, CAT, and GSH (Bhowmick et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In this work, we chronicled how the beginning of diabetes distressed the oxidative stress marker. A crucial function of the antioxidant enzyme SOD is to protecting cells from oxidative damage. Oxidative stress causes the superoxide radical to transfigured to hydrogen peroxide, which is subsequently bring down prior to water molecules (Das et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). GSH lowers peroxidative stress by averting free radicals from oxidizing through lipid peroxidation. As fatty acid disassembles releases MDA, our bodies exhibit higher levels of lipid peroxidation, which leads to increased oxidative stress, and higher MDA congregation (Bhowmick et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). According to our findings, the rats' oxidative stress from the disease's progression has decreased. Rats receiving various dosages of FPH escaped from death. Huang and Wu have documented the function of shark fish liver in lowering oxidative stress during the incident of diabetes (Huang et al., 2010)\u003c/p\u003e\u003cp\u003eAO and Et-Br fluorescent staining enables the detection of morphological changes associated with apoptosis, allowing for the discrimination between normal cells, early and late apoptotic cells, and necrotic cells. AO penetrates cells with intact membranes, including normal and early apoptotic cells, emitting green fluorescence in untreated pancreatic cells. In contrast, Et-Br only enters cells with compromised membranes, such as late apoptotic and dead cells, producing orange-red fluorescence upon binding to condensed DNA fragments or apoptotic bodies. Notably, dual AO/Et-Br staining can detect mild DNA damage. Our results show that rats fed 50 mg/kg FPH exhibit similar characteristics to control rats and vehicle control rats, suggesting a potential protective effect of FPH (Dutta et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Muscle Blaze Biozyme Iso-Zero, 2023).\u003c/p\u003e\u003cp\u003eMoreover, DAPI staining revealed that pancreatic cells from T2DM rats treated with FPH displayed characteristic apoptotic features, including chromatin condensation and nuclear fragmentation, indicative of cellular apoptosis (Dutta et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eHistopathological examination of the pancreas further substantiated this hypothesis, revealing that FPH protected against the detrimental effects of STZ-induced T2DM. Notably, FPH displayed the comparable mode of action to that of the vehicle control and control groups, specifically, enhancing insulin secretion and promoting pancreatic function.\u003c/p\u003e\u003cp\u003eThis hypothesis has been further confirmed by the basic morphological changes in apoptotic cells and identifies the distinction between normal cells, early and late apoptotic cells, and necrotic cells. The apoptotic cell death results obtained by flow cytometer were shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e8\u003c/span\u003e The results show scattered plot, which was created fluorescence colour to generate through FITC and PI staining. It shows early cell apoptosis followed by late apoptosis due to induction of STZ in T2DM rats which coincidentally converts into necrotic cell death. Supplementation of FPH at the dose of 50 mg/kg b. w. shown the lowest apoptosis.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eSupposition of this study that many therapeutic peptides present in FPH and they play important role to manage ROS and elevation of GLP-1 level with impediment of DPP-4 action in pancreas, thereby, lower the hyperglycaemia (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e10\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe emergence of the population with diabetes has been climbing day by day. Hence, the exploration for an acceptable food gleaned integrant is in great demand. Fish protein hydrolysates (FPH) of volavetki (\u003cem\u003ePanna microdon\u003c/em\u003e) plays a key role in controlling and the management of type 2 diabetes mellitus (T2DM) through inhibiting pancreatic DPP-4 activity and hepatic glucose 6- phosphatase activity also enhancing pancreatic glucokinase activity with increasing the action of GLP-1. Various peptides in FPH presupposed to up-down regulation in cellular enzymes which approbates anti-diabetic activity. Principally, the FPH exemplified their potential to improve glycemia and ought to be contemplated in the admonishment of T2DM.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003cdiv id=\"Sec14\" class=\"Section3\"\u003e\u003ch2\u003eChemicals and instruments\u003c/h2\u003e\u003cp\u003eMajor biochemical parameter was measured using diagnostic kit of glycosylated haemoglobin (HbA1c) purchased from agappe diagnostics LTD, Hills, Ernakulam, Kerala, India. Fasting blood glucose (FBG) performed from tail vein of rats by using blood glucose monitoring system with strips (Apollo pharmacy, APG01). We assayed enzyme-linked immunosorbent assay (ELISA) kits like insulin, and c-peptides purchased from Weldon biotech Pvt. Ltd, Patparganj, Mayur Vihar, Delhi, India, glucose 6-phosphatase, glucagon like peptide-1 (GLP-1), and DPP-4, purchased from wuxidonglin sci \u0026amp; tech development Co. Ltd, Wuxi, Jiangsu, India. Anti-diabetic drug and Streptozotocin (STZ) were come by sisco research laboratories Pvt. Ltd, Taloja, Maharashtra, India. We brought the Protease enzyme (LOT 0000562173) from HIMEDIA laboratories Pvt. Ltd., Nashik, India. All other chemicals and enzyme like calcium chloride (Cacl\u003csub\u003e2\u003c/sub\u003e), magnesium chloride (MgCl\u003csub\u003e2\u003c/sub\u003e), hank\u0026rsquo;s balanced salt solution (HBSS), phosphate buffer saline (PBS) solution, potassium dihydrogen phosphate (KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e), potassium chloride (KCL), D- glucose, sodium chloride (NaCl), magnesium sulphate (MgSo\u003csub\u003e4\u003c/sub\u003e), sodium hydrogen phosphate (Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e), and bicarbonate of soda (NaHco\u003csub\u003e3\u003c/sub\u003e) were picked from SRL, India, and MERCK, India, HiMedia Laboratories Pvt. Ltd. Mumbai, India, and Crest Biosystems Goa, India and protease enzyme was purchased from Caltech life science, Bhubaneswar, Odisha, India. Anti-oxidant enzyme parameters like glutathione (GSH), superoxide dismutase (SOD), catalase (CAT) was determined by absorbance UV-VIS double beam Spectrophotometer (systronics, India). Protein hydrolysate from volavetki (\u003cem\u003ePanna microdon\u003c/em\u003e) was performed using tissue homogenizer (Eltek), pH matter (Digital), Incubator with shaker (YONA\u003csup\u003eR\u003c/sup\u003e), cold centrifuge (Eltek), freeze dryer (ICC-INSTIND). And other instruments were used for various examination like fluorescence inverted microscope (Magnus), flow cytometer (Beckman coulter), semiautoanalizer (MISPA VIVA) and fluorescence microscope (Olympus, Model No. BX43F). Digital weight balance (accuracy-0.1mg) Adhair Dutta and Sons was used for weighted all materials.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003ePreparation of volavetki fish protein hydrolysates (FPH)\u003c/h2\u003e\u003cp\u003eSea fish volavetki (\u003cem\u003ePanna microdon\u003c/em\u003e, Bleeker, 1949) sea fish was freshly purchased from the local market of Digha mohona (21\u0026deg;38\u0026prime;18\u0026Prime;N 87\u0026deg;30\u0026prime;35\u0026Prime;E), coastal area which is located along the Bay of Bengal, Purba Medinipur, West Bengal, India. The samples were packed in ice container. The sample were identified by Assistant professor, Department of Zoology, Belda College and a specimen was deposited at department of Zoology Belda College, Belda, Paschim Medinipur, West Bengal, India. FPH was prepared by step by step, muscles were separated from volavetki sea fish then rinsed with cold distilled water and make fine chopped. Next, 1 kg of fish muscle were mixed in 10L HBSS buffer keeping on ice container and completed the homogenization process. Then, enzymatic hydrolysis was conducted in protease at an enzyme-to-substrate ratio (wt/wt) of 1:100 at 37\u0026deg;C (pH 7.4) under constant stirring in incubator cum shaker (CareLab) for 30 minutes. Next, the samples were centrifuged at 5,000 \u0026times; g for 10 min at 4\u0026deg;C, and the supernatants were collected and freeze-dried (Harnedy et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Bunsroem et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Asokan et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eIdentification of peptides and amino acid sequences by nano liquid chromatography mass spectrometry (nLC‒MS/MS)\u003c/h2\u003e\u003cp\u003eFinding the bioactive substances that are contained in the FPH by nLC‒MS/MS. In briefly, 50 mM iodoacetamide alkylated with 5 mM and tris 2- carboxyethyl phosphine (TCEP) then breakdown with Trypsin (1:50, Trypsin/lysate ratio) at 37\u0026deg;C for 16 hours. A speed vacuum was used to dry the digests after they had been cleaned with C18 silica cartridge for elimination of any salt. After dying the pellet was re-suspended with buffer A, concentrated at 0.1% formic acid and 5% acetonitrile. Thermo QE and nano LC 1200 were used for the experiments. On a Thermo Fisher Scientific C18 column measuring 50 cm and 3.0\u0026micro;m Easy-spray, one unit was loaded. Peptides were injected for MS analysis after being eluted using a 0\u0026ndash;40% gradient of buffer B (80% acetonitrile, 0.1% formic acid) at a flow rate of 300 nl/min. The LC gradients ran for 60 minutes. At 70k resolution, MS1 spectra were obtained using the Orbitrap. Dynamic exclusion was used to exclude all charge states for a given precursor for 10 seconds. At 17500 resolutions MS2 spectra were obtained (Harnedy et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Elam et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Asokan et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eSelection of animal\u0026rsquo;s care, and handling\u003c/h2\u003e\u003cp\u003eHealthy adult male Wistar strain albino rats weighing about 150\u0026thinsp;\u0026plusmn;\u0026thinsp;20 g were purchased from Saha Enterprise, 386/2, Nilachal, Birati, Kolkata-700051, Registration number: 1828/PO/BT/S/15/CPCSEA. Experimental process was approved by Institutional animal ethics committee (IAEC-CPCSEA) registration number of 1905/PO/Re/S/16/CCSEA for Raja Narendra Lal Khan Women\u0026rsquo;s College (Autonomous). The rats were housed inside a well-ventilated room in metal wire cages with free access to food and water, at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C, humidity 60\u0026thinsp;\u0026plusmn;\u0026thinsp;5%- and 12-hours dark-light cycle and throughout the experimental period. We strictly followed the scientific procedure using experimental animal as per CPCSEA (Approval No. 01/IAEC (1)/S/RNLKWC/2023) and ARRIVE guideline.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eT2DM induction\u003c/h2\u003e\u003cp\u003eAnimals were acclimatized on high lipid diet (HLD) diet (Pyne et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), which included the following ingredients: cholesterol 0.5g, corn starch 40g, mineral mixture 0.5g, casein 20g, milk powder, sucrose 10g, bengal gram flour 15g, vitamin premix 0.5g, coconut oil 13ml, and salt 0.5g for 180 days. After acclimatization, for all rats other than the vehicle control rats and control rats, a single intraperitoneal injection of freshly made STZ (0.5 mL of pH 4.5, 0.01 M citrate buffer; 40 mg/kg b.w.) was used to induce T2DM. Vehicle control rats were injected with 0.5 mL of 0.01 M citrate buffer (pH 4.5). After 72 hours of injection, animals exhibiting the blood glucose levels greater than 250 mg/dL were considered as T2DM and sorted into 8 groups of 5 animals per group.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eExperimental design\u003c/h2\u003e\u003cp\u003eRandomly allocated total 40 rats and divided into 8 groups (n\u0026thinsp;=\u0026thinsp;5) as follows:\u003c/p\u003e\u003cp\u003eGroup 1: Control rats fed HLD.\u003c/p\u003e\u003cp\u003eGroup 2: Vehicle control rats fed HLD and a single intraperitoneal injection of 0.5 mL of 0.01 M citrate buffer.\u003c/p\u003e\u003cp\u003eGroup 3: T2DM rats fed HLD without any supplementation.\u003c/p\u003e\u003cp\u003eGroup 4: T2DM rats fed HLD and FPH (12.5mg/kg of b.w.) supplementation for 28 days.\u003c/p\u003e\u003cp\u003eGroup 5: T2DM rats fed HLD and FPH (25mg/kg of b.w.) supplementation for 28 days.\u003c/p\u003e\u003cp\u003eGroup 6: T2DM rats fed HLD and FPH (50mg/kg of b.w.) supplementation for 28 days.\u003c/p\u003e\u003cp\u003eGroup 7: T2DM rats fed HLD and FPH (75mg/kg of b.w.) supplementation for 28 days.\u003c/p\u003e\u003cp\u003eGroup 8: T2DM rats fed HLD and standard anti-T2DM oral medicine, namely, gliptin dose of 12mg/kg of b.w. for 28 days through oral gavage\u003c/p\u003e\u003cp\u003eAfter completion of the experimental period, the rats were decapitated to avoid stress. Blood was collected from all rats, and centrifuged at 5000 \u0026times; g for 10 min to separate the plasma. Then plasma and the organs i.e., pancreas and liver were stored at \u0026minus;\u0026thinsp;20\u0026deg;C for further analysis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eMeasurement of FBG and HbA1c levels\u003c/h2\u003e\u003cp\u003eFBG and HbA1c from whole blood was measured 5 times to confirm sustained hyperglycemia during the experimental period at 0, 7, and 14, 21, 28 days from tail vein of rats, using a glucometer and FBG level was expressed as mg/dL. The standard kit method was utilised to assay HbA1c. Briefly mixing 10 \u0026micro;L of whole blood with 0.5 mL of haemolyzed reagent and allow at room temperature for 10 minutes to ensure complete lysis. Next, 300 \u0026micro;L latex and 10 \u0026micro;L hemolysate sample were combined, and the mixture was incubated at 37\u003csup\u003e◦\u003c/sup\u003eC for 5 minutes. Next, 100 \u0026micro;L anti-human HbA1c mouse monoclonal antibody was added, and the mixture was again incubated at 37\u003csup\u003e◦\u003c/sup\u003eC for 5 minutes. The output was then measured using a semiautoanalyser at 360 nm wavelengths. The expression for HbA1c was g% (Pyne et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003eEstimation of plasma insulin, c-peptide, and GLP-1\u003c/h2\u003e\u003cp\u003eThe plasma insulin and c-peptide were estimated by an ELISA plate reader with standard kit method. In brief, 50 \u0026micro;L of plasma was combined with 100 \u0026micro;L of insulin and c-peptides enzyme reagent to each well and allow for 120 mins at 25\u003csup\u003e◦\u003c/sup\u003eC -27\u003csup\u003e◦\u003c/sup\u003eC. Then discard the contents and added 350 \u0026micro;L, 0.01 M of PBS buffer (pH 7.2) with deionized water and washed for two times. Then added 100 \u0026micro;L tetramethylbenzidine substrate and incubated for 15 mins at room temperature and finally added 50 \u0026micro;L hydrogen peroxide (H2O2) in buffer (0.01M PBS buffer, pH 7.4 containing deionized water) and read the absorbance at 450 nm in ELISA plate reader within 30 mins. Unit of c-peptide and insulin were expressed as ng/mL of plasma (Das et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Pyne et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eGLP-1 was estimated by an ELISA plate reader with standard kit method. In short, 50 \u0026micro;L of plasma was combined with 50 \u0026micro;L of biotin labelled GLP-1 to each well and allow for 60 mins at 37\u003csup\u003e◦\u003c/sup\u003eC. Then discard the contents and added 300 \u0026micro;L, 0.01 M of PBS buffer (pH 7.2) containing deionized water and washed three times. Then added 100 \u0026micro;L substrate and incubated for 60 mins at 37\u003csup\u003e◦\u003c/sup\u003eC. Finally added 50 \u0026micro;L hydrogen peroxide (H2O2) in buffer (0.01M PBS buffer, pH 7.4 containing deionized water) and read the absorbance at 450nm in ELISA plate reader within 30 mins. GLP-1 was expressed as ng/mL of plasma (Das et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003ePancreas and liver tissue preparation for determination of in vivo antioxidant activities\u003c/h2\u003e\u003cp\u003eHomogenates of pancreas and liver tissues were prepared using a 150 mM, pH 7.4 phosphate buffer solution and then used to measure the activities of enzymes (Das et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Das et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2016\u003c/span\u003e;).\u003c/p\u003e\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\u003ch2\u003eDetermination of DPP-4 and glucokinase activity in pancreas and glucose 6- phosphatase activity in liver\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eActivities of DPP-4 and glucokinase in pancreatic tissue, and activities of glucose 6-phosphatase in liver were estimated by using ELISA kit method. Briefly, 20 \u0026micro;L of pancreatic tissue sample, 30 \u0026micro;L of 0.02 M Tris-HCl buffer (pH 8.0), 0.10 M NaCl and 1 mM EDTA were mixed with 50 \u0026micro;L of DPP-4 standard and incubated at 37\u003csup\u003e◦\u003c/sup\u003eC for 60 minutes. Then discard the contents and 300 \u0026micro;L of 0.02 M Tris-HCl buffer (pH 8.0) was added and washed for three times. Then 100 \u0026micro;L substrate of 200 \u0026micro;M H-Gly-Pro-AMC was added and incubated at 37\u003csup\u003e◦\u003c/sup\u003eC for 60 minutes. Finally, we measured the absorbance at 450nm in ELISA plate reader within 30 mins. DPP-4 was expressed as pg/g of tissue (Pyne et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eFor determination of glucokinase activity- 20 \u0026micro;L of pancreatic tissue sample, 30 \u0026micro;L of 0.02 M Tris-HCl buffer (pH 8.0), 0.10 M NaCl and 1 mM EDTA were combined with 50 \u0026micro;L of glucokinase standard and incubated at 37\u003csup\u003e◦\u003c/sup\u003eC for 60 minutes. Then discard the contents and 300 \u0026micro;L of 0.02 M Tris-HCl buffer (pH 8.0) was added and washed for three times. Then added 100 \u0026micro;L substrate (200 \u0026micro;M of H-Gly-Pro-AMC) and incubated for 60 mins at 37\u003csup\u003e◦\u003c/sup\u003eC. Absorbance was measured at 360nm and 460 nm wavelengths respectively in ELISA plate reader within 30 min. Glucokinase activity was expressed as ng/\u0026micro;g (Das et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Pyne et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eFor activities of glucose 6- phosphatase in brief, 20 \u0026micro;L of hepatic tissue sample, 30 \u0026micro;L of 0.02 M Tris-HCl buffer (pH 8.0), 0.10 M NaCl and 1 mM EDTA were combined with 50 \u0026micro;L of glucose 6- phosphatase standard and incubated at 37\u003csup\u003e◦\u003c/sup\u003eC for 60 minutes. Then discard the contents and 300 \u0026micro;L of 0.02 M Tris-HCl buffer (pH 8.0) was added and washed for three times. Then 100 \u0026micro;L of 200 \u0026micro;M H-Gly-Pro-AMC substrate was added and incubated for 60 minutes at 37\u003csup\u003e◦\u003c/sup\u003eC. Then measured the absorbance at 360nm and 460 nm wavelengths respectively in ELISA plate reader within 30 mins. Glucose 6- phosphatase activity was demonstrated as U/g of tissue (Das et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Pyne et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003eDetermination of antioxidant enzyme activities and oxidative stress marker\u003c/h2\u003e\u003cp\u003eAt first pancreatic tissues were homogenised with 100 mM ice-cold Tris-cacodylate buffer with 50 mg/mL of tissue concentration and centrifuged at 4\u0026ordm;C for 20 minutes at 10,000 rpm. Utilising a UV-VIS double beam spectrophotometer set at 240 nm wavelength. 50% reduction of pyrogallol's auto-oxidation was demonstrated by one unit of SOD enzyme activity. The activity of SOD was defined just as Unit/mg of tissue/min (Giri et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Bhowmick et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e50 mg per each mL of pancreatic tissues were homogenised with 0.05 M Tris-HCl (pH 7.0) buffer. Separate centrifugations of these homogenates were conducted at 4\u0026deg;C for 10 minutes with 4,000 rpm. CAT was determined in tissue supernatant using the protocol developed in our laboratory, and measured the absorbance at 240nm using a UV-VIS double beam spectrophotometer. The CAT activity was demonstrated as mmol of H2O2 consumption/mg of tissue/ min (Giri et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Bhowmick et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eOur standard laboratory procedure was used to determine the MDA level. At 535nm of spectrophotometer the measurement was carried out including moral values are intimate as nmol/dL of pancreatic tissue (Pyne et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eQuantification of GSH was done by pancreatic tissue homogenate of 0.2 M sodium phosphate (pH 8.0) buffer and combined with dithio-bis-nitrobenzoic acid and measured using a UV-VIS double beam spectrophotometer set to 405 nm according to the standard method. The levels of GSH were demonstrated as Units/mg protein (Das et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e\u003cdiv id=\"Sec25\" class=\"Section3\"\u003e\u003ch2\u003eApoptotic morphological changes by acridine orange (AO) and ethidium bromide (AO/ EtBr) staining detection\u003c/h2\u003e\u003cp\u003eTwo amazing DNA-binding dyes called AO and Et-Br were used to detect apoptotic and necrotic cells. Pancreatic cells were washed with 50 mM of cold PBS, pH 7.4. Then AO and Et-Br mixture were used for staining. The staining procedure was taking place for 5 minutes at 25\u0026deg;C. After that, the cells were washed for three times with PBS, then under a fluorescence microscope it was examined at 40x magnifications (Dutta et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec26\" class=\"Section3\"\u003e\u003ch2\u003eDetermination of nuclear integrity and apoptosis by 4\u0026rsquo;,6-diamidino-2-phenylindole (DAPI) stanning\u003c/h2\u003e\u003cp\u003eFor preparation of DAPI staining, cold PBS buffer was used for cells washed, 2.5% glutaraldehyde was used for cells fixing for 15 minutes, permeabilized with 0. 1% Triton X-100, and then stained with 1 mg/mL DAPI for 5 minutes at 37\u0026deg;C. After that, the cells were washed again with PBS and examined under fluorescence microscopy at 40x (Dutta et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec27\" class=\"Section3\"\u003e\u003ch2\u003ePancreatic cell apoptosis assay by flow cytometry\u003c/h2\u003e\u003cp\u003eTo assess the mechanism of pancreas tissue cell death, the apoptosis and necrosis assay were measured by flow cytometer using Annexin A5 - FITC apoptosis detection kits. Briefly, cells were placed in cultured solution (DNase100 mg in HBSS) then centrifuged at 4˚ C for 5 minutes at 5,000 rpm. Next, afloat was collected and add 200\u0026micro;l binding buffer for cell suspension. Before analysis, 5\u0026micro;L of propidium iodide and 1\u0026micro;L of annexin A5-FITC was mixed and incubate at room temperature in dark place for 15 minutes. After that 400\u0026micro;L 1x ice cold binding buffer was added. With the help of Flow Cytometer, the quantity of apoptosis and necrosis cells were analysed (Dutta et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec28\" class=\"Section2\"\u003e\u003ch2\u003eHistological preparation\u003c/h2\u003e\u003cp\u003eThe pancreas specimen was fixed in 5% formaldehyde solution and surrounded in paraffin. In the histopathological analysis, 3 sections were evaluated from each rat and 3 fields were evaluated from each section. Sections were cut at 5\u0026micro;m thick with the microtome machine consequently deparaffined with xylene. Every piece of the rat pancreas paraffin block was sectioned (5\u0026micro;m) prior to staining with haematoxylin and eosin staining. Then slides were observed by using invert microscope with digital camera (Das et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Pyne et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec29\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eData are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of means obtained from 5 rats per group to evaluate variances between the groups. All assays were tested in 3 replicates by one way ANOVA test the statistical analysis was performed by originpro 6.1 software, following the Bonferroni's modification for determination of statistical significance (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) among all groups compared to diabetic group (Das et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eT1DM \u0026nbsp;Type 1 diabetes mellitus\u003c/p\u003e\n\u003cp\u003eT2DM \u0026nbsp;Type 2 diabetes mellitus\u003c/p\u003e\n\u003cp\u003eGLP-1 Glucagon like peptide-1\u003c/p\u003e\n\u003cp\u003eDPP4 \u0026nbsp;Dipeptidyl peptidase-4\u003c/p\u003e\n\u003cp\u003eIFD International Diabetes Federation\u003c/p\u003e\n\u003cp\u003eHLD High lipid diet\u003c/p\u003e\n\u003cp\u003eFPH Fish protein hydrolysates\u003c/p\u003e\n\u003cp\u003eSTZ \u0026nbsp;Streptozotocin\u003c/p\u003e\n\u003cp\u003eHbA1c Glycosylated haemoglobin\u003c/p\u003e\n\u003cp\u003eFBG Fasting blood glucose\u003c/p\u003e\n\u003cp\u003eGSH Glutathione\u003c/p\u003e\n\u003cp\u003eSOD Superoxide dismutase\u003c/p\u003e\n\u003cp\u003eCAT Catalase\u003c/p\u003e\n\u003cp\u003enLC-MS/MS Nano liquid\u0026nbsp;\u003cem\u003emass spectrometric\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eDAPI \u0026nbsp;4’,6-diamidino-2-phenylindole\u003c/p\u003e\n\u003cp\u003eAO/ Et‑Br Acridine orange and ethidium bromide\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eDeclaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Dr. Koushik Das reports financial support was provided by Belda College. Koushik Das reports a relationship with Belda College that includes: employment. No If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported\u0026nbsp;in\u0026nbsp;this\u0026nbsp;paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStudy was designed on Wister stain albino rat model which was approved by the Institutional Animal Ethics Committee (Approval No. 01/IAEC (1)/S/RNLKWC/2023) dated 15.06.2023 of Raja Narendra Lal Khan Women’s College (Autonomous).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnimal Ethics declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experimental procedures involving animals were conducted in accordance with the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) and the ARRIVE guidelines. The study protocol was approved by the Institutional Animal Ethics Committee (Approval No. 01/IAEC (1)/S/RNLKWC/2023) dated 15.06.2023 of Raja Narendra Lal Khan Women’s College (Autonomous).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRaw data were generated by the Raja Narendra Lal Khan Women’s College (Autonomous) and data supporting the finding of the study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo fund\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSupriya Bhowmick: Writing – original draft, Methodology, Data analysis\u003c/p\u003e\n\u003cp\u003eShayan Panda: Methodology, Data analysis, Investigation, Writing – original draft\u003c/p\u003e\n\u003cp\u003eMadhumita Pal: Methodology, Investigation, Writing – original draft\u003c/p\u003e\n\u003cp\u003eSanjay Das: Methodology, Investigation\u003c/p\u003e\n\u003cp\u003eMeghamala Mandal: Writing – review \u0026amp; editing\u003c/p\u003e\n\u003cp\u003eShrabanti Pyne: Writing – review \u0026amp; editing\u003c/p\u003e\n\u003cp\u003eDeblina Giri: Methodology, Investigation\u003c/p\u003e\n\u003cp\u003eKoushik Das: Conceptualization, Writing – review \u0026amp; editing\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank the DST-FIST (Memo no. SR/FST/College-2017/03 (C), 29\u003csup\u003eth\u003c/sup\u003e August 2018) for equipment and infrastructure facility for Raja Narendra Lal Khan Women’s College (Autonomous).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAli, A. M., Gabbar, M. A., Abdel-Twab, S. M., Fahmy, E. M., Ebaid, H., Alhazza, I. M., et al. (2020). 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Hypoglycemic effect of hydrophobic BCAA peptides is associated with altered PI3K/Akt protein expression. \u003cem\u003eJournal of Agricultural and Food Chemistry\u003c/em\u003e, \u003cem\u003e69\u003c/em\u003e(15), 4446\u0026ndash;4452. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/acs.jafc.1c00726\u003c/span\u003e\u003cspan address=\"10.1021/acs.jafc.1c00726\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"FPH, DPP-4, GLP-1, T2DM, Panna microdon and FBG","lastPublishedDoi":"10.21203/rs.3.rs-7469537/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7469537/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eIn the present study, the antidiabetic properties of \u003cem\u003ePanna microdon\u003c/em\u003e sea fish protein hydrolysates (FPH) in high lipid diet and streptozotocin-induced type 2 diabetes mellitus (T2DM) rats were investigated through the mechanistic way using glucagon like peptide-1 (GLP-1) agonistic and dipeptidyl peptidase-4 (DPP-4) inhibitory activities. There were eight groups of 5 rats each: control, vehicle control, and six T2DM groups out of these one T2DM control, four T2DM groups supplemented with FPH at the doses of 12.5, 25, 50, and 75 mg/ kg b.w./day respectively, and one T2DM group treated with gliptin at the dose of 12 mg/kg b.w./day for 28 days.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eAfter 28 days of experiment the T2DM group presented a significant increase in fasting blood glucose (FBG) and glycated hemoglobin (HbA1c) levels in plasma, hepatic glucose-6 phosphatase activities in liver, level of lipid peroxidation, pancreatic DPP-4 activities as well as a significant decrease in the levels of plasma insulin, c-peptides, GLP-1, and antioxidant activities of superoxide dismutase, catalase, glutathione, and glucokinase activity in pancreas compared to control group. However, the daily supplementation of FPH for 28 days improved plasma insulin, c-peptides, GLP-1 levels and as well as significantly decrease plasma FBG and HbA1c levels, DPP-4 activities, lipid peroxidation and hepatic glucose-6 phosphatase activities compared to T2DM group. FPH supplementation reduced pancreatic ROS generation and pancreatic necrosis.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eResults have been presumed that antidiabetes activities were shown by various potential peptides in FPH identified by nLC‒MS/MS analysis.\u003c/p\u003e","manuscriptTitle":"Antidiabetic potential of volavetki sea fish protein hydrolysates in high lipid diet and streptozotocin-induced diabetes rats","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-25 19:32:47","doi":"10.21203/rs.3.rs-7469537/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"deb5ff0c-c774-4a37-9f3d-b659aaf66aaa","owner":[],"postedDate":"September 25th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-01-26T16:00:27+00:00","versionOfRecord":{"articleIdentity":"rs-7469537","link":"https://doi.org/10.1186/s41936-026-00546-w","journal":{"identity":"the-journal-of-basic-and-applied-zoology","isVorOnly":false,"title":"The Journal of Basic and Applied Zoology"},"publishedOn":"2026-01-19 15:57:12","publishedOnDateReadable":"January 19th, 2026"},"versionCreatedAt":"2025-09-25 19:32:47","video":"","vorDoi":"10.1186/s41936-026-00546-w","vorDoiUrl":"https://doi.org/10.1186/s41936-026-00546-w","workflowStages":[]},"version":"v1","identity":"rs-7469537","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7469537","identity":"rs-7469537","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}