Modelling type 2 diabetes in rats via high-lipid diet and streptozotocin-induced insulin resistance and β-cell dysfunction assessed by C-peptide

Modelling type 2 diabetes in rats via high-lipid diet and streptozotocin-induced insulin resistance and β-cell dysfunction assessed by C-peptide | 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 Modelling type 2 diabetes in rats via high-lipid diet and streptozotocin-induced insulin resistance and β-cell dysfunction assessed by C-peptide Dr. Koushik Das, Sanjay Das, Shrabanti Pyne, Sayan Panda, Madhumita Pal, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8953734/v2 This work is licensed under a CC BY 4.0 License Status: Posted Version 2 posted You are reading this latest preprint version Show more versions Abstract Streptozotocin, that has a selective pharmacological toxicity toward pancreatic beta cells, in addition to high lipid diet (HLD) has been widely used to induce T2DM. However, no evidence has shown that superior dose of streptozotocin (STZ) to establish T2DM. This study was initiated to develop an animal model (Wister Albino rats) of T2DM with suitable dose of STZ. Total fifty male rats (210 ± 20 g) were arbitrarily divided into 10 groups (n = 5). Two groups were control group fed normal diet (ND) and high lipid diet (HLD). The remaining rats were induced with STZ at 20, 40, 60 and 80 mg/kg body weight, with each dose tested under ND and HLD conditions. Body weight, blood glucose, HbA1c, serum insulin, C-peptide, pancreatic glucokinase, serum triglycerides (TG), total cholesterol (TC), antioxidant enzymes (SOD, CAT, GSH, MDA) and pancreatic histology. 80 mg/kg STZ group rats expired within 7 days. After 28 days of experiment, Blood glucose was markedly raised up. Insulin and C-peptide levels were lower in STZ/HLD fed groups (P < 0.001) rats. Pancreatic glucokinase activity significantly decreased (P < 0.05). SOD, CAT, GSH and TG, TC increased significantly in STZ/HLD treated groups (P < 0.01). We observed that, β- cells were present in STZ/HLD fed rats pancreas and insulin secretion is higher than ND fed rats. We concluded that, HLD with 40mg STZ/Kg b.w. rats provide a novel animal model for T2DM without any contradictions and is suitable for the testing of antidiabetic compounds. Endocrinology & Metabolism Insulin Type 2 diabetes mellitus High lipid diet Streptozotocin C- peptide Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 1. Introduction According to IDF 2024, 9.2 million people with diabetes mellitus (DM) were living with the condition, with 1.8 million under the age of 20 (Brito et al., 2025 ). According to the World Health Organization, DM is classified into Type 1 diabetes mellitus (T1DM), Type 2 diabetes mellitus (T2DM), gestational diabetes mellitus (GDM), and other specific types caused by genetic, pancreatic, or drug-related factors (World Health Organization, 2019 ). Plasma C-peptide not only classified the DM types but also crucial in formulating criteria to safeguard its better management (Upadhyay et al., 2021 ). C-peptide is a polypeptide secreted alongside insulin by pancreatic β cells through enzymatic cleavage of proinsulin. Both insulin and C-peptide are released in equimolar amounts. Nevertheless, for the reason that C-peptide has a longer half life than insulin, it assists as a more stable and consistent marker for evaluating insulin secretion in DM condition (Apostolopoulou et al., 2025 ). T1DM is a multifactorial, heterogeneous, chronic disease, and autoimmune, considered by the distraction of β cells in pancreatic islets of Langerhans (Akinlade et al., 2021a ). T1DM occurs gradually and can be divided into three distinct stages. In the 1st stage, persons develop DM related autoantibodies, which specify an autoimmune (insulin autoantibodies (IAA), glutamic acid decarboxylase autoantibodies (GADA), IA-2 autoantibodies (IA-2A), and zinc transporter 8 autoantibodies (ZnT8A) response contrary to the pancreatic beta cells (Go et al., 2024 ; Wan et al., 2023 ). In this stage not only pancreatic β cells function normal but also plasma C-peptide secretion normal and blood glucose levels characteristically remain within the normal range. In the 2nd stage, progressively β cell losses from the pancreas (Akinlade et al., 2021a , p. 1; Buettner et al., 2007 ). As a result, insulin secretion is diminished. Side by side, plasma C- peptide level continuously decreased. Finally, in the 3rd stage, GADA can trigger the immune system to secret autoantibodies against pancreatic β cells and destructed the pancreas. In this stage plasma C-peptide level undetectable (Chen et al., 2023 ). Alongside, blood glucose level also higher than normal range. The risk of progression from 1st stage to clinical T1DM is 35%–50% within five years, rising to 75% for 2nd Stage within 2 years (Baxter and Schofield, 1980 ; Buettner et al., 2007 ). On the other hand, T2DM is an enduring metabolic disease characterized by hyperglycaemia ensuing from a combination of insulin resistance and liberal β cell dysfunction (Akash et al., 2014 , p. 2). In the early stages of T2DM, additional adiposity and chronic low grade inflammation blight insulin signalling in skeletal muscle and adipose tissue, ferment a state of insulin resistance in which cells involve higher condensations of insulin to maintain glucose uptake. Pancreatic β cells primarily expiate for this diminished insulin sensitivity via increasing insulin secretion, resultant in hyperinsulinaemia (Kilari et al., 2021 ). For the reason that, proinsulin is lacerate into insulin and C-peptide in equimolar quantities, this compensatory response is reflected by elevated circulating C-peptide concentrations. Insulin acts on its tyrosine kinase receptor to activate insulin receptor substrates, phosphoinositide 3-kinase (PI3K) and Akt (called Protein Kinase B) while, activation of Akt triggers phosphorylation of the Rab GTPase-activating protein AS160 (Akt Substrate of 160 kDa) and activates Rac1 (small GTPase), leading to the mobilization of GLUT 4 containing vesicles to the cell surface and facilitating glucose transport. In individuals with insulin resistance, defects in IRS (Insulin Receptor Substrate) signalling and actin remodelling impair GLUT4 translocation, consequently postprandial glucose uptake is abridged despite high insulin levels (Baxter and Schofield, 1980 ; Buettner et al., 2007 ; Jiang et al., 2020 ). With insistent glucotoxicity, lipotoxicity and metabolic stress induce β-cell dedifferentiation, oxidative damage and apoptosis, leading to progressive loss of insulin secretory capacity (Abdel-Megeid et al., 2008 ). C-peptide levels table land and eventually decline as β-cell mass decreases, signalling a transition from compensatory hyperinsulinaemia to relative and then absolute insulin deficiency. This evolution from insulin resistance with elevated endogenous insulin and C-peptide to eventual β-cell failure with declining C-peptide underpins of T2DM (Aboismaiel et al., 2024 ; Imagawa et al., 2012 ). T2DM has become one of the most pervasive and serious public health challenges of our time. Animal models, especially rats (Wister albino), are widely used in DM research due to their physiological similarities to humans, ease of handling, and reproducibility of experimental outcomes (Abdel-Megeid et al., 2008 ). STZ present in glucosamine nitrosourea antibiotic which is toxic to pancreatic β cells. STZ induction is one of the most commonly used for DM experiment. Following intraperitoneal injection, STZ models are used to study both T1DM and T2DM. STZ enters pancreatic β-cells through the high capacity glucose transporter GLUT2. STZ also releases nitric oxide and generates reactive oxygen species (ROS), leading to oxidative and nitrosative stress (Aboulmagd et al., 2020 ; Furman, 2021 ). β-cells are especially vulnerable because they express low levels of antioxidant enzymes, and STZ administration decreases superoxide dismutase and glutathione peroxidase activities. the β cell damage induced by STZ results in hypoinsulinemia and loss of glucose stimulated insulin secretion. In rats, STZ administration leads to decreased insulin stimulated glucose transport and lipogenesis in adipocytes and severe hypoinsulinemia, indicating both impaired insulin production and peripheral insulin resistance. STZ exposure reduces pancreatic GLUT2 mRNA level and dramatically lowers plasma insulin concentrations as well as C-peptide (Jaishree and Narsimha, 2020 ). Fat mice retain higher GLUT2 expression and near-normal insulin and c peptide levels despite STZ challenge. These functional deficits arise because the DNA alkylation and oxidative stress induced by STZ lead to β cell death and loss of insulin-secretory capacity. Collectively, STZ selective uptake by GLUT2 expressing β-cells, its DNA-alkylating and ROS-generating actions, and the consequent morphological and functional destruction of pancreatic islets underpin its widespread use in experimental models of insulin deficient diabetes (Alfheeaid et al., 2023 ). The lack of uniformity poses challenges for reproducibility, data interpretation, and comparison between studies. Although STZ has been widely employed to establish experimental DM models, considerable variation exists in the doses administered across studies (A-Elgadir et al., 2024 ; Bagheripour et al., 2024 ; Cai et al., 2019 ; Gharaat et al., 2024 ; Salem et al., 2024 ). Therefore, there is a need to systematically evaluate and optimize the dose of STZ in combination with different dietary regimens to establish a more reliable rat model of T2DM. A well-characterized and standardized model would not only improve the understanding of disease mechanisms but also enhance the translational value of preclinical studies for developing antidiabetic drugs and nutritional interventions. 2. Methodology 2.1. Chemicals and apparatus Streptozotocin (STZ) was purchased from sisco research laboratories Pvt.Ltd, Maharashtra, India. All other chemicals were purchased from SRL and MERCK, HiMedia Laboratories, and Crest Biosystems, India. We used diagnostic kit of glycosylated haemoglobin (HbA1c) were purched from agappe diagnostics LTD, India and enzyme-linked immune sorbent assay (ELISA) kits like insulin, and c-peptides and glucokinase purchased from Weldon biotech Pvt. Ltd, wuxidonglin sci & tech development Co. Ltd, India. Triglyceride and total cholesterol were measured by semi auto analyzer standard kit and purchased from Agappe Hills, Kerala, India. We used many instruments like digital weight balance (accuracy-0.1mg) (Adhair Dutta and Sons), semi autoanalizer (MISPA VIVA). pH matter (Digital), incubator with shaker (YONA R ), cold centrifuge (Eltek), freeze dryer (ICC-INSTIND), light inverted microscope (Magnus). 2.2. Care of animal Total fifty adult male eight weeks old Albino Wister rats were used in the study and were obtained from the Saha Enterprise, 386/2, Nilachal, Birati, Kolkata-700051, West Bengal, India (Regd. No. 1828/PO/Bt/S/15/CPCSEA). All animal trials were supervised by the Institutional Animal Ethical Committee (IAEC), which was approved by Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Environment, Forest and Climate Change, Government of India, approval no 02/IAEC/1/S/RNLKWC/2023. We accurately followed the scientific way using animal as per CPCSEA and ARRIVE guidelines (Das et al., 2024 ). Rats were randomly divided into ten groups each consist five rats (n = 5) and on condition that the normal diet (ND) and water ad libitum for one week for conformation with comfortable temperature of 25 ± 2̊ C with the perfect 12-hour cycle of light and dark. After one week all rats were provided high lipid diet (HLD) for 45 days for weight gain. We were used 210 ± 20g rats for this experiment. 2.3. Experimental design Total fifty male rats were randomly divided into ten groups (n = 5). Control normal diet (CND) group fed (n = 5) fed only normal diet and control high lipid diet (CHLD) group (n = 5) fed only high lipid diet (Table 1 ). Composition of normal diet (ND) and high lipid diet (HLD) diet were given in Table 2 . Duration of experiment was 28 days. After the end of experiment period all rats were sacrificed and blood were collected from vein and stored at -20°C deep freeze. We closely knit blood from the dorsal aorta and segregated the plasma using centrifugation at 3000 rpm for 5 minutes. The serum was kept at -20°C for the biochemical assay. Important organs like pancreas, liver and kidney were collected and stored at -20°C for further experiment. Body weight was measured initial and before the sacrificed of rats. Table 1 Dietary intervention of treatment groups: Group name Treatment CND Control group fed only normal diet. CHLD Control group fed only high lipid diet (HLD). 20 mg STZ ND 20 mg/Kg body weight STZ induced rats fed only normal diet. 40 mg STZ ND 40 mg/Kg body weight STZ induced rats fed only normal diet. 60 mg STZ ND 60 mg/Kg body weight STZ induced rats fed only normal diet. 80 mg STZ ND 80 mg/Kg body weight STZ induced rats fed only normal diet. 20 mg STZ HLD 20 mg/Kg body weight STZ induced rats fed only HLD. 40 mg STZ HLD 40 mg/Kg body weight STZ induced rats fed only HLD. 60 mg STZ HLD 60 mg/Kg body weight STZ induced rats fed only HLD. 80 mg STZ HLD 80 mg/Kg body weight STZ induced rats fed only HLD. Table 2 Composition of food ingredients of normal diet and HLD (Das et al., 2024 ; Khatun et al., 2019 ) Foods ingredients (g) Normal diet (n = 5) (ND) High lipid diet diet (n = 5) (HLD) Corn Starch 40 40 Casin 15 20 Bengal gram flour 18 15 Sucrose 25 10 Coconut Oil - 13 Mineral Mixture 0.5 0.5 Cholesterol 0.5 0.5 Vitamin Premix 0.5 0.5 Salt 0.5 0.5 Total (g) 100 100 2.4. Induction of streptozotocin Total forty rats and ten groups (n = 5) were induced streptozotocin (STZ). The drug was dissolved right before the injection in saline at in 0.1 mol/L citrate buffer (pH 4.5), because STZ degrades within 15 to 20 min after dissolving in the citrate buffer, the STZ solution should be prepared immediately before use and injected within 5 min of dissolution and administered intraperitoneally as single dose to rats groups (Jiang et al., 2020 ; Pyne et al., 2023 ). Rats were kept deprived of their feed and water for twelve hours before administration of STZ. STZ at different concentrations were induced, for instance, 20 mg/Kg b.w, 40 mg/Kg b.w, 60 mg/Kg b.w and 80 mg/Kg b.w. Blood glucose levels were obtained after 72 hours by using commercially available glucometer, blood was pinched from lateral tail vein. Rats with blood glucose level > 126 mg/dl were considered as DM (Almalki et al., 2024 ). 2.5. Assessment of blood glucose level All rats blood glucose level were measure from the tip of their tail vein by a single touch glucometer (Apollo pharmacy; model No. APG01) which were available in local markets. Blood glucose level was measured at 0 day, 3rd day, 7th day, 14th day, 21st day and 28th day. The blood glucose level was expressed by g/dL. 2.6. Estimation of glycosylated haemoglobin (HbA1c) This process was accomplished indiscriminately across all experimental groups. HbA1c quantification was propulsion using a standardized kit-based protocol analyzed with a semi-automated analyzer (Maiti et al., 2005 ). The HbA1c level was expressed by g/dL. 2.7. Estimation of plasma insulin and C-peptide level and pancreatic glucokinase Plasma insulin and C- peptide levels and pancreatic glucokinase were quantified using a rat specific enzyme linked immunosorbent assay (ELISA) kit according to the protocol. Plasma insulin and C-peptide levels was measured at 450 nm using an ELISA plate reader within 30 minutes. Insulin concentrations and C-peptide level were calculated against a standard curve and expressed as ng/mL. Pancreatic glucokinase was measured at 360 nm (excitation) and 460 nm (emission) using a microplate reader within 30 minutes of reaction completion. Activity was quantified against a standard curve and expressed as ng of glucokinase per µg of total protein (ng/µg) (Mallick et al., 2007 ). 2.8. Estimation of total cholesterol and triglyceride of plasma Total cholesterol (TC) and triglyceride (TG) were measured using standard kit method by semi-autoanalyzer. TC and TG were expressed in mg/dL (Das et al., 2010 ). Estimation of antioxidant enzyme Superoxide dismutase (SOD), Catalase (CAT) and reduced glutathione (GSH) activity of pancreatic tissue SOD and CAT were measured in tissue supernatant by our laboratory established method and reading of absorbance was noted using a UV-VIS double beam spectrophotometer at 240 nm. Quantification of GSH was performed by pancreatic tissue homogenate mixed with dithio-bis-nitrobenzoic acid according to the standard method using UV-VIS double beam spectrophotometer at 405 nm. The levels of GSH were expressed as µg of GSH/mg pancreatic tissue (Zhu et al., 2025 ). 2.10. Quantification of malondialdehyde (MDA) level of pancreatic tissue Measurements were carried out by a UV-VIS double beam spectrophotometer at 535 nm and values are expressed nmol/mg of pancreatic tissue (Qiu et al., 2025 ). 2.11 Histopathological examination of pancreatic tissue Upon finishing the experiment, the pancreatic tissues were fixed in a 10% prepared formalin solution, three sliced into sections approximately 4 µm thick. These covers were then transferred to a tissue processor, where they underwent automated dehydration using alcohol, clearing with xylene, and wax infiltration, followed by saturation for approximately 14 hours overnight. The tissues were afterward embedded in heated paraffin, which was permitted to cool and solidify into paraffin blocks. Each block was cut and sectioned to about 4 µm thickness via a rotary microtome. The thin sections were floated in a water bath at 45°C for 5 seconds, carefully retrieved, and mounted onto microscopic glass slides. The slides were then stained with haematoxylin and eosin (H&E), mounted with DPX, and observed under an inverted light microscope for evaluation (Wu et al., 2025 ). Magnification − 10x. 2.12 Data analysis All experimental data are obtainable as the mean ± standard error (SE) and were analyzed using GraphPad Prism version 8.0.2. Each assay was performed in triplicate. Statistical assessments were conducted using one way analysis of variance (ANOVA) followed by the Bonferroni post hoc test for multiple comparisons. All groups rats were compared each other for best observation. Differences were measured statistically significant at p < 0.05 (Das et al., 2024 ; Tian et al., 2025 ). 3. Results We noted that, 80mg STZ ND and 80mg STZ HLD group all rats were completely died within seven days and both groups were excluded from the experiment. Experiments were continued rest group of rats. 3.1. Comparation of body weight among the groups The body weight of the rats must to be measured during the experiment. The HLD fed rats dramatically augmented in body weight compared to the ND fed rats after 4 weeks (P < 0.05). By week 4, the HLD fed group rats gained more weight than the CND group rats and showed characteristics of obesity (Table 3 ). Statistically no significant difference was showed in the final values of the body weights among other groups (P < 0.05). Table 3 Body weight measured on 0-day, 3rd days, 7th days, 14th days, 21st days and 28th days of experiment. Data were expressed as Mean ± SE (n = 5) and analysed by t-test (ns: Not significant; *P < 0.05, **P < 0.01 and ***P < 0.001). Groups 0 Day 3 Day 7 Day 14 days 21 days 28 days CND 253.7± 1.37* 250.46 ± 1.49* 256.87 ± 1.66* 254.21 ± 2.98** 256.02 ± 3.87* 257.01 ± 4.56** CHLD 255.73 ± 1.63* 256.74 ± 1.48** 261.34 ± 1.64* 264.78 ± 2.97* 267.84 ± 3.84* 277.36 ± 4.42* 20 mg STZ ND 236.59 ± 1.45* 237 ± 1.44* 233.54 ± 1.63* 231.24 ± 2.95* 234.65 ± 3.84* 233.68 ± 4.42 40 mg STZ ND 263.4 ± 1.73* 265.05 ± 1.45* 260.95 ± 1.65* 167.14 ± 2.99* 129.54 ± 3.21 * 126.65 ± 4.56** 60 mg STZ ND 255.56 ± 1.23* 256.02 ± 1.49** 251 ± 1.67** 225 ± 2.96* 172 ± 3.84* 132 ± 4.32* 20 mg STZ HLD 255.97 ± 1.73** 257.018 ± 1.43* 258.36 ± 1.62* 256.74 ± 2.98* 259.54 ± 3.21 * 265.05 ± 4.12* 40 mg STZ HLD 253.61 ± 1.75* 252.53 ± 1.42** 160.46 ± 1.64* 145.67 ± 2.97* 122.53 ± 3.82* 123.64 ± 4.12* 60 mg STZ HLD 253 ± 1.47* 256.02± 1.41* 137 ± 1.65* 136 ± 2.96* 128 ± 3.88* 115 ± 4.41** 3.2. Assessment of blood glucose level Follow up procedure was continued for 28 days. Blood glucose level was no significant different between CND and CHLD group rats. According to our experiment, It has been noted that 20mg STZ ND and 20 STZ HLD groups blood glucose level was normal at the end of the experiment, which no significantly different with CND and CHLD group rats (P < 0.05). On the other hand, 40mg STZ ND, 60mg STZ ND, 40mg STZ HLD and 60mg STZ HLD groups rats blood glucose level was bluffy elevated. At the end of the experiment, we observed that blood glucose level was constant (P < 0.05). Which is strongly indicated that hyperglycaemia (Fig. 1 ) . 3.3. Estimation of glycated haemoglobin (HbA1c) After the end of the experiment duration HbA1c was measured. The CND group and CHLD group rats showed normal HbA1c levels throughout the study (P < 0.05). HbA1c level of CND group was 4.988 mg/dL, HLD group was 5.014 mg/dL, 20mg STZ ND group was 5.642 mg/dL, 40mg STZ ND was 7.548mg/dL, 60mg STZ ND was 11.242 mg/dL, 20mg STZ HLD group was 7.737 mg/dL, 40mg STZ HLD was 9.338 mg/dL, 60mg STZ HLD was 10.886 mg/dL. 40mg STZ ND, 60mg STZ ND, 40mg STZ HLD and 60mg STZ HLD strongly indicate hyperglycaemia (P < 0.05; Fig. 2 ). All groups were statistically compared each other. 3.4. Estimation of plasma insulin End of the experiment, we observed that plasma insulin level was normal at CND and CHLD group of rats. Side by side, plasma insulin level progressively decreased with various doses of STZ. CND group, CHLD group, 20mg STZ ND group, 40mg STZ ND group, 60mg STZ ND group, 20mg STZ HLD group, 40mg STZ HLD group and 60mg STZ HLD group insulin level was 6.504 ng/mL, 6.820 ng/mL, 4.291 ng/mL, 0.618 ng/mL,0.666 mg/dL, 5.303 ng/mL, 3.362 ng/mL and 2.482 ng/mL, respectively. All groups were statistically compared each other. During the experiment insulin level was increased at 20mg STZ ND and 20mg STZ HLD group of rats and significantly parallel with CND and CHLD group. On the other hand, 40mg STZ ND and 60mg STZ ND groups was insulin secretion level lower than 40mg STZ HLD and 60mg STZ HLD groups. 40mg STZ ND, 60mg STZ ND, 40mg STZ HLD and 60mg STZ HLD groups showed strongly imply 3.5. Estimation of plasma C-peptide level End of the experiment, we observed that plasma C-peptide level was normal at CND and CHLD group of rats. Side by side, plasma c peptide level progressively decreased with induced doses of STZ. CND group, CHLD group, 20mg STZ ND group, 40mg STZ ND group, 60mg STZ ND group, 20mg STZ HLD group, 40mg STZ HLD group and 60mg STZ HLD group c peptide level was 0.533 ng/mL, 0.543 ng/mL, 0.479 ng/mL, 0.059 ng/mL, 0.042 ng/mL, 0.508 ng/mL, 0.224 mg/dL and 0.212 ng/mL, respectively. All groups were statistically compared each other. During the experiment plasma c peptide level was increased at 20mg STZ ND and 20mg STZ HLD group of rats and parallel with CND and CHLD group. On the other hand, 40mg STZ ND and 60mg STZ ND groups was C-peptide secretion level amazement inferior than 40mg STZ HLD and 60mg STZ HLD groups (P < 0.01). Remarkably, 40mg STZ ND and 60mg STZ ND group rats showed significantly lowest C-peptide secretion level when compare to the other treatment group (P < 0.05; Fig. 4 ). 3.6. Assessment of pancreatic glucokinase enzyme activity End of the experiment, we observed that pancreatic glucokinase enzyme activity was normal at CND and CHLD group of rats. Side by side, pancreatic glucokinase enzyme activity progressively decreased with different doses of STZ. CND group, CHLD group, 20mg STZ ND group, 40mg STZ ND group, 60mg STZ ND group, 20mg STZ HLD group, 40mg STZ HLD group and 60mg STZ HLD group glucokinase enzyme activity was 0.262 units/h/mg of protein, 0.263 units/h/mg of protein, 0.155 units/h/mg of protein, 0.053 units/h/mg of protein, 0.059 units/h/mg of protein, 0.177 units/h/mg of protein, 0.087 units/h/mg of protein and 0.081units/h/mg of protein, respectively. All groups were statistically compared each other. During the experiment glucokinase enzyme activity was increased at 20mg STZ ND and 20mg STZ HLD group of rats. On the other hand, 40mg STZ ND and 60mg STZ ND groups was glucokinase enzyme activity secretion level lower than 40mg STZ HLD and 60mg STZ HLD groups. Interestingly, 40mg STZ ND and 60mg STZ ND group rats showed significantly lowest glucokinase enzyme activity when compare to the other treatment group (P < 0.05; Fig. 5 ). 3.7. Estimation of plasma total cholesterol End of the experiment, we observed that plasma TC was CND group (116.62 mg/dL) and CHLD group (230.044 mg/dL) plasma TC level (230.04 mg/dL) was higher than CND group. Side by side, other treatment group rats, such as, 20mg STZ ND group, 40mg STZ ND group, 60mg STZ ND group, 20mg STZ HLD group, 40mg STZ HLD group and 60mg STZ HLD group cholesterol level were 126.22 mg/dL, 134.21 mg/dL, 140.03 mg/dL, 203.27 mg/dL, 238.03 mg/dL and 232.33 mg/dL, respectively. During the experiment cholesterol was decreased at ND fed rats with STZ (P < 0.05: Fig. 6 ). 3.8. Estimation of plasma triglyceride End of the experiment, we observed that TG level of plasma was normal at CND group (154.192 mg/dL) and CHLD group of rats fed HLD, cholesterol level (272.02mg/dL) was higher than CND group. Side by side, other treatment group rats, such as, 20mg STZ ND group, 40mg STZ ND group, 60mg STZ ND group, 20mg STZ HLD group, 40mg STZ HLD group and 60mg STZ HLD group cholesterol level were 155.75 mg/dL, 161.15 mg/dL, 164.34 mg/dL, 276.34 mg/dL, 279.33 mg/dL and 282.01 mg/dL, respectively. During the experiment TG level was decreased at normal diet with STZ induced group of rats. On the other hand, HLD with STZ induced treatment group rats cholesterol level was higher (P < 0.05: Fig. 7 ). Estimation of antioxidant enzyme Superoxide dismutase (SOD), Catalase (CAT) and reduced glutathione (GSH) activity of pancreatic tissue End of the experiment, we observed that SOD enzyme activity was normal at CND and CHLD group of rats. Side by side, SOD enzyme activity progressively decreased with different doses of STZ. CND group, CHLD group, 20mg STZ ND group, 40mg STZ ND group, 60mg STZ ND group, 20mg STZ HLD group, 40mg STZ HLD group and 60mg STZ HLD group SOD enzyme activity was 11.578 units/mg protein, 12.712 units/mg protein, 7.466 units/mg protein, 7.934 units/mg protein, 6.486 units/mg protein, 6.496 units/mg protein, 6.448 units/mg protein and 5.726 units/mg protein, respectively. Antioxidant enzyme SOD lowest at 60mg STZ HLD group rats (P < 0.05; Fig. 8 ). End of the experiment, we observed that CAT enzyme activity was normal at CND and CHLD group of rats. Side by side, CAT enzyme activity progressively decreased with different doses of STZ. CND group, CHLD group, 20mg STZ ND group, 40mg STZ ND group, 60mg STZ ND group, 20mg STZ HLD group, 40mg STZ HLD group and 60mg STZ HLD group CAT enzyme activity was 18.536 m mol of H 2 O 2 consumption/mg of tissue/min, 18.528 m mol of H 2 O 2 consumption/mg of tissue/min, 11.862 m mol of H 2 O 2 consumption/mg of tissue/min, 10.496 m mol of H 2 O 2 consumption/mg of tissue/min, 9.718 m mol of H 2 O 2 consumption/mg of tissue/min, 11.0892 m mol of H 2 O 2 consumption/mg of tissue/min, 9.05 m mol of H 2 O 2 consumption/mg of tissue/min and 8.738 m mol of H 2 O 2 consumption/mg of tissue/min, respectively. Antioxidant enzyme CAT lowest at 60mg STZ HLD group rats (P < 0.05; Fig. 9 ). End of the experiment, we observed that GSH enzyme activity was normal at CND and CHLD group of rats. Side by side, GSH enzyme activity progressively decreased with different doses of STZ. CND group, CHLD group, 20mg STZ ND group, 40mg STZ ND group, 60mg STZ ND group, 20mg STZ HLD group, 40mg STZ HLD group and 60mg STZ HLD group GSH enzyme activity was 3.16 unit /mg of protein, 3.224 unit /mg of protein, 2.862 unit /mg of protein, 1.01 unit /mg of protein, 0.948 unit /mg of protein, 1.4074 unit /mg of protein, 1.026 unit /mg of protein and 0.952 unit /mg of protein, respectively. Antioxidant enzyme GSH lowest at 60mg STZ ND group rats (P < 0.05; Fig. 10 ). 3.10. Estimation of MDA End of the experiment, we observed that MDA enzyme activity was normal at CND and CHLD group of rats. Side by side, MDA enzyme activity progressively increased with different doses of STZ. CND group, CHLD group, 20mg STZ ND group, 40mg STZ ND group, 60mg STZ ND group, 20mg STZ HLD group, 40mg STZ HLD group and 60mg STZ HLD group MDA enzyme activity was 9.398 n mol/dL of pancreatic tissue, 9.448 n mol/dL of pancreatic tissue, 18.036 n mol/dL of pancreatic tissue, 35.124 n mol/dL of pancreatic tissue, 39.46 n mol/dL of pancreatic tissue, 29.58 n mol/dL of pancreatic tissue, 39.666 n mol/dL of pancreatic tissue and 40.506 n mol/dL of pancreatic tissue, respectively. Antioxidant enzyme MDA highest at 60mg STZ HLD group rats (P < 0.05; Fig. 11 ). 3.11. Histological analysis of pancreatic tissues STZ with normal diet shows reduced islet size, beta cell loss, some inflammation. STZ with high lipid diet shows more severe beta cell loss, maybe fibrosis, increased inflammation. According to Fig. 3.11. we observed that 40mg STZ ND and 60 STZ ND group rats pancreas showed that, reduction in pancreatic islet size and irregular islet architecture. Significant beta cell loss (~ 60–70%) with scattered apoptotic bodies. Mild lymphocytic infiltration observed in peri-islet regions. Minimal collagen deposition, indicating early fibrosis. On the other hand, 40mg STZ HLD and 60 STZ HLD group rats pancreas showed that Severe macro vesicular steatosis (> 50% hepatocyte involvement) with lipid droplet coalescence. Diffuse hepatocellular ballooning, necro inflammatory foci, and Kupffer cell hyperplasia. Early perisinusoidal fibrosis and elevated oxidative stress markers (Fig. 12 ) 4. Discussion In STZ induced normal diet fed rats exhibit rapid weight loss (within 1–2 weeks) due to insulin deficiency, promoting lipolysis, muscle catabolism, and polyuria or polyphagia without efficient nutrient utilization (Guo et al., 2018 ; Skovsø, 2014a ). On the other hand, HLD with STZ causes obesity, reflecting insulin resistance and hyperphagia. Partial β cell loss offsets HLD effects, mimicking DM progression (Akinlade et al., 2021b ). According to Table 3 , We saw that HLD fed rats weight higher than normal fed rats. STZ induced normal fed rats blood glucose surges rapidly within days due to complete beta-cell destruction and absent insulin, persisting without exogenous treatment (Akinlade et al., 2021c ). Side by side, HLD primes insulin resistance, upregulating SREBP-2 transcription factor in liver, boosting HMG-CoA reductase (rate-limiting cholesterol synthesis enzyme) and decreasing LDL receptor expression reducing cholesterol clearance (Pozzo et al., 2016 ).​ Dose of STZ 40 mg/kg b.w. then impairs residual beta-cell function, worsening hyperglycaemia and hyperinsulinemia, which further activates SREBP-1c, promoting de novo lipogenesis and secretion loaded with TC and TG (Srinivasan et al., 2005 ). ​According to our results, blood glucose level was raised in both fed rats groups, but STZ with HLD groups rats present hyperlipidaemia also present hyperglycaemia (Fig. 1 ). STZ with HLD through glucolipotoxicity showing excess lipids overwhelm peroxisomal β-oxidation, accumulating toxic species that inflame hepatocytes, perpetuating dyslipidaemia similar to T2DM. STZ with normal diet severe hyperglycaemia and HbA1c higher indicate that absolute insulin deficiency which is strongly indicate that T1DM (Singh et al., 2024a ; Skovsø, 2014b ). On the other hand, STZ with HLD shows moderate hyperglycaemia and HbA1c reflecting insulin resistance with residual secretion which is strongly indicate that T2DM (Singh et al., 2024b ). In Fig. 2 showed that, ND fed rats HbA1c level was higher than HLD fed rats, which is strongly indicate that T2DM. Insulin and C-peptide level was very low (undetectable) causing complete beta cell destruction in STZ with ND fed rats indicate that T1DM (Singh et al., 2024c ). But then again, STZ with HLD group rats low insulin with detectable C-peptide indicate that T2DM (Singh et al., 2024c ). In Fig. 3 showing that, insulin level was lower in STZ induced ND fed rats than STZ induced HLD fed rats groups. Side by side, Fig. 4 indicate that C- peptide level was very low which is undetectable in STZ induced ND fed rats than STZ induced HLD fed rats, especially 40mg STZ HLD group and 60mg STZ HLD group rats. This result strongly indicates that STZ with HLD is the mimic of T2DM. STZ with ND rats, pancreatic glucokinase keeps high activity in beta cells, serving as glucose sensor (Yoshida et al., 2007 ). It phosphorylates glucose, glycolysis, ATP rise, Ca 2+ influx, and insulin secretion, which is mimic T1DM (Abu Aqel et al., 2024 ). On the other hand, STZ with HLD help to downregulates glucokinase expression, through SREBP-1c and lipotoxicity inhibition. Excess lipids impair glucokinase translocation, reducing glucose sensing and GSIS despite hyperinsulinemia—hallmark of early insulin resistance (Kim et al., 1995 ; Li et al., 2023 ). According to our results, Fig. 5 indicated that glucokinase activity was lower in STZ ND groups. Particularly, 40 mg STZ ND and 60 mg STZ ND groups rats. In contrast, 40mg STZ HLD and 60mg STZ HLD group rats pancreatic glucokinase levels strongly indicate that hyperglycaemia with T2DM. STZ with ND fed rats, insulin deficiency impairs lipoprotein lipase, causing moderate TG elevation and TC rise from reduced clearance, without obesity indicating T1DM (Gylling et al., 2004 ). STZ with HLD fed rats showed marked dyslipidaemia, TG and TC flows due to HLD induced resistance, excess LDL secretion and hepatic SREBP activation. HLD amplifies through lipogenesis which is mimic T2DM (Skovsø, 2014c ). In Figs. 6 and 7 showing that TG and TC level were higher in STZ induced HLD fed rats than ND fed rats groups. This result strongly indicated that 40mg STZ HLD and 60 mg STZ HLD group rats were mimicking T2DM. STZ with ND induces hyperglycaemia ambitious ROS through auto oxidation of glucose, elevating MDA while depleting SOD and CAT drop and GSH reduction from beta-cell destruction represent that T1DM (Singh et al., 2024d ).​ STZ with HLD amplifies oxidative burden through glucolipotoxicity and MDA surges higher, SOD and CAT fall, GSH drops due to lipid peroxidation and inflammation from HLD excess (Cecerska-Heryć et al., 2025 ).​ In Fig. 8 showed that SOD level was lower in 40mg STZ HLD and 60mg STZ HLD group fed rats, which mimicking T2DM. Side by side, in Fig. 9 CAT activity lower in 40mg STZ HLD group than any other group. On the other hand, Fig. 10 showing the GSH activity is higher in 40 mg STZ HLD group rats which is indicating T2DM. MDA level was increased in STZ induced in 40mg STZ HLD and 60mg STZ HLD group rats. This result strong indicate establishing hyperglycaemia with T2DM. In STZ induced DM with normal diet, pancreas histology reveals shrunken islets with near-total beta-cell depletion, absent insulin immunoreactivity, increased alpha-cell prominence, and periductal fibrosis from chronic hyperglycaemia.​ STZ induced HLD shows hypertrophied islets, amyloid deposition, beta-cell hypertrophy with degranulation, fibrosis, and inflammatory infiltrates amplified by HFD lipotoxicity. In Fig. 12 showed that 60mg STZ ND pancreatic beta cells is lowest which is strongly indicate that T1DM. Conversely, STZ induced HLD fed rats 40mg STZ HLD and 60mg STZ HLD group present beta cells which is strongly indicate that T2DM. 5. Conclusion The present investigation demonstrates that The STZ induced model on normal diet precipitated catastrophic β-cell destruction, evidenced by undetectable circulating insulin and C-peptide levels alongside severe, persistent hyperglycaemia and elevated glycosylated haemoglobin. This profound glucose dysregulation reflects complete loss of glucose sensing capacity and insulin secretory function, mirroring the autoimmune-driven β-cell destruction characteristic of type 1 diabetes mellitus. In contrast, the high lipid dietary context attenuated the severity of β-cell obliteration, preserving detectable C-peptide despite reduced insulin secretion. This residual endocrine function, coupled with moderate rather than severe hyperglycaemia and blunted glycaemic control, phenotypically resembles the progressive insulin insufficiency and peripheral insulin resistance defining type 2 diabetes mellitus. The combination of high-fat diet with STZ at 40 mg/kg body weight represents an optimal balance between cost effectiveness, animal welfare, diabetogenic efficacy, and pathophysiological relevance for type 2 diabetes research. produces moderate and stable hyperglycaemia characteristic of human T2DM, preserves partial β cell function and insulin resistance, and minimizes off target organ toxicity and ethically superior choice compared to the 60 mg/kg dose. It has been concluded that the combination of HLD and 40mg STZ/Kg body weight treated rats serves an authenticate animal model for T2DM without any contradictions. Declarations CRediT authorship contribution statement Sanjay Das - Writing – original draft, Visualization, Validation, Methodology, Software, Investigation, Formal analysis, Data curation. Supriya Bhowmick - Formal analysis, Data curation. Sayan Panda - Formal analysis, Data curation. Madhumita Pal - Formal analysis, Data curation. Shrabanti Pyne- Writing- Resources, Methodology. Koushik Das- Review and editing, Supervision, Funding acquisition, Conceptualization. Mrinal Kanti Paira- Review & editing, Supervision, Resources, Methodology, Funding acquisition, Conceptualization. Competing interests The authors declare that no conflict of interest. Compliance with ethical standards Conflict of interest the authors declare that they have no conflicts of interest. Funding sources No funding. Acknowledgment The authors sincerely acknowledge Dr. Chandra Sekhar Hajra, Principal, Belda College, West Bengal, India for his invaluable support, constant encouragement, and for providing the necessary facilities to carry out this research work. Data availability Data supporting the finding of the study are available from the corresponding author upon reasonable request. References Abdel-Megeid AA, Attia AE-RM, Elmarasy SS, Ibrahim A, A.M (2008) Effect of Different Types of Fish on Rats Suffering from Diabetes. 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Supplementary Files floatimage1.jpeg Graphical abstract: Mechanism of STZ with high lipid diet for mimicking T2DM Cite Share Download PDF Status: Posted Version 2 posted You are reading this latest preprint version Show more versions Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8953734","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":596862055,"identity":"b43ead33-2b9b-42d9-bae7-87bccc9ef345","order_by":0,"name":"Dr. Koushik Das","email":"data:image/png;base64,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","orcid":"","institution":"Belda College","correspondingAuthor":true,"prefix":"Dr.","firstName":"Koushik","middleName":"","lastName":"Das","suffix":""},{"id":614773218,"identity":"84fa4d50-ac03-49bf-8551-7f81ff596197","order_by":1,"name":"Sanjay Das","email":"","orcid":"","institution":"Raja Narendra Lal Khan Women’s College","correspondingAuthor":false,"prefix":"","firstName":"Sanjay","middleName":"","lastName":"Das","suffix":""},{"id":614774015,"identity":"3e9fd314-2c26-40b5-9c71-6b62de24866a","order_by":2,"name":"Shrabanti Pyne","email":"","orcid":"","institution":"Raja Narendra Lal Khan Women’s College","correspondingAuthor":false,"prefix":"","firstName":"Shrabanti","middleName":"","lastName":"Pyne","suffix":""},{"id":614774301,"identity":"1d926072-4c79-494f-abeb-19cdbdd73c6e","order_by":3,"name":"Sayan Panda","email":"","orcid":"","institution":"Belda College","correspondingAuthor":false,"prefix":"","firstName":"Sayan","middleName":"","lastName":"Panda","suffix":""},{"id":614774426,"identity":"9b3451e6-74f4-4255-a060-5cdb77425f6b","order_by":4,"name":"Madhumita Pal","email":"","orcid":"","institution":"Belda College","correspondingAuthor":false,"prefix":"","firstName":"Madhumita","middleName":"","lastName":"Pal","suffix":""},{"id":614774586,"identity":"b075c5c5-50be-4f89-91a2-08ca32593350","order_by":5,"name":"Supriya Bhowmik","email":"","orcid":"","institution":"Belda College","correspondingAuthor":false,"prefix":"","firstName":"Supriya","middleName":"","lastName":"Bhowmik","suffix":""},{"id":614774925,"identity":"a6ad0ea2-8e68-4b2f-858e-67a9631522a2","order_by":6,"name":"Dr. Mrinal Kanti Paira","email":"","orcid":"","institution":"Raja Narendra Lal Khan Women’s College","correspondingAuthor":false,"prefix":"Dr.","firstName":"Mrinal","middleName":"Kanti","lastName":"Paira","suffix":""}],"badges":[],"createdAt":"2026-02-24 07:05:47","currentVersionCode":2,"declarations":{"humanSubjects":false,"vertebrateSubjects":true,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":true},"doi":"10.21203/rs.3.rs-8953734/v2","doiUrl":"https://doi.org/10.21203/rs.3.rs-8953734/v2","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":106821687,"identity":"8aabbdcf-7d48-46a9-916a-07913c57c7a3","added_by":"auto","created_at":"2026-04-13 19:00:56","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":74667,"visible":true,"origin":"","legend":"\u003cp\u003eFig. 1 Continue observation of hyperglycaemia of T2DM rats (n =5) were observed for 28 d after a STZ injection. Blood glucose levels were increased after induced STZ injection of treatment group. 40mg STZ ND, 60mg STZ ND, 40mg STZ HLD and 60mg STZ HLD group rats consistently increased which is indicated that hyperglycaemia. Each point represents the Mean ± SE (n=5). CND group rats fed only normal diet. CHLD group rats fed only HLD; 20mg STZ ND, 40mg STZ ND and 60mg STZ ND group rats fed only ND; 20mg/Kg STZ HLD, 40mg STZ HLD and 60mg STZ HLD group rats fed only HLD (P\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"Fig.1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8953734/v2/56c964981d42f9937ec11045.jpg"},{"id":106821686,"identity":"7d68da89-6062-420f-b080-2425549628c9","added_by":"auto","created_at":"2026-04-13 19:00:56","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":102216,"visible":true,"origin":"","legend":"\u003cp\u003eFig. 2 Continue observation of hyperglycaemia of T2DM rats (n =5) were observed for 28 d after a STZ injection. HbA1c levels were increased of treatment group rats after a STZ injection. 40mg STZ ND, 60mg STZ ND, 40mg STZ HLD and 60mg STZ HLDgroup rats consistently increased hyperglycaemia. Each point represents the Mean ± SE (n=5). CND group rats fed only normal diet; CHLD group rats fed only HLD; 20mg STZ N, 40mg STZ ND and 60mg STZ ND group rats fed normal diet; 20mg/Kg STZ, , 40mg STZ HLD and 60mg STZ HLD group rats fed HLD. Data were expressed as Mean ± SE (n=5) and analyzed by one way ANOVA (Bonferroni post hoc statistical test: * ns: Not significant; *P\u0026lt;0.05, **P\u0026lt;0.01 and ***P\u0026lt;0.001).\u003c/p\u003e","description":"","filename":"Fig.2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8953734/v2/12e93d083f63e66e1dba48d9.jpg"},{"id":106821695,"identity":"3a3315a2-fa33-4305-bcfa-1e765f002390","added_by":"auto","created_at":"2026-04-13 19:00:58","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":97083,"visible":true,"origin":"","legend":"\u003cp\u003eFig. 3 Continue observation of hyperglycaemia of T2DM rats (n =5) were observed for 28 d after STZ injection. 20 mg STZ ND group, 20mg STZ HLD group plasma insulin levels were normal. Plasma insulin levels also decreased sharply after induced STZ injection. CND group rats fed only normal diet. CHLD group rats fed only HLD. 20mg STZ ND, 40mg STZ ND, 60mg STZ ND group rats fed normal diet; 20mg/Kg STZ, 40mg STZ HLD, 60mg STZ HLD group rats fed HLD induced rats fed HLD (P\u0026lt;0.05). Data were expressed as Mean ± SE (n=5) and analyzed by one way ANOVA (Bonferroni post hoc statistical test: * ns: Not significant; *P\u0026lt;0.05, **P\u0026lt;0.01 and ***P\u0026lt;0.001).\u003c/p\u003e","description":"","filename":"Fig.3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8953734/v2/905d23f81c4fa171d7b971fc.jpg"},{"id":106821696,"identity":"8b8df18b-64b5-4c3a-9523-c4d375d12064","added_by":"auto","created_at":"2026-04-13 19:00:58","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":83877,"visible":true,"origin":"","legend":"\u003cp\u003eFig. 4 Continue observation of hyperglycaemia of T2DM rats (n =5) were observed for 28 d after STZ injection. 20 mg STZ ND group, 20mg STZ HLD group plasma c peptide levels were normal and other treatment groups c peptide levels also decreased sharply. CND group rats fed only normal diet; CHLD group rats fed only HLD; 20mg STZ ND, 40mg STZ ND and 60mg STZ ND group rats fed normal diet. 20mg STZ HLD, 40mg STZ HLD and 60mg STZ HLD group rats fed HLD (P\u0026lt;0.05).Data were expressed as Mean ± SE (n=5) and analyzed by one way ANOVA (Bonferroni post hoc statistical test: * ns: Not significant; *P\u0026lt;0.05, **P\u0026lt;0.01 and ***P\u0026lt;0.001).\u003c/p\u003e","description":"","filename":"Fig.4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8953734/v2/24015f449188db1cfb4a8143.jpg"},{"id":106821689,"identity":"70b66830-fd36-43df-bd5f-1a04e9e826cf","added_by":"auto","created_at":"2026-04-13 19:00:56","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":83386,"visible":true,"origin":"","legend":"\u003cp\u003eFig. 5 Continue observation of hyperglycaemia of T2DM rats (n =5) were observed for 28 d after STZ injection. 20 mg STZ ND group, 20mg STZ HLD group pancreatic glucokinase enzyme activity was normal. Glucokinase enzyme activity also decreased sharply after induced STZ injection. CND group rats fed only normal diet; CHLD group rats fed only HLD; 20mg STZ ND, 40mg STZ ND and 60mg STZ ND group rats fed normal diet. 20mg STZ HLD, 40mg STZ HLD and 60mg STZ HLD group rats fed HLD (P\u0026lt;0.05). Data were expressed as Mean ± SE (n=5) and analyzed by one way ANOVA (Bonferroni post hoc statistical test: * ns: Not significant; *P\u0026lt;0.05, **P\u0026lt;0.01 and ***P\u0026lt;0.001).\u003c/p\u003e","description":"","filename":"Fig.5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8953734/v2/c4c8b9cf66c4d30960194f4c.jpg"},{"id":106821688,"identity":"cd30019b-b0ef-461d-b2d1-e03113aef59b","added_by":"auto","created_at":"2026-04-13 19:00:56","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":86497,"visible":true,"origin":"","legend":"\u003cp\u003eFig. 6 Continue observation of hyperglycaemia of T2DM rats (n =5) were observed for 28 d after STZ injection. CND group total cholesterol was normal. Plasma cholesterol decreased sharply STZ induced with ND fed rats groups (P\u0026lt;0.05). Plasma cholesterol level was increased STZ induced HLD fed rats. CND group rats fed only normal diet; CHLD group rats fed only HLD; 20mg STZ ND, 40mg STZ ND and 60mg STZ ND group rats fed normal diet. 20mg STZ HLD, 40mg STZ HLD and 60mg STZ HLD group rats fed HLD. Data were expressed as Mean ± SE (n=5) and analyzed by one way ANOVA (Bonferroni post hoc statistical test: * ns: Not significant; *P\u0026lt;0.05, **P\u0026lt;0.01 and ***P\u0026lt;0.001).\u003c/p\u003e","description":"","filename":"Fig.6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8953734/v2/2431aca24d242d4f34d77405.jpg"},{"id":106821691,"identity":"7e556344-80f8-45dd-91fe-3b983479fcf0","added_by":"auto","created_at":"2026-04-13 19:00:56","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":88531,"visible":true,"origin":"","legend":"\u003cp\u003eFig. 7 Continue observation of hyperglycaemia of T2DM rats (n =5) were observed for 28 d after STZ injection. CND group triglyceride level was normal. Plasma triglyceride level decreased sharply STZ induced with ND fed rats groups and increased STZ induced HLD fed rats. CND group rats fed only normal diet; CHLD group rats fed only HLD; 20mg STZ ND, 40mg STZ ND and 60mg STZ ND group rats fed ND. 20mg STZ HLD, 40mg STZ HLD and 60mg STZ HLD group rats fed HLD. Data were expressed as Mean ± SE (n=5) and analyzed by one way ANOVA (Bonferroni post hoc statistical test: * ns: Not significant; *P\u0026lt;0.05, **P\u0026lt;0.01 and ***P\u0026lt;0.001).\u003c/p\u003e","description":"","filename":"Fig.7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8953734/v2/9cee28259f0101097db5f3f1.jpg"},{"id":106821692,"identity":"b37acd10-a8b9-4182-8940-5bbac594a012","added_by":"auto","created_at":"2026-04-13 19:00:56","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":89457,"visible":true,"origin":"","legend":"\u003cp\u003eFig. 8 Continue observation of hyperglycaemia of T2DM rats (n =5) were observed for 28 d after STZ injection. SOD enzyme activity progressively decreased with different doses of STZ of treatment group. CND group rats fed only normal diet; CHLD group rats fed only HLD; 20mg STZ ND, 40mg STZ ND and 60mg STZ ND group rats fed ND. 20mg STZ HLD, 40mg STZ HLD and 60mg STZ HLD group rats fed HLD. Data were expressed as Mean ± SE (n=5) and analyzed by one way ANOVA (Bonferroni post hoc statistical test: * ns: Not significant; *P\u0026lt;0.05, **P\u0026lt;0.01 and ***P\u0026lt;0.001).\u003c/p\u003e","description":"","filename":"Fig.8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8953734/v2/71b173c6605dca25696868d7.jpg"},{"id":106821694,"identity":"7209e461-7d24-4f95-9f76-5bc4b8daa7a1","added_by":"auto","created_at":"2026-04-13 19:00:57","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":82340,"visible":true,"origin":"","legend":"\u003cp\u003eFig. 9 Continue observation of hyperglycaemia of T2DM rats (n =5) were observed for 28 d after STZ injection. CAT enzyme activity progressively decreased with different doses of STZ of treatment group. CND group rats fed only normal diet; CHLD group rats fed only HLD; 20mg STZ ND, 40mg STZ ND and 60mg STZ ND group rats fed ND. 20mg STZ HLD, 40mg STZ HLD and 60mg STZ HLD group rats fed HLD. Data were expressed as Mean ± SE (n=5) and analyzed by one way ANOVA (Bonferroni post hoc statistical test: * ns: Not significant; *P\u0026lt;0.05, **P\u0026lt;0.01 and ***P\u0026lt;0.001).\u003c/p\u003e","description":"","filename":"Fig.9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8953734/v2/54b01294befd476004f02d85.jpg"},{"id":106821690,"identity":"ce731d70-ba37-4a31-ac1d-4de008870b08","added_by":"auto","created_at":"2026-04-13 19:00:56","extension":"jpg","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":86991,"visible":true,"origin":"","legend":"\u003cp\u003eFig. 10Continue observation of hyperglycaemia of T2DM rats (n =5) were observed for 28 d after STZ injection. GSH enzyme activity progressively decreased with different doses of STZ of treatment group. CND group rats fed only normal diet; CHLD group rats fed only HLD; 20mg STZ ND, 40mg STZ ND and 60mg STZ ND group rats fed ND. 20mg STZ HLD, 40mg STZ HLD and 60mg STZ HLD group rats fed HLD. Data were expressed as Mean ± SE (n=5) and analyzed by one way ANOVA (Bonferroni post hoc statistical test: * ns: Not significant; *P\u0026lt;0.05, **P\u0026lt;0.01 and ***P\u0026lt;0.001).\u003c/p\u003e","description":"","filename":"Fig.10.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8953734/v2/0ee15dc09caa2b1d915dd049.jpg"},{"id":106821693,"identity":"dc4fce59-58b6-4008-a17e-9239c1cb9add","added_by":"auto","created_at":"2026-04-13 19:00:57","extension":"jpg","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":86817,"visible":true,"origin":"","legend":"\u003cp\u003eFig. 11 Continue observation of hyperglycaemia of T2DM rats (n =5) were observed for 28 d after STZ injection. MDA enzyme activity progressively increased with different doses of STZ of treatment group. CND group rats fed only normal diet; CHLD group rats fed only HLD; 20mg STZ ND, 40mg STZ ND and 60mg STZ ND group rats fed ND. 20mg STZ HLD, 40mg STZ HLD and 60mg STZ HLD group rats fed HLD. Data were expressed as Mean ± SE (n=5) and analyzed by one way ANOVA (Bonferroni post hoc statistical test: * ns: Not significant; *P\u0026lt;0.05, **P\u0026lt;0.01 and ***P\u0026lt;0.001).\u003c/p\u003e","description":"","filename":"Fig.11.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8953734/v2/60cdda3e2db6b5dd6279efb6.jpg"},{"id":106821701,"identity":"b0791c20-4e8e-4ea0-8338-186d5452adcb","added_by":"auto","created_at":"2026-04-13 19:00:58","extension":"jpg","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":359555,"visible":true,"origin":"","legend":"\u003cp\u003eFig. 12 HLD with STZ group exhibited exacerbated histological damage across all examined organs compared to the ND with STZ group, highlighting the synergistic detrimental effects of hyperlipidaemia and STZ-induced diabetes on pancreatic tissues. Lipid-driven oxidative stress and inflammation likely accelerated tissue degeneration in the HLD. Shown that HLD fed rats groups such as 40mg STZ HLD and 60mg STZ HLDpancreatic beta cells were less than any other treatments groups rats. A- CND, B- CHLD, C-20mg STZ ND, D- 40mg STZ ND, E- 60mg STZ ND,F- 20mg STZ HLD, G- 40mg STZ HLD and H- 60mg STZ HLD group.\u003c/p\u003e","description":"","filename":"Fig.12.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8953734/v2/43826c7d1e24c250d8978ac2.jpg"},{"id":106960627,"identity":"6dfba162-1c60-428d-a2eb-6e13a033e43c","added_by":"auto","created_at":"2026-04-15 09:22:15","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2640720,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8953734/v2/42315e57-0c30-4030-8256-9b12ed24ae33.pdf"},{"id":106821685,"identity":"a68d8fb6-989e-41ee-ae4e-ebbedb8fb8ee","added_by":"auto","created_at":"2026-04-13 19:00:56","extension":"jpeg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":268938,"visible":true,"origin":"","legend":"\u003cp\u003eGraphical abstract: Mechanism of STZ with high lipid diet for mimicking T2DM\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8953734/v2/4ca9c78273e54234b5944e79.jpeg"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"Modelling type 2 diabetes in rats via high-lipid diet and streptozotocin-induced insulin resistance and β-cell dysfunction assessed by C-peptide","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAccording to IDF 2024, 9.2\u0026nbsp;million people with diabetes mellitus (DM) were living with the condition, with 1.8\u0026nbsp;million under the age of 20 (Brito et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). According to the World Health Organization, DM is classified into Type 1 diabetes mellitus (T1DM), Type 2 diabetes mellitus (T2DM), gestational diabetes mellitus (GDM), and other specific types caused by genetic, pancreatic, or drug-related factors (World Health Organization, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Plasma C-peptide not only classified the DM types but also crucial in formulating criteria to safeguard its better management (Upadhyay et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). C-peptide is a polypeptide secreted alongside insulin by pancreatic β cells through enzymatic cleavage of proinsulin. Both insulin and C-peptide are released in equimolar amounts. Nevertheless, for the reason that C-peptide has a longer half life than insulin, it assists as a more stable and consistent marker for evaluating insulin secretion in DM condition (Apostolopoulou et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). T1DM is a multifactorial, heterogeneous, chronic disease, and autoimmune, considered by the distraction of β cells in pancreatic islets of Langerhans (Akinlade et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021a\u003c/span\u003e). T1DM occurs gradually and can be divided into three distinct stages. In the 1st stage, persons develop DM related autoantibodies, which specify an autoimmune (insulin autoantibodies (IAA), glutamic acid decarboxylase autoantibodies (GADA), IA-2 autoantibodies (IA-2A), and zinc transporter 8 autoantibodies (ZnT8A) response contrary to the pancreatic beta cells (Go et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Wan et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In this stage not only pancreatic β cells function normal but also plasma C-peptide secretion normal and blood glucose levels characteristically remain within the normal range. In the 2nd stage, progressively β cell losses from the pancreas (Akinlade et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021a\u003c/span\u003e, p. 1; Buettner et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). As a result, insulin secretion is diminished. Side by side, plasma C- peptide level continuously decreased. Finally, in the 3rd stage, GADA can trigger the immune system to secret autoantibodies against pancreatic β cells and destructed the pancreas. In this stage plasma C-peptide level undetectable (Chen et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Alongside, blood glucose level also higher than normal range. The risk of progression from 1st stage to clinical T1DM is 35%\u0026ndash;50% within five years, rising to 75% for 2nd Stage within 2 years (Baxter and Schofield, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1980\u003c/span\u003e; Buettner et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). On the other hand, T2DM is an enduring metabolic disease characterized by hyperglycaemia ensuing from a combination of insulin resistance and liberal β cell dysfunction (Akash et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2014\u003c/span\u003e, p. 2). In the early stages of T2DM, additional adiposity and chronic low grade inflammation blight insulin signalling in skeletal muscle and adipose tissue, ferment a state of insulin resistance in which cells involve higher condensations of insulin to maintain glucose uptake. Pancreatic β cells primarily expiate for this diminished insulin sensitivity via increasing insulin secretion, resultant in hyperinsulinaemia (Kilari et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). For the reason that, proinsulin is lacerate into insulin and C-peptide in equimolar quantities, this compensatory response is reflected by elevated circulating C-peptide concentrations. Insulin acts on its tyrosine kinase receptor to activate insulin receptor substrates, phosphoinositide 3-kinase (PI3K) and Akt (called Protein Kinase B) while, activation of Akt triggers phosphorylation of the Rab GTPase-activating protein AS160 (Akt Substrate of 160 kDa) and activates Rac1 (small GTPase), leading to the mobilization of GLUT 4 containing vesicles to the cell surface and facilitating glucose transport. In individuals with insulin resistance, defects in IRS (Insulin Receptor Substrate) signalling and actin remodelling impair GLUT4 translocation, consequently postprandial glucose uptake is abridged despite high insulin levels (Baxter and Schofield, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1980\u003c/span\u003e; Buettner et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Jiang et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). With insistent glucotoxicity, lipotoxicity and metabolic stress induce β-cell dedifferentiation, oxidative damage and apoptosis, leading to progressive loss of insulin secretory capacity (Abdel-Megeid et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). C-peptide levels table land and eventually decline as β-cell mass decreases, signalling a transition from compensatory hyperinsulinaemia to relative and then absolute insulin deficiency. This evolution from insulin resistance with elevated endogenous insulin and C-peptide to eventual β-cell failure with declining C-peptide underpins of T2DM (Aboismaiel et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Imagawa et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). T2DM has become one of the most pervasive and serious public health challenges of our time. Animal models, especially rats (Wister albino), are widely used in DM research due to their physiological similarities to humans, ease of handling, and reproducibility of experimental outcomes (Abdel-Megeid et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). STZ present in glucosamine nitrosourea antibiotic which is toxic to pancreatic β cells. STZ induction is one of the most commonly used for DM experiment. Following intraperitoneal injection, STZ models are used to study both T1DM and T2DM. STZ enters pancreatic β-cells through the high capacity glucose transporter GLUT2. STZ also releases nitric oxide and generates reactive oxygen species (ROS), leading to oxidative and nitrosative stress (Aboulmagd et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Furman, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). β-cells are especially vulnerable because they express low levels of antioxidant enzymes, and STZ administration decreases superoxide dismutase and glutathione peroxidase activities. the β cell damage induced by STZ results in hypoinsulinemia and loss of glucose stimulated insulin secretion. In rats, STZ administration leads to decreased insulin stimulated glucose transport and lipogenesis in adipocytes and severe hypoinsulinemia, indicating both impaired insulin production and peripheral insulin resistance. STZ exposure reduces pancreatic GLUT2 mRNA level and dramatically lowers plasma insulin concentrations as well as C-peptide (Jaishree and Narsimha, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Fat mice retain higher GLUT2 expression and near-normal insulin and c peptide levels despite STZ challenge. These functional deficits arise because the DNA alkylation and oxidative stress induced by STZ lead to β cell death and loss of insulin-secretory capacity. Collectively, STZ selective uptake by GLUT2 expressing β-cells, its DNA-alkylating and ROS-generating actions, and the consequent morphological and functional destruction of pancreatic islets underpin its widespread use in experimental models of insulin deficient diabetes (Alfheeaid et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The lack of uniformity poses challenges for reproducibility, data interpretation, and comparison between studies. Although STZ has been widely employed to establish experimental DM models, considerable variation exists in the doses administered across studies (A-Elgadir et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Bagheripour et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Cai et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Gharaat et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Salem et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Therefore, there is a need to systematically evaluate and optimize the dose of STZ in combination with different dietary regimens to establish a more reliable rat model of T2DM. A well-characterized and standardized model would not only improve the understanding of disease mechanisms but also enhance the translational value of preclinical studies for developing antidiabetic drugs and nutritional interventions.\u003c/p\u003e"},{"header":"2. Methodology","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Chemicals and apparatus\u003c/h2\u003e \u003cp\u003eStreptozotocin (STZ) was purchased from sisco research laboratories Pvt.Ltd, Maharashtra, India. All other chemicals were purchased from SRL and MERCK, HiMedia Laboratories, and Crest Biosystems, India. We used diagnostic kit of glycosylated haemoglobin (HbA1c) were purched from agappe diagnostics LTD, India and enzyme-linked immune sorbent assay (ELISA) kits like insulin, and c-peptides and glucokinase purchased from Weldon biotech Pvt. Ltd, wuxidonglin sci \u0026amp; tech development Co. Ltd, India. Triglyceride and total cholesterol were measured by semi auto analyzer standard kit and purchased from Agappe Hills, Kerala, India. We used many instruments like digital weight balance (accuracy-0.1mg) (Adhair Dutta and Sons), semi autoanalizer (MISPA VIVA). pH matter (Digital), incubator with shaker (YONA\u003csup\u003eR\u003c/sup\u003e), cold centrifuge (Eltek), freeze dryer (ICC-INSTIND), light inverted microscope (Magnus).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Care of animal\u003c/h2\u003e \u003cp\u003eTotal fifty adult male eight weeks old Albino Wister rats were used in the study and were obtained from the Saha Enterprise, 386/2, Nilachal, Birati, Kolkata-700051, West Bengal, India (Regd. No. 1828/PO/Bt/S/15/CPCSEA). All animal trials were supervised by the Institutional Animal Ethical Committee (IAEC), which was approved by Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Environment, Forest and Climate Change, Government of India, approval no 02/IAEC/1/S/RNLKWC/2023. We accurately followed the scientific way using animal as per CPCSEA and ARRIVE guidelines (Das et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Rats were randomly divided into ten groups each consist five rats (n\u0026thinsp;=\u0026thinsp;5) and on condition that the normal diet (ND) and water ad libitum for one week for conformation with comfortable temperature of 25\u0026thinsp;\u0026plusmn;\u0026thinsp;2̊ C with the perfect 12-hour cycle of light and dark. After one week all rats were provided high lipid diet (HLD) for 45 days for weight gain. We were used 210\u0026thinsp;\u0026plusmn;\u0026thinsp;20g rats for this experiment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Experimental design\u003c/h2\u003e \u003cp\u003eTotal fifty male rats were randomly divided into ten groups (n\u0026thinsp;=\u0026thinsp;5). Control normal diet (CND) group fed (n\u0026thinsp;=\u0026thinsp;5) fed only normal diet and control high lipid diet (CHLD) group (n\u0026thinsp;=\u0026thinsp;5) fed only high lipid diet (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Composition of normal diet (ND) and high lipid diet (HLD) diet were given in Table \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Duration of experiment was 28 days. After the end of experiment period all rats were sacrificed and blood were collected from vein and stored at -20\u0026deg;C deep freeze. We closely knit blood from the dorsal aorta and segregated the plasma using centrifugation at 3000 rpm for 5 minutes. The serum was kept at -20\u0026deg;C for the biochemical assay. Important organs like pancreas, liver and kidney were collected and stored at -20\u0026deg;C for further experiment. Body weight was measured initial and before the sacrificed of rats.\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\u003eDietary intervention of treatment groups:\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTreatment\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl group fed only normal diet.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCHLD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl group fed only high lipid diet (HLD).\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e20 mg STZ ND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e20 mg/Kg body weight STZ induced rats fed only normal diet.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e40 mg STZ ND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e40 mg/Kg body weight STZ induced rats fed only normal diet.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e60 mg STZ ND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e60 mg/Kg body weight STZ induced rats fed only normal diet.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e80 mg STZ ND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e80 mg/Kg body weight STZ induced rats fed only normal diet.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e20 mg STZ HLD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e20 mg/Kg body weight STZ induced rats fed only HLD.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e40 mg STZ HLD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e40 mg/Kg body weight STZ induced rats fed only HLD.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e60 mg STZ HLD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e60 mg/Kg body weight STZ induced rats fed only HLD.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e80 mg STZ HLD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e80 mg/Kg body weight STZ induced rats fed only HLD.\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 \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComposition of food ingredients of normal diet and HLD (Das et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Khatun et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2019\u003c/span\u003e)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFoods ingredients (g)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNormal diet (n\u0026thinsp;=\u0026thinsp;5) (ND)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHigh lipid diet diet (n\u0026thinsp;=\u0026thinsp;5) (HLD)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCorn Starch\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCasin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBengal gram flour\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSucrose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCoconut Oil\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMineral Mixture\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCholesterol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVitamin Premix\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSalt\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal (g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Induction of streptozotocin\u003c/h2\u003e \u003cp\u003eTotal forty rats and ten groups (n\u0026thinsp;=\u0026thinsp;5) were induced streptozotocin (STZ). The drug was dissolved right before the injection in saline at in 0.1 mol/L citrate buffer (pH 4.5), because STZ degrades within 15 to 20 min after dissolving in the citrate buffer, the STZ solution should be prepared immediately before use and injected within 5 min of dissolution and administered intraperitoneally as single dose to rats groups (Jiang et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Pyne et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Rats were kept deprived of their feed and water for twelve hours before administration of STZ. STZ at different concentrations were induced, for instance, 20 mg/Kg b.w, 40 mg/Kg b.w, 60 mg/Kg b.w and 80 mg/Kg b.w. Blood glucose levels were obtained after 72 hours by using commercially available glucometer, blood was pinched from lateral tail vein. Rats with blood glucose level\u0026thinsp;\u0026gt;\u0026thinsp;126 mg/dl were considered as DM (Almalki et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Assessment of blood glucose level\u003c/h2\u003e \u003cp\u003eAll rats blood glucose level were measure from the tip of their tail vein by a single touch glucometer (Apollo pharmacy; model No. APG01) which were available in local markets. Blood glucose level was measured at 0 day, 3rd day, 7th day, 14th day, 21st day and 28th day. The blood glucose level was expressed by g/dL.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Estimation of glycosylated haemoglobin (HbA1c)\u003c/h2\u003e \u003cp\u003eThis process was accomplished indiscriminately across all experimental groups. HbA1c quantification was propulsion using a standardized kit-based protocol analyzed with a semi-automated analyzer (Maiti et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). The HbA1c level was expressed by g/dL.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Estimation of plasma insulin and C-peptide level and pancreatic glucokinase\u003c/h2\u003e \u003cp\u003ePlasma insulin and C- peptide levels and pancreatic glucokinase were quantified using a rat specific enzyme linked immunosorbent assay (ELISA) kit according to the protocol. Plasma insulin and C-peptide levels was measured at 450 nm using an ELISA plate reader within 30 minutes. Insulin concentrations and C-peptide level were calculated against a standard curve and expressed as ng/mL. Pancreatic glucokinase was measured at 360 nm (excitation) and 460 nm (emission) using a microplate reader within 30 minutes of reaction completion. Activity was quantified against a standard curve and expressed as ng of glucokinase per \u0026micro;g of total protein (ng/\u0026micro;g) (Mallick et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2007\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Estimation of total cholesterol and triglyceride of plasma\u003c/h2\u003e \u003cp\u003eTotal cholesterol (TC) and triglyceride (TG) were measured using standard kit method by semi-autoanalyzer. TC and TG were expressed in mg/dL (Das et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003col\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eEstimation of antioxidant enzyme Superoxide dismutase (SOD), Catalase (CAT) and reduced glutathione (GSH) activity of pancreatic tissue\u003c/b\u003e \u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e \u003c/p\u003e \u003cp\u003eSOD and CAT were measured in tissue supernatant by our laboratory established method and reading of absorbance was noted using a UV-VIS double beam spectrophotometer at 240 nm. Quantification of GSH was performed by pancreatic tissue homogenate mixed with dithio-bis-nitrobenzoic acid according to the standard method using UV-VIS double beam spectrophotometer at 405 nm. The levels of GSH were expressed as \u0026micro;g of GSH/mg pancreatic tissue (Zhu et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.10. Quantification of malondialdehyde (MDA) level of pancreatic tissue\u003c/h2\u003e \u003cp\u003eMeasurements were carried out by a UV-VIS double beam spectrophotometer at 535 nm and values are expressed nmol/mg of pancreatic tissue (Qiu et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.11 Histopathological examination of pancreatic tissue\u003c/h2\u003e \u003cp\u003eUpon finishing the experiment, the pancreatic tissues were fixed in a 10% prepared formalin solution, three sliced into sections approximately 4 \u0026micro;m thick. These covers were then transferred to a tissue processor, where they underwent automated dehydration using alcohol, clearing with xylene, and wax infiltration, followed by saturation for approximately 14 hours overnight. The tissues were afterward embedded in heated paraffin, which was permitted to cool and solidify into paraffin blocks. Each block was cut and sectioned to about 4 \u0026micro;m thickness via a rotary microtome. The thin sections were floated in a water bath at 45\u0026deg;C for 5 seconds, carefully retrieved, and mounted onto microscopic glass slides. The slides were then stained with haematoxylin and eosin (H\u0026amp;E), mounted with DPX, and observed under an inverted light microscope for evaluation (Wu et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Magnification \u0026minus;\u0026thinsp;10x.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.12 Data analysis\u003c/h2\u003e \u003cp\u003eAll experimental data are obtainable as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error (SE) and were analyzed using GraphPad Prism version 8.0.2. Each assay was performed in triplicate. Statistical assessments were conducted using one way analysis of variance (ANOVA) followed by the Bonferroni post hoc test for multiple comparisons. All groups rats were compared each other for best observation. Differences were measured statistically significant at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 (Das et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Tian et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003eWe noted that, 80mg STZ ND and 80mg STZ HLD group all rats were completely died within seven days and both groups were excluded from the experiment. Experiments were continued rest group of rats.\u003c/p\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Comparation of body weight among the groups\u003c/h2\u003e \u003cp\u003eThe body weight of the rats must to be measured during the experiment. The HLD fed rats dramatically augmented in body weight compared to the ND fed rats after 4 weeks (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). By week 4, the HLD fed group rats gained more weight than the CND group rats and showed characteristics of obesity (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Statistically no significant difference was showed in the final values of the body weights among other groups (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eBody weight measured on 0-day, 3rd days, 7th days, 14th days, 21st days and 28th days of experiment. Data were expressed as Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SE (n\u0026thinsp;=\u0026thinsp;5) and analysed by t-test (ns: Not significant; *P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **P\u0026thinsp;\u0026lt;\u0026thinsp;0.01 and ***P\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" 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 \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\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\u003e0 Day\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3 Day\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7 Day\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e14 days\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e21 days\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e28 days\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e253.7\u0026plusmn;\u003c/p\u003e \u003cp\u003e1.37*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e250.46\u0026thinsp;\u0026plusmn;\u0026thinsp;1.49*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e256.87\u0026thinsp;\u0026plusmn;\u0026thinsp;1.66*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e254.21\u0026thinsp;\u0026plusmn;\u0026thinsp;2.98**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e256.02\u0026thinsp;\u0026plusmn;\u0026thinsp;3.87*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e257.01\u0026thinsp;\u0026plusmn;\u0026thinsp;4.56**\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCHLD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e255.73\u0026thinsp;\u0026plusmn;\u0026thinsp;1.63*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e256.74\u0026thinsp;\u0026plusmn;\u0026thinsp;1.48**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e261.34\u0026thinsp;\u0026plusmn;\u0026thinsp;1.64*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e264.78\u0026thinsp;\u0026plusmn;\u0026thinsp;2.97*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e267.84\u0026thinsp;\u0026plusmn;\u0026thinsp;3.84*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e277.36\u0026thinsp;\u0026plusmn;\u0026thinsp;4.42*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e20 mg STZ ND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e236.59\u0026thinsp;\u0026plusmn;\u0026thinsp;1.45*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e237\u0026thinsp;\u0026plusmn;\u0026thinsp;1.44*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e233.54\u0026thinsp;\u0026plusmn;\u0026thinsp;1.63*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e231.24\u0026thinsp;\u0026plusmn;\u0026thinsp;2.95*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e234.65\u0026thinsp;\u0026plusmn;\u0026thinsp;3.84*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e233.68\u0026thinsp;\u0026plusmn;\u0026thinsp;4.42\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e40 mg STZ ND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e263.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.73*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e265.05\u0026thinsp;\u0026plusmn;\u0026thinsp;1.45*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e260.95\u0026thinsp;\u0026plusmn;\u0026thinsp;1.65*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e167.14\u0026thinsp;\u0026plusmn;\u0026thinsp;2.99*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e129.54\u0026thinsp;\u0026plusmn;\u0026thinsp;3.21 *\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e126.65\u0026thinsp;\u0026plusmn;\u0026thinsp;4.56**\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e60 mg STZ ND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e255.56\u0026thinsp;\u0026plusmn;\u0026thinsp;1.23*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e256.02\u0026thinsp;\u0026plusmn;\u0026thinsp;1.49**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e251\u0026thinsp;\u0026plusmn;\u0026thinsp;1.67**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e225\u0026thinsp;\u0026plusmn;\u0026thinsp;2.96*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e172\u0026thinsp;\u0026plusmn;\u0026thinsp;3.84*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e132\u0026thinsp;\u0026plusmn;\u0026thinsp;4.32*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e20 mg STZ HLD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e255.97\u0026thinsp;\u0026plusmn;\u0026thinsp;1.73**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e257.018\u0026thinsp;\u0026plusmn;\u0026thinsp;1.43*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e258.36\u0026thinsp;\u0026plusmn;\u0026thinsp;1.62*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e256.74\u0026thinsp;\u0026plusmn;\u0026thinsp;2.98*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e259.54\u0026thinsp;\u0026plusmn;\u0026thinsp;3.21 *\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e265.05\u0026thinsp;\u0026plusmn;\u0026thinsp;4.12*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e40 mg STZ HLD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e253.61\u0026thinsp;\u0026plusmn;\u0026thinsp;1.75*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e252.53\u0026thinsp;\u0026plusmn;\u0026thinsp;1.42**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e160.46\u0026thinsp;\u0026plusmn;\u0026thinsp;1.64*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e145.67\u0026thinsp;\u0026plusmn;\u0026thinsp;2.97*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e122.53\u0026thinsp;\u0026plusmn;\u0026thinsp;3.82*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e123.64\u0026thinsp;\u0026plusmn;\u0026thinsp;4.12*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e60 mg STZ HLD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e253\u0026thinsp;\u0026plusmn;\u0026thinsp;1.47*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e256.02\u0026plusmn;\u003c/p\u003e \u003cp\u003e1.41*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e137\u0026thinsp;\u0026plusmn;\u0026thinsp;1.65*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e136\u0026thinsp;\u0026plusmn;\u0026thinsp;2.96*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e128\u0026thinsp;\u0026plusmn;\u0026thinsp;3.88*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e115\u0026thinsp;\u0026plusmn;\u0026thinsp;4.41**\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Assessment of blood glucose level\u003c/h2\u003e \u003cp\u003eFollow up procedure was continued for 28 days. Blood glucose level was no significant different between CND and CHLD group rats. According to our experiment, It has been noted that 20mg STZ ND and 20 STZ HLD groups blood glucose level was normal at the end of the experiment, which no significantly different with CND and CHLD group rats (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). On the other hand, 40mg STZ ND, 60mg STZ ND, 40mg STZ HLD and 60mg STZ HLD groups rats blood glucose level was bluffy elevated. At the end of the experiment, we observed that blood glucose level was constant (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Which is strongly indicated that hyperglycaemia (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.3. \u003cb\u003eEstimation of glycated haemoglobin (HbA1c)\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eAfter the end of the experiment duration HbA1c was measured. The CND group and CHLD group rats showed normal HbA1c levels throughout the study (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). HbA1c level of CND group was 4.988 mg/dL, HLD group was 5.014 mg/dL, 20mg STZ ND group was 5.642 mg/dL, 40mg STZ ND was 7.548mg/dL, 60mg STZ ND was 11.242 mg/dL, 20mg STZ HLD group was 7.737 mg/dL, 40mg STZ HLD was 9.338 mg/dL, 60mg STZ HLD was 10.886 mg/dL. 40mg STZ ND, 60mg STZ ND, 40mg STZ HLD and 60mg STZ HLD strongly indicate hyperglycaemia (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003e). All groups were statistically compared each other.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Estimation of plasma insulin\u003c/h2\u003e \u003cp\u003eEnd of the experiment, we observed that plasma insulin level was normal at CND and CHLD group of rats. Side by side, plasma insulin level progressively decreased with various doses of STZ. CND group, CHLD group, 20mg STZ ND group, 40mg STZ ND group, 60mg STZ ND group, 20mg STZ HLD group, 40mg STZ HLD group and 60mg STZ HLD group insulin level was 6.504 ng/mL, 6.820 ng/mL, 4.291 ng/mL, 0.618 ng/mL,0.666 mg/dL, 5.303 ng/mL, 3.362 ng/mL and 2.482 ng/mL, respectively. All groups were statistically compared each other. During the experiment insulin level was increased at 20mg STZ ND and 20mg STZ HLD group of rats and significantly parallel with CND and CHLD group. On the other hand, 40mg STZ ND and 60mg STZ ND groups was insulin secretion level lower than 40mg STZ HLD and 60mg STZ HLD groups. 40mg STZ ND, 60mg STZ ND, 40mg STZ HLD and 60mg STZ HLD groups showed strongly imply\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Estimation of plasma C-peptide level\u003c/h2\u003e \u003cp\u003eEnd of the experiment, we observed that plasma C-peptide level was normal at CND and CHLD group of rats. Side by side, plasma c peptide level progressively decreased with induced doses of STZ. CND group, CHLD group, 20mg STZ ND group, 40mg STZ ND group, 60mg STZ ND group, 20mg STZ HLD group, 40mg STZ HLD group and 60mg STZ HLD group c peptide level was 0.533 ng/mL, 0.543 ng/mL, 0.479 ng/mL, 0.059 ng/mL, 0.042 ng/mL, 0.508 ng/mL, 0.224 mg/dL and 0.212 ng/mL, respectively. All groups were statistically compared each other. During the experiment plasma c peptide level was increased at 20mg STZ ND and 20mg STZ HLD group of rats and parallel with CND and CHLD group. On the other hand, 40mg STZ ND and 60mg STZ ND groups was C-peptide secretion level amazement inferior than 40mg STZ HLD and 60mg STZ HLD groups (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Remarkably, 40mg STZ ND and 60mg STZ ND group rats showed significantly lowest C-peptide secretion level when compare to the other treatment group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.6. Assessment of pancreatic glucokinase enzyme activity\u003c/h2\u003e \u003cp\u003eEnd of the experiment, we observed that pancreatic glucokinase enzyme activity was normal at CND and CHLD group of rats. Side by side, pancreatic glucokinase enzyme activity progressively decreased with different doses of STZ. CND group, CHLD group, 20mg STZ ND group, 40mg STZ ND group, 60mg STZ ND group, 20mg STZ HLD group, 40mg STZ HLD group and 60mg STZ HLD group glucokinase enzyme activity was 0.262 units/h/mg of protein, 0.263 units/h/mg of protein, 0.155 units/h/mg of protein, 0.053 units/h/mg of protein, 0.059 units/h/mg of protein, 0.177 units/h/mg of protein, 0.087 units/h/mg of protein and 0.081units/h/mg of protein, respectively. All groups were statistically compared each other. During the experiment glucokinase enzyme activity was increased at 20mg STZ ND and 20mg STZ HLD group of rats. On the other hand, 40mg STZ ND and 60mg STZ ND groups was glucokinase enzyme activity secretion level lower than 40mg STZ HLD and 60mg STZ HLD groups. Interestingly, 40mg STZ ND and 60mg STZ ND group rats showed significantly lowest glucokinase enzyme activity when compare to the other treatment group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.7. Estimation of plasma total cholesterol\u003c/h2\u003e \u003cp\u003eEnd of the experiment, we observed that plasma TC was CND group (116.62 mg/dL) and CHLD group (230.044 mg/dL) plasma TC level (230.04 mg/dL) was higher than CND group. Side by side, other treatment group rats, such as, 20mg STZ ND group, 40mg STZ ND group, 60mg STZ ND group, 20mg STZ HLD group, 40mg STZ HLD group and 60mg STZ HLD group cholesterol level were 126.22 mg/dL, 134.21 mg/dL, 140.03 mg/dL, 203.27 mg/dL, 238.03 mg/dL and 232.33 mg/dL, respectively. During the experiment cholesterol was decreased at ND fed rats with STZ (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05: Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.8. Estimation of plasma triglyceride\u003c/h2\u003e \u003cp\u003eEnd of the experiment, we observed that TG level of plasma was normal at CND group (154.192 mg/dL) and CHLD group of rats fed HLD, cholesterol level (272.02mg/dL) was higher than CND group. Side by side, other treatment group rats, such as, 20mg STZ ND group, 40mg STZ ND group, 60mg STZ ND group, 20mg STZ HLD group, 40mg STZ HLD group and 60mg STZ HLD group cholesterol level were 155.75 mg/dL, 161.15 mg/dL, 164.34 mg/dL, 276.34 mg/dL, 279.33 mg/dL and 282.01 mg/dL, respectively. During the experiment TG level was decreased at normal diet with STZ induced group of rats. On the other hand, HLD with STZ induced treatment group rats cholesterol level was higher (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05: Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003col\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eEstimation of antioxidant enzyme Superoxide dismutase (SOD), Catalase (CAT) and reduced glutathione (GSH) activity of pancreatic tissue\u003c/b\u003e \u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e \u003c/p\u003e \u003cp\u003eEnd of the experiment, we observed that SOD enzyme activity was normal at CND and CHLD group of rats. Side by side, SOD enzyme activity progressively decreased with different doses of STZ. CND group, CHLD group, 20mg STZ ND group, 40mg STZ ND group, 60mg STZ ND group, 20mg STZ HLD group, 40mg STZ HLD group and 60mg STZ HLD group SOD enzyme activity was 11.578 units/mg protein, 12.712 units/mg protein, 7.466 units/mg protein, 7.934 units/mg protein, 6.486 units/mg protein, 6.496 units/mg protein, 6.448 units/mg protein and 5.726 units/mg protein, respectively. Antioxidant enzyme SOD lowest at 60mg STZ HLD group rats (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eEnd of the experiment, we observed that CAT enzyme activity was normal at CND and CHLD group of rats. Side by side, CAT enzyme activity progressively decreased with different doses of STZ. CND group, CHLD group, 20mg STZ ND group, 40mg STZ ND group, 60mg STZ ND group, 20mg STZ HLD group, 40mg STZ HLD group and 60mg STZ HLD group CAT enzyme activity was 18.536 m mol of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e consumption/mg of tissue/min, 18.528 m mol of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e consumption/mg of tissue/min, 11.862 m mol of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e consumption/mg of tissue/min, 10.496 m mol of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e consumption/mg of tissue/min, 9.718 m mol of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e consumption/mg of tissue/min, 11.0892 m mol of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e consumption/mg of tissue/min, 9.05 m mol of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e consumption/mg of tissue/min and 8.738 m mol of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e consumption/mg of tissue/min, respectively. Antioxidant enzyme CAT lowest at 60mg STZ HLD group rats (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eEnd of the experiment, we observed that GSH enzyme activity was normal at CND and CHLD group of rats. Side by side, GSH enzyme activity progressively decreased with different doses of STZ. CND group, CHLD group, 20mg STZ ND group, 40mg STZ ND group, 60mg STZ ND group, 20mg STZ HLD group, 40mg STZ HLD group and 60mg STZ HLD group GSH enzyme activity was 3.16 unit /mg of protein, 3.224 unit /mg of protein, 2.862 unit /mg of protein, 1.01 unit /mg of protein, 0.948 unit /mg of protein, 1.4074 unit /mg of protein, 1.026 unit /mg of protein and 0.952 unit /mg of protein, respectively. Antioxidant enzyme GSH lowest at 60mg STZ ND group rats (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e10\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.10. Estimation of MDA\u003c/h2\u003e \u003cp\u003eEnd of the experiment, we observed that MDA enzyme activity was normal at CND and CHLD group of rats. Side by side, MDA enzyme activity progressively increased with different doses of STZ. CND group, CHLD group, 20mg STZ ND group, 40mg STZ ND group, 60mg STZ ND group, 20mg STZ HLD group, 40mg STZ HLD group and 60mg STZ HLD group MDA enzyme activity was 9.398 n mol/dL of pancreatic tissue, 9.448 n mol/dL of pancreatic tissue, 18.036 n mol/dL of pancreatic tissue, 35.124 n mol/dL of pancreatic tissue, 39.46 n mol/dL of pancreatic tissue, 29.58 n mol/dL of pancreatic tissue, 39.666 n mol/dL of pancreatic tissue and 40.506 n mol/dL of pancreatic tissue, respectively. Antioxidant enzyme MDA highest at 60mg STZ HLD group rats (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e11\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e3.11. Histological analysis of pancreatic tissues\u003c/h2\u003e \u003cp\u003eSTZ with normal diet shows reduced islet size, beta cell loss, some inflammation. STZ with high lipid diet shows more severe beta cell loss, maybe fibrosis, increased inflammation. According to Fig.\u0026nbsp;3.11. we observed that 40mg STZ ND and 60 STZ ND group rats pancreas showed that, reduction in pancreatic islet size and irregular islet architecture. Significant beta cell loss (~\u0026thinsp;60\u0026ndash;70%) with scattered apoptotic bodies. Mild lymphocytic infiltration observed in peri-islet regions. Minimal collagen deposition, indicating early fibrosis. On the other hand, 40mg STZ HLD and 60 STZ HLD group rats pancreas showed that Severe macro vesicular steatosis (\u0026gt;\u0026thinsp;50% hepatocyte involvement) with lipid droplet coalescence. Diffuse hepatocellular ballooning, necro inflammatory foci, and Kupffer cell hyperplasia. Early perisinusoidal fibrosis and elevated oxidative stress markers (Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e12\u003c/span\u003e)\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eIn STZ induced normal diet fed rats exhibit rapid weight loss (within 1\u0026ndash;2 weeks) due to insulin deficiency, promoting lipolysis, muscle catabolism, and polyuria or polyphagia without efficient nutrient utilization (Guo et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Skovs\u0026oslash;, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2014a\u003c/span\u003e). On the other hand, HLD with STZ causes obesity, reflecting insulin resistance and hyperphagia. Partial β cell loss offsets HLD effects, mimicking DM progression (Akinlade et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2021b\u003c/span\u003e). According to Table \u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, We saw that HLD fed rats weight higher than normal fed rats.\u003c/p\u003e \u003cp\u003eSTZ induced normal fed rats blood glucose surges rapidly within days due to complete beta-cell destruction and absent insulin, persisting without exogenous treatment (Akinlade et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2021c\u003c/span\u003e). Side by side, HLD primes insulin resistance, upregulating SREBP-2 transcription factor in liver, boosting HMG-CoA reductase (rate-limiting cholesterol synthesis enzyme) and decreasing LDL receptor expression reducing cholesterol clearance (Pozzo et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).​ Dose of STZ 40 mg/kg b.w. then impairs residual beta-cell function, worsening hyperglycaemia and hyperinsulinemia, which further activates SREBP-1c, promoting de novo lipogenesis and secretion loaded with TC and TG (Srinivasan et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). ​According to our results, blood glucose level was raised in both fed rats groups, but STZ with HLD groups rats present hyperlipidaemia also present hyperglycaemia (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003e). STZ with HLD through glucolipotoxicity showing excess lipids overwhelm peroxisomal β-oxidation, accumulating toxic species that inflame hepatocytes, perpetuating dyslipidaemia similar to T2DM.\u003c/p\u003e \u003cp\u003eSTZ with normal diet severe hyperglycaemia and HbA1c higher indicate that absolute insulin deficiency which is strongly indicate that T1DM (Singh et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2024a\u003c/span\u003e; Skovs\u0026oslash;, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2014b\u003c/span\u003e). On the other hand, STZ with HLD shows moderate hyperglycaemia and HbA1c reflecting insulin resistance with residual secretion which is strongly indicate that T2DM (Singh et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2024b\u003c/span\u003e). In Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003e showed that, ND fed rats HbA1c level was higher than HLD fed rats, which is strongly indicate that T2DM.\u003c/p\u003e \u003cp\u003eInsulin and C-peptide level was very low (undetectable) causing complete beta cell destruction in STZ with ND fed rats indicate that T1DM (Singh et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2024c\u003c/span\u003e). But then again, STZ with HLD group rats low insulin with detectable C-peptide indicate that T2DM (Singh et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2024c\u003c/span\u003e). In Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003e showing that, insulin level was lower in STZ induced ND fed rats than STZ induced HLD fed rats groups. Side by side, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003e indicate that C- peptide level was very low which is undetectable in STZ induced ND fed rats than STZ induced HLD fed rats, especially 40mg STZ HLD group and 60mg STZ HLD group rats. This result strongly indicates that STZ with HLD is the mimic of T2DM.\u003c/p\u003e \u003cp\u003eSTZ with ND rats, pancreatic glucokinase keeps high activity in beta cells, serving as glucose sensor (Yoshida et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). It phosphorylates glucose, glycolysis, ATP rise, Ca\u003csup\u003e2+\u003c/sup\u003e influx, and insulin secretion, which is mimic T1DM (Abu Aqel et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). On the other hand, STZ with HLD help to downregulates glucokinase expression, through SREBP-1c and lipotoxicity inhibition. Excess lipids impair glucokinase translocation, reducing glucose sensing and GSIS despite hyperinsulinemia\u0026mdash;hallmark of early insulin resistance (Kim et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). According to our results, Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003e indicated that glucokinase activity was lower in STZ ND groups. Particularly, 40 mg STZ ND and 60 mg STZ ND groups rats. In contrast, 40mg STZ HLD and 60mg STZ HLD group rats pancreatic glucokinase levels strongly indicate that hyperglycaemia with T2DM.\u003c/p\u003e \u003cp\u003eSTZ with ND fed rats, insulin deficiency impairs lipoprotein lipase, causing moderate TG elevation and TC rise from reduced clearance, without obesity indicating T1DM (Gylling et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). STZ with HLD fed rats showed marked dyslipidaemia, TG and TC flows due to HLD induced resistance, excess LDL secretion and hepatic SREBP activation. HLD amplifies through lipogenesis which is mimic T2DM (Skovs\u0026oslash;, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2014c\u003c/span\u003e). In Figs.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e6\u003c/span\u003e and \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e7\u003c/span\u003e showing that TG and TC level were higher in STZ induced HLD fed rats than ND fed rats groups. This result strongly indicated that 40mg STZ HLD and 60 mg STZ HLD group rats were mimicking T2DM.\u003c/p\u003e \u003cp\u003eSTZ with ND induces hyperglycaemia ambitious ROS through auto oxidation of glucose, elevating MDA while depleting SOD and CAT drop and GSH reduction from beta-cell destruction represent that T1DM (Singh et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2024d\u003c/span\u003e).​ STZ with HLD amplifies oxidative burden through glucolipotoxicity and MDA surges higher, SOD and CAT fall, GSH drops due to lipid peroxidation and inflammation from HLD excess (Cecerska-Heryć et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).​ In Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e8\u003c/span\u003e showed that SOD level was lower in 40mg STZ HLD and 60mg STZ HLD group fed rats, which mimicking T2DM. Side by side, in Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e9\u003c/span\u003e CAT activity lower in 40mg STZ HLD group than any other group. On the other hand, Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e10\u003c/span\u003e showing the GSH activity is higher in 40 mg STZ HLD group rats which is indicating T2DM. MDA level was increased in STZ induced in 40mg STZ HLD and 60mg STZ HLD group rats. This result strong indicate establishing hyperglycaemia with T2DM.\u003c/p\u003e \u003cp\u003eIn STZ induced DM with normal diet, pancreas histology reveals shrunken islets with near-total beta-cell depletion, absent insulin immunoreactivity, increased alpha-cell prominence, and periductal fibrosis from chronic hyperglycaemia.​ STZ induced HLD shows hypertrophied islets, amyloid deposition, beta-cell hypertrophy with degranulation, fibrosis, and inflammatory infiltrates amplified by HFD lipotoxicity. In Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e12\u003c/span\u003e showed that 60mg STZ ND pancreatic beta cells is lowest which is strongly indicate that T1DM. Conversely, STZ induced HLD fed rats 40mg STZ HLD and 60mg STZ HLD group present beta cells which is strongly indicate that T2DM.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThe present investigation demonstrates that The STZ induced model on normal diet precipitated catastrophic β-cell destruction, evidenced by undetectable circulating insulin and C-peptide levels alongside severe, persistent hyperglycaemia and elevated glycosylated haemoglobin. This profound glucose dysregulation reflects complete loss of glucose sensing capacity and insulin secretory function, mirroring the autoimmune-driven β-cell destruction characteristic of type 1 diabetes mellitus. In contrast, the high lipid dietary context attenuated the severity of β-cell obliteration, preserving detectable C-peptide despite reduced insulin secretion. This residual endocrine function, coupled with moderate rather than severe hyperglycaemia and blunted glycaemic control, phenotypically resembles the progressive insulin insufficiency and peripheral insulin resistance defining type 2 diabetes mellitus. The combination of high-fat diet with STZ at 40 mg/kg body weight represents an optimal balance between cost effectiveness, animal welfare, diabetogenic efficacy, and pathophysiological relevance for type 2 diabetes research. produces moderate and stable hyperglycaemia characteristic of human T2DM, preserves partial β cell function and insulin resistance, and minimizes off target organ toxicity and ethically superior choice compared to the 60 mg/kg dose. It has been concluded that the combination of HLD and 40mg STZ/Kg body weight treated rats serves an authenticate animal model for T2DM without any contradictions.\u003c/p\u003e"},{"header":"Declarations","content":" \u003cp\u003e \u003cb\u003eCRediT authorship contribution statement\u003c/b\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eSanjay Das\u003c/b\u003e - Writing \u0026ndash; original draft, Visualization, Validation, Methodology, Software, Investigation, Formal analysis, Data curation. \u003cb\u003eSupriya Bhowmick -\u003c/b\u003e Formal analysis, Data curation. \u003cb\u003eSayan Panda\u003c/b\u003e - Formal analysis, Data curation. \u003cb\u003eMadhumita Pal\u003c/b\u003e- Formal analysis, Data curation. \u003cb\u003eShrabanti Pyne-\u003c/b\u003e Writing- Resources, Methodology. \u003cb\u003eKoushik Das-\u003c/b\u003e Review and editing, Supervision, Funding acquisition, Conceptualization. \u003cb\u003eMrinal Kanti Paira-\u003c/b\u003e Review \u0026amp; editing, Supervision, Resources, Methodology, Funding acquisition, Conceptualization.\u003c/p\u003e\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare that no conflict of interest.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eCompliance with ethical standards\u003c/h2\u003e \u003cp\u003eConflict of interest the authors declare that they have no conflicts of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding sources\u003c/h2\u003e \u003cp\u003eNo funding.\u003c/p\u003e\u003ch2\u003eAcknowledgment\u003c/h2\u003e \u003cp\u003eThe authors sincerely acknowledge Dr. Chandra Sekhar Hajra, Principal, Belda College, West Bengal, India for his invaluable support, constant encouragement, and for providing the necessary facilities to carry out this research work.\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eData supporting the finding of the study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbdel-Megeid AA, Attia AE-RM, Elmarasy SS, Ibrahim A, A.M (2008) Effect of Different Types of Fish on Rats Suffering from Diabetes. 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PLoS ONE 20:e0326897. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1371/journal.pone.0326897\u003c/span\u003e\u003cspan address=\"10.1371/journal.pone.0326897\" 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":true,"hideJournal":true,"highlight":"","institution":"Raja Narendra Lal Khan Women's College","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"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":"Insulin, Type 2 diabetes mellitus, High lipid diet, Streptozotocin, C- peptide","lastPublishedDoi":"10.21203/rs.3.rs-8953734/v2","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8953734/v2","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eStreptozotocin, that has a selective pharmacological toxicity toward pancreatic beta cells, in addition to high lipid diet (HLD) has been widely used to induce T2DM. However, no evidence has shown that superior dose of streptozotocin (STZ) to establish T2DM. This study was initiated to develop an animal model (Wister Albino rats) of T2DM with suitable dose of STZ. Total fifty male rats (210\u0026thinsp;\u0026plusmn;\u0026thinsp;20 g) were arbitrarily divided into 10 groups (n\u0026thinsp;=\u0026thinsp;5). Two groups were control group fed normal diet (ND) and high lipid diet (HLD). The remaining rats were induced with STZ at 20, 40, 60 and 80 mg/kg body weight, with each dose tested under ND and HLD conditions. Body weight, blood glucose, HbA1c, serum insulin, C-peptide, pancreatic glucokinase, serum triglycerides (TG), total cholesterol (TC), antioxidant enzymes (SOD, CAT, GSH, MDA) and pancreatic histology. 80 mg/kg STZ group rats expired within 7 days. After 28 days of experiment, Blood glucose was markedly raised up. Insulin and C-peptide levels were lower in STZ/HLD fed groups (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) rats. Pancreatic glucokinase activity significantly decreased (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). SOD, CAT, GSH and TG, TC increased significantly in STZ/HLD treated groups (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). We observed that, β- cells were present in STZ/HLD fed rats pancreas and insulin secretion is higher than ND fed rats. We concluded that, HLD with 40mg STZ/Kg b.w. rats provide a novel animal model for T2DM without any contradictions and is suitable for the testing of antidiabetic compounds.\u003c/p\u003e","manuscriptTitle":"Modelling type 2 diabetes in rats via high-lipid diet and streptozotocin-induced insulin resistance and β-cell dysfunction assessed by C-peptide","msid":"","msnumber":"","nonDraftVersions":[{"code":2,"date":"2026-04-13 19:00:48","doi":"10.21203/rs.3.rs-8953734/v2","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}},{"code":1,"date":"2026-02-25 10:46:13","doi":"10.21203/rs.3.rs-8953734/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":"April 13th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":63509105,"name":"Endocrinology \u0026 Metabolism"}],"tags":[],"updatedAt":"2026-02-25T10:46:13+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-13 19:00:48","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v2","identity":"rs-8953734","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8953734","identity":"rs-8953734","version":["v2"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}