Vitamin C Supplementation Mitigates Diabetes-Associated Skeletal Muscle Atrophy

Vitamin C Supplementation Mitigates Diabetes-Associated Skeletal Muscle Atrophy | 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 Article Vitamin C Supplementation Mitigates Diabetes-Associated Skeletal Muscle Atrophy Vinay Kumar Rao, Supriya Bevinakoppamath, Parika Kala, Swarnaseetha Adusumalli, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8342512/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 9 You are reading this latest preprint version Abstract Background Skeletal muscle atrophy is a major complication of diabetes linked with poor prognosis and reduced quality of life. Antioxidant vitamin C has shown promise in alleviating diabetic complications in rodents and mammals. Whether vitamin C is effective in mitigating diabetic muscle atrophy and the underlying molecular mechanisms remains unclear. The Drosophila larval body wall muscle offers a powerful system to identify interventions that target muscle atrophy and hypertrophy. Methods To induce diabetic conditions, wild-type Canton-S Drosophila larvae were fed with High Sugar Diet (HSD) with a final concentration of 1 M sucrose. Control larvae (CT) were fed a diet containing 0.1 M sucrose. Body wall muscles of mid-third instar larvae were used for molecular analysis. The effect of vitamin C on skeletal muscle regeneration was assessed using a streptozotocin-induced diabetic mouse model. Results HSD-fed larvae exhibited severe growth inhibition and developed hyperglycemia, accompanied by increased triglycerides in the hemolymph. Ventral longitudinal VL3 and VL4 body wall myofibers were reduced in size, exhibited decreased expression of mef2 and mhc, along with increased expression of FOXO target genes, indicating muscle atrophy. Supplementation of Vitamin C (Ascorbic acid) to HSD (HSD + AA) rescued growth inhibition, reduced food aversion, and alleviated diabetic phenotypes. Transcriptomic analysis of HSD + AA larval muscles revealed enhanced expression of genes linked to metabolism, muscle development, and differentiation. Vitamin C reduced oxidative stress and, interestingly, rescued the expression of epigenetic regulator Ten-eleven-translocation (Tet), which utilizes vitamin C as a cofactor for its activity. Furthermore, vitamin C improved skeletal muscle regeneration in the injured diabetic mice. Conclusion Collectively, our data demonstrate that vitamin C mitigates muscle atrophy and enhances skeletal muscle regeneration in diabetic muscles. These results suggest that vitamin C intake, in combination with anti-diabetic medications, may offer promising strategy to mitigate long-term diabetic complications. Health sciences/Diseases/Endocrine system and metabolic diseases/Diabetes/Diabetes complications Biological sciences/Physiology/Metabolism/Metabolic diseases/Diabetes/Diabetes complications Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Diabetes is generally accompanied with complications of various organs including heart, kidney, eye, skeletal muscles and tend to worsen over time [ 1 ]. Skeletal muscle is a vital metabolic organ that helps coordinate the body’s energy and protein balance primarily through glucose uptake, storage of amino acids making it a key tissue in maintaining insulin sensitivity and protein synthesis for tissue repair. Both type 1 and type 2 diabetes cause atrophy of skeletal muscles, characterised by shrinking of muscle fibres, altered metabolic activity, and significant loss of muscle mass and function ultimately resulting in reduced quality of life of diabetic patients [ 2 – 4 ]. Mechanistically, insulin resistance in diabetic muscle alters PI3K–AKT–mTOR signalling, which is critical for muscle anabolism [ 5 ]. In addition, increased expression MuRF1 and Atrogin-1, which promote protein degradation, are activated concurrently with insulin resistance [ 6 ]. When combined, these changes tilt the balance towards catabolism gradually leading to the loss of skeletal muscle mass and function. Additionally oxidative stress and mitochondrial dysfunction, contributes to energy imbalance and decreased muscle regeneration[ 7 ]. Although the current therapeutics for diabetes focuses on controlling blood glucose, they fall short in addressing the gradual loss of skeletal muscle mass and strength in diabetic patients. Nutritional supplements that not only restore normal glycemia but also counteract muscle atrophy linked to diabetes are therefore becoming increasingly necessary. Interestingly, diabetic patients tend to have lower levels of antioxidant vitamins such as vitamin C, E and A [ 8 , 9 ]. Vitamin C is an essential nutrient critical for maintaining various metabolic and physiological functions. It serves as a cofactor for many enzymes notably for Fe(II) and 2-oxoglutarate-dependent dioxygenases including Tet enzymes, which catalyze the oxidation of DNA 5-methylcytosine into 5-hydroxymethylcytosine and regulate gene expression[ 10 , 11 ]. Increasing evidence indicates a critical role for vitamin C in skeletal muscle regeneration [ 12 – 14 ]. Vitamin C uptake reduced oxidative stress and improved insulin sensitivity in diabetic patients [ 15 ]. Deficiency of vitamin C causes increased ROS and muscle wasting with concomitant increase in catabolic genes [ 16 ]. Recent mechanistic insights have shown that the vitamin C increases myoblast differentiation marked by reduction in global H3K9 methylation [ 17 ]. While these studies demonstrate the role of vitamin C in skeletal muscle homeostasis, whether vitamin C counteracts muscle atrophy in diabetes and the underlying mechanisms remains to be further investigated. In this study, using a Drosophila melanogaster (Here after referred as Drosophila) model of HSD-induced diabetes, which showed characteristics of diabetic-like phenotypes, we examined the effect of vitamin C on the reversal of diabetic-like phenotypes and skeletal muscle atrophy. Vitamin C supplementation led to rescue in larval myofiber size compared to the HSD group. Vitamin C increased food uptake by HSD-fed larvae, indicating reversal of food aversion. Transcriptomic analysis of VC-HSD larval muscles revealed enhanced expression of metabolic genes, and increased expression of genes linked to muscle development and differentiation. Vitamin C supplementation reduced oxidative stress and, interestingly, dTet expression is rescued in diabetic muscles. We extend our findings to the diabetic mouse model and find that vitamin C helps improve skeletal muscle regeneration in diabetic mice. Altogether, the data suggests that vitamin C could be beneficial in mitigating muscle atrophy in diabetes condition. Materials and methods Fly stock Canton-S wild type flies were obtained from the National Drosophila Stock Centre facility, University of Mysore. 100ml of media contained Semolina (10g), Agar (1g), propionic acid (0.75ml), Sucrose (5.13 g = 0.15 M for CT, 34.2g = 1.0 M for HSD) [ 18 ], Ascorbic acid (AA, 0.05g = 10mM). Experimental larvae were reared on one of the following diets: control (CT), High-Sugar Diet (HSD), or HSD supplemented with either vitamin C (ascorbic acid) (HSD + AA), or CT diet supplemented with ascorbic acid (CT + AA). Glucose estimation Hemolymph was pooled from 50–100 third instar larvae to obtain 2µl for the assay. Glucose was measured by adding 2µl of the hemolymph to 198µl of Erba Glucose Reagent (# 120235, Erba Mannheim, Germany) in a 96-well plate. The plate was incubated at 37 0 C for 15min. The absorbance of standards and the samples were measured against blank at 505nm. Triglyceride (TAG) estimation For whole body TAG, 5 larvae were homogenized in PBS + 0.1% Tween and centrifuged at 5000rpm for 5 min at 4 0 C. The supernatant was heated for 5 min at 65°C to inactivate lipases [ 18 ]. 2µl of the homogenate was mixed with 198µl of triglyceride reagent (# 120237, Erba Mannheim, Germany) and analysed as per the manufacturer's instructions. Larval feeding assay Larval feeding assay was performed according to published protocols [ 19 ]. Third instar larvae were starved for 2h and transferred into the 6 well plates containing the specific diets with 0.05% (wt/vol) bromophenol blue dye (#93676, SRL, India). After 10min of feeding, the larvae were washed with PBS and homogenized in Milli-Q water. The lysates were centrifuged and 100µl of supernatant was transferred to 96-well plate and absorbance was measured at 595 nm. Size assessment and crawling assay Wandering third instar larvae were heat fixed and imaged using Trinocular stereo zoom (Model: SZX7, Olympus). Area was calculated using Gryphax software. For crawling assay, larvae were allowed to crawl on a 90mm petri dish containing 2% agarose over graph paper with a 0.2 cm 2 grid. Number of grid lines crossed in 90s were recorded with the camera attached to stereo zoom microscope [ 20 ]. Phalloidin staining Larval muscle dissection and staining were performed as described previously [ 21 ]. Mid third instar larvae were dissected PBS. The muscle fillets were fixed in 4% formaldehyde for 20min at room temperature. The fillets were washed using 0.3% PBS with Triton X-100 stained with Phalloidin (#P1951, Sigma, USA) for 1h at room temperature. The fillets were washed with PBST, and the nuclei were counterstained with DAPI (#TC229, Himedia, India) for 5min. The fillets were washed and mounted onto a glass slide in mounting media. High-resolution images of VL3 and VL4 muscles were captured at 64× magnification using a Leica Stellaris confocal microscope. Muscle area quantification was performed using ImageJ (NIH). Quantitative real-time PCR (qPCR) Total RNA was isolated from 10 third instar larvae or larval body wall muscles using Qiazol reagent (#79306, Qiagen, USA). First-strand cDNA was synthesized from 2µg of total RNA with high-capacity cDNA synthesis kit (#4368814, ThermoFisher, USA). qPCRs were performed in Quant studio 5Dx System (Applied Bio systems, USA). Each 20µl reaction mixture contained 2µl of diluted cDNA, 10µM each of the forward and reverse primers, and 10µl 2 × DyNamo color flash SYBR green (Thermo Fisher, USA). Reactions for each sample were carried out in triplicates. PCR amplification was performed with standard machine settings. Melting curves were generated for testing single product after amplification. Relative gene expression was quantified using the 2 −ΔCt method. Data was normalized to endogenous control RPL32 [ 22 ]. All the primers used are listed in the Supplementary Table 1. Chromatin Immunoprecipitation Chromatin Immunoprecipitation was performed using SimpleChIP® Enzymatic Chromatin (#9003S, CST, USA) with slight modifications. Briefly, 300–600 larval body wall muscles were dissected and fixed in 1.8% formaldehyde for 15min at room temperature. The muscles were incubated in Buffer A for 5min followed by centrifugation at 4000rpm for 5min at 4 0 C. The muscles were then homogenized in Buffer B using micro pestles and subjected to micrococcal nuclease digestion (3ul for 100 larval body muscles) for 8min at 37 0 C. The samples were then sonicated using 4mm probe with 30% Amplitude with 20sec ON and 30sec OFF for 10 cycles (Sonics Vibracell 500). The lysates were centrifuged at 10000rpm for 10min at 4 0 C, the supernatant was subjected to chromatin immunoprecipitation. 10% of the lysate was kept as input. To the rest of the lysate H3K9me2 (1:600) (#9753S, CST, USA) or IgG antibody (1:1200) were added and incubated overnight at 4 0 C. 30µl of Protein G Magnetic Beads were added to each IP reaction and incubated for 2h at 4°C. After the elution of chromatin from antibody/protein G magnetic beads, the chromatin samples were subjected to reverse crosslinking, and the DNA was purified from both input and ChIP samples. The relative enrichment was calculated by normalizing values of ChIP-qPCR with the input. Primers used for ChIP-qPCR and the promoter region details are listed in Table 1. Muscle injury and ascorbic acid administration in diabetic mice All mice experiments were approved by the institutional animal ethical committee (IAEC). C57BL/6 male mice (6–8-week-old) were housed at 24°C ± 1°C and 55% ± 5% humidity, with a 12hr light-dark cycle and free access to food and freshwater. Hyperglycemia was induced by administration of low doses of Streptozotocin (STZ) (#S0130 Sigma, USA) dissolved in sodium citrate buffer (50mM, pH 4.5) via intra-peritoneal route (50 mg/kg body weight per day) with a 27G needle for 5 consecutive days [ 23 ]. Control group mice were injected with an equal volume of citrate buffer. Fasting blood glucose was estimated seven days after the last STZ injection. Mice with > 150mg/dL glucose levels were considered to be diabetic. TA muscles of hind limbs were unilaterally injected with 50µl of either 1.2% BaCl2 in 0.9% NaCl or 0.9%NaCl (vehicle control). Post-injury hyperglycemic and injured mice were randomly divided into 3 groups. One group received ascorbic acid (AA) prepared as detailed in previous reports [ 24 ]. Mice were administered with AA at 4g/kg body weight concentration per day via intra-peritoneal route for the next 7 days [ 25 ]. Other two groups received only saline as vehicle control. After 7 days, mice from all the groups were euthanized and TA muscles were collected, fixed in paraformaldehyde for further analysis. Haematoxylin and Eosin staining Muscles were fixed in 4% paraformaldehyde. Fixed tissues were processed overnight using a Leica automated tissue processor. Tissues were embedded in paraffin, and 4µm sections were cut and mounted onto glass slides, followed by heat fixation. Sections were stained with haematoxylin for 5min, differentiated in acid alcohol, and rinsed with water. Eosin staining (1%) was performed for 30s, followed by dehydration in 100% ethanol. Bright-field images were acquired using a Leica light microscope with LAS EZ software. Quantitative image analysis was performed using ImageJ (NIH). Cell Culture C2C12 murine myoblast cells were maintained in growth medium consisting of DMEM (#11960044, Gibco, USA) supplemented with 20% fetal bovine serum (#10270106, Gibco, USA) and 1% anti-anti (#15240062, Gibco, USA). Cells were cultured at 37°C in a humidified atmosphere with 5% CO₂. Differentiation was induced by switching to differentiation medium composed of DMEM supplemented with 2% horse serum. For treatment groups, AA was added to the differentiation medium at a final concentration of 100µM. Western Blot Samples were resolved on 10% SDS-PAGE gels and subsequently transferred onto PVDF membrane. Membrane was blocked in 5% non-fat dry milk for 1h at room temperature and incubated overnight at 4°C with anti-myosin heavy chain (#M4276, Sigma, USA), anti-myogenin (#sc-52903, Santa Cruz, USA) 1:1000 dilution and anti- β-actin (#CAB340Mi22, Cloud clone, USA) 1:10000 dilution. After washing, membranes were incubated with HRP-conjugated secondary antibodies for 1h at room temperature. Bands were visualized using Clarity™ Western ECL Substrate (#1705060, Bio-Rad, USA), and images were captured using ChemiDoc Imaging System (Bio-Rad, USA). Statistical analysis Data are expressed as mean ± standard error (SEM) or standard deviation (SD) unless otherwise mentioned. Means of different groups were compared and analysed using unpaired student’s t-test with Welch’s corrections. Statistical analysis for multiple groups was compared using ANOVA. Differences were reported as statistically significant when p < 0.05. GraphPad Prism 10 software was used for statistical analysis and generation of graphs. Asterisks indicate degrees of significance: * p -value < 0.05; ** p -value < 0.01; *** p -value < 0.001; **** p -value < 0.0001. Results HSD induces type 2 diabetes in Drosophila larvae Drosophila larvae reared on HSD display similar pathophysiology of type 2 diabetes in humans [ 18 , 26 ]. To examine whether high sugar feeding to wild type Canton- S larvae induce hyperglycemia and displays characteristics of diabetes, larvae in the HSD condition were fed media with a final concentration of 1 M sucrose. In contrast, control larvae (CT) were fed media containing 0.1 M sucrose. HSD-fed larvae displayed a significant reduction in body size and delayed development ( Fig. 1 A, B &C) . Our observations are consistent with the phenotypes observed in IPC ablated flies or insulin receptor mutant flies [ 27 , 28 ]. We next tested the larval feeding behaviour in control and HSD diet by larval feeding assay. We found that HSD-fed larvae had reduced food intake as evidenced by lower blue stains, which were also quantified ( Fig. 1 C &D) . Despite eating less, HSD-fed larvae developed hyperglycemia and elevated triglyceride levels in the hemolymph suggesting a higher calorie intake ( Fig. 1 E &F) . These results indicate that HSD-fed larvae are hyperglycemic and display characteristics of type 2 diabetes. HSD-fed diabetic larvae exhibit muscle atrophy We examined whether HSD-fed diabetic larvae exhibited some of the characteristics of muscle atrophy. We analyzed myofiber size of ventral longitudinal VL3 and VL4 body wall muscles, which are each comprised of single myofibers. A significant reduction in the size of VL3 and VL4 muscles was evident from the phalloidin staining ( Fig. 2 A &B) . In addition, locomotory function was significantly impaired in HSD-fed larvae compared to the controls ( Fig. 2 C ) . We investigated the impact of HSD on the expression of FOXO target genes in the body wall muscles. HSD larval muscles had significantly higher expression of InR and Thor, indicating insulin resistance and increased catabolism ( Fig. 2 D &E) . While the expression of muscle anabolic genes mef2 and mhc were reduced ( Fig. 2 F &G) . These results suggest that HSD-fed diabetic larvae had significant impact on the muscles and display muscle atrophy phenotype. Vitamin C supplementation rescues diabetic-like phenotypes in HSD-fed larvae Next, we tested the beneficial effect of vitamin C on diabetic-like phenotypes. Consistent with the earlier data, HSD-fed larvae had reduced body size and growth inhibition. Interestingly, vitamin C-supplementation to HSD-fed larvae (HSD + AA) showed a rescue from the diabetic-like phenotype ( Fig. 3 A ) . A significant rescue in body size was observed in HSD + AA larvae ( Fig. 3 B ) . Vitamin C supplementation led to increased food consumption, as evidenced by increased blue stain and dye absorbance ( Fig. 3 C &D) . Despite higher calorie in HSD, a reduction in hemolymph glucose levels was seen; however, the triglyceride levels remained unchanged in the HSD + AA group ( Fig. 3 E &F) . HSD causes peripheral insulin resistance in Drosophila, marked by changes in the expression of circulating Drosophila insulin-like peptides (DILPs). DILPs are released from insulin-producing cells (IPCs) in the central nervous system (CNS) and regulate glucose homeostasis [ 29 , 30 ]. Similarly, Adipokinetic hormones (akh), expressed in the fat body, helps mobilize stored fat and is increased in diabetic flies [ 31 , 32 ]. We observed a significant increase in the expression of dilp2 and akh in HSD-fed larvae, indicating insulin resistance ( Fig. 3 G &H) . A substantial reduction in both dilp2 and akh expression was evident in HSD + AA larvae, indicating maintenance of glucose metabolism homeostasis. Next, we performed RNA-seq to understand the transcriptional changes in control, HSD, and HSD + AA larval body wall muscles. Compared to controls, 1,144 genes were differentially regulated in HSD larval muscles. In comparison, 2881 genes were differentially regulated in HSD + AA larval muscle compared to HSD ( Fig. 3 I &J Supplementary files) . KEGG pathways and biological process analysis showed enrichment for genes involved in various metabolic pathways in HSD + AA muscles ( Fig. 3 K &L) . In contrast, the HSD group had enrichment for genes involved in cuticle development, suggesting that HSD-fed larvae had a delayed developmental program and were metabolically less active. In addition, genes linked to muscle development were significantly enriched in HSD + AA compared to HSD ( Fig. 3 M ) . Furthermore, the expression of mef2 target genes, Mhc, Arp8, Act87E, Act57B were enriched in the HSD + AA compared to the HSD, indicating enhanced muscle anabolism with vitamin C ( Supplementary Figure S1 ) . Overall, the data suggest vitamin C supplementation improved metabolism and promoted muscle gene expression in diabetic larvae. Vitamin C reduces oxidative stress and rescues Tet expression in HSD-fed larvae Vitamin C is a potent antioxidant. Since vitamin C supplementation rescued diabetic- like phenotypes, we measured the total antioxidant capacity (TAC). Compared to HSD, HSD + AA larvae showed increased TAC indicating reduced oxidative stress upon vitamin C supplementation ( Fig. 4 A ) . Consistent with this the expression of antioxidant genes SOD1, SOD2 and Catalase were higher in HSD + AA fed larvae ( Fig. 4 B, C &D) . In addition to its role as an antioxidant, vitamin C functions as a co-factor for several enzymes including epigenetic modifier Tet whose expression and activity are critical for muscle differentiation and development [ 33 ]. To this end, we examined the expression of Drosophila Tet (dTet) in the muscles of control, HSD, and HSD + AA larvae. dTet was significantly downregulated in HSD larval muscles compared to controls. In contrast, dTet expression was rescued upon vitamin C supplementation ( Fig. 4 E ) . Alternatively, we also checked if dTet is altered in controls upon vitamin C supplementation. We did not see changes in dTet expression or in the larval size ( Fig. 4 F ) . Consistent with its reduced expression, we found higher repressive H3K9me2 on the dTet promoter in HSD larval muscles, while it was reversed in HSD + AA ( Fig. 4 G ) . These results confirm that dTet is transcriptionally downregulated in HSD larval muscles. Vitamin C mitigates muscle atrophy in diabetic larvae Since muscle genes were enriched in RNA-seq analysis in the HSD + AA group, we analyzed the myofiber size in all three groups. Phalloidin staining showed increased size of VL3 and VL4 muscles in the HSD + AA group compared to HSD (Fig. 5 A &B ). Improved locomotion was evident in the HSD + AA larvae compared to the HSD larvae ( Fig. 5 C ) . Consistently, expression of mef2 and mhc was significantly higher in the HSD + AA compared to HSD group (Fig. 5 C &D) . These findings indicate that vitamin C supplementation increases muscle size and function and alleviates muscle atrophy in diabetic larvae. Vitamin C improves skeletal muscle regeneration in diabetic mice We furthered our findings on a diabetic mouse model to examine the effect of vitamin C on skeletal muscle regeneration. Five low doses of Streptozotocin (STZ) were injected intraperitoneally to induce a type 2 diabetes in C57BL/6 mice (Fig. 6 A) [ 34 ]. Mice were divided into three groups: controls (No STZ), STZ, and STZ with vitamin C (4g/kg body weight) (STZ + AA). The STZ group had significantly higher blood glucose, indicating that the mice were hyperglycemic ( Fig. 6 B). Surprisingly, STZ + AA mice did not exhibit changes in glucose levels, compared to STZ mice. Consistently, we did not observe changes in insulin levels in the pancreatic β-cells between the STZ and STZ + AA groups, indicating that vitamin C did not improve the pancreatic β-cells function in the diabetic mice (Supplementary Figure S2 ) . To further assess the effect of vitamin C on skeletal muscle regeneration, we injured the Tibialis Anterior (TA) muscle with barium chloride [ 35 ]. Interestingly, seven days after the injury, mice treated with AA intraperitoneally had improved muscle regeneration compared to STZ diabetic mice ( Fig. 6 C ) . Increased myofiber size and enhanced fusion index were evident in the STZ + AA diabetic mice ( Fig. 6 D &E) . In addition, presence of vitamin C led to enhanced in vitro myogenic differentiation of C2C12 mouse myoblasts as evidenced by increased expression myogenin and myosin heavy chain ( Fig. 6 F ). Overall, these results suggest a beneficial role of vitamin C in skeletal muscle differentiation and regeneration. Discussion In this study we demonstrate the beneficial effect of vitamin C in mitigating muscle atrophy and improving skeletal muscle regeneration in diabetic larvae and mouse respectively. These results highlight the critical role of essential micronutrient vitamin C, in maintaining muscle homeostasis in diabetes. Importantly, in addition to its role as an antioxidant, our findings highlight a role for vitamin C in modulating epigenetic regulators in diabetic skeletal muscles. We used Drosophila larvae for several reasons: 1. In Drosophila, the conserved insulin signaling regulates constant feeding behaviour and energy storage mechanisms, making it an effective model to study metabolic dysfunction [ 26 ]. 2. Hyperglycemia in Drosophila causes insulin resistance, leading to developmental delay and reduced body size, which mimics a key feature of diabetes pathology in mammals [ 26 ]. 3. It eliminates sex specific variabilities due to hyperglycemia which otherwise could act as a confounding factor [ 36 ]. 4. Larval body wall muscle undergoes a 50-fold increase in muscle mass during larval development, which provides an excellent window to study muscle atrophy and hypertrophy in vivo [ 37 , 38 ]. HSD-fed larvae exhibited increased expression of FOXO target genes, decreased mef2 and mhc, and reduced myofiber size, all of which reflect increased catabolic signaling. Such changes are evident in diabetic mammals where FOXO activity drives proteasomal and autophagy degradation pathways [ 39 ]. Vitamin C supplementation alleviated these changes in diabetic larvae. In a randomized, crossover study involving individuals with type 2 diabetes, vitamin C supplementation resulted in improved glucose uptake and reduced oxidative stress in skeletal muscles (Mason et al., 2016). Another trial with diabetic patients on metformin showed that supplementation with vitamin C (and/or vitamin E) improved fasting blood glucose, HbA1c, and insulin resistance [ 40 ]. Our findings support a beneficial role for vitamin C in mitigating muscle atrophy in diabetes. A rescue in dTet expression in HSD + AA larvae indicates that in addition to its antioxidant and metabolic effects, vitamin C may also induce epigenetic changes in diabetic muscle. Vitamin C’s ability to modulate the epigenome through Tet dioxygenases is emerging as an important gene regulatory mechanism. Tet catalyze oxidation of 5-methylcytosine (5mc) to 5-hydroxymethylcytosine (5hmc), facilitating DNA demethylation and activation of gene expression. Vitamin C functions as a cofactor for Tet and enhances their catalytic activity [ 41 ]. In the context of myogenesis, vitamin C induces expression of myogenic genes through Tet-dependent epigenetic mechanisms [ 17 ]. During development, dTet has been shown to co-localize with mef2 in muscle precursor cells and depletion of dTet in flies causes defects in locomotion and pupal lethality [ 33 ]. Since vitamin C alleviated the diabetic phenotype in HSD-fed larvae, our findings suggest that the expression and activity of dTet may be crucial in the larvals muscle. A significant limitation of our study is that, although vitamin C restored dTet expression in HSD-fed larvae, we did not assess dTet enzymatic activity or its downstream epigenetic effects. Notably, DNA methylation is not evident in Drosophila; however, dTet is implicated in RNA hydroxymethylation [ 42 ]. A further understanding of the consequences of dTet-mediated RNA hydroxymethylation, particularly on muscle specific mRNA, will help determine whether increased dTet expression upon vitamin C translates into functional epigenetic regulation in diabetic muscle.Furthermore, Tet loss-of-function studies under diabetic conditions would help clarify the muscle-protective effects of vitamin C. The observed effect of vitamin C on muscle regeneration in injured diabetic mice may be due to its direct interaction with PAX7 thereby by activating myogenic genes or through the transport of vitamin C to the injured site, which enhances cell adhesion and migration, both of which are essential for regeneration [ 43 , 44 ]. However, the vitamin C-mediated epigenetic mechanisms underlying skeletal muscle regeneration warrant further investigation. Overall, this study demonstrates that vitamin C mitigates muscle atrophy, and supplementation of vitamin C with anti-diabetic medications may be beneficial in controlling diabetic complications. Declarations Author information Manipal Institute of Regenerative Medicine, Manipal Academy of Higher Education, Manipal, India. Parika Kala, Vinay Kumar Rao Department of Medical Genetics, JSS Medical College, JSS Academy of Higher Education and Research, Mysuru, India. Supriya Bevinakoppamath, Akila Prashant Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore. Swarnaseetha Adusumalli Department of Developmental Biology and Genetics (DBG), Indian Institute of Science (IISc), India Vishnu Ramada, Aishwarya A Makam, Nikhil R Gandasi Conflict of Interest The authors declare no conflict of interest Ethical declaration All animal experiments were conducted after the approval of Institutional animal ethical committee clearance (JSS AHER, Mysore). Author contribution SB: conceptualization, methodology, validation, writing. PK: methodology and validation. SA: RNA-seq analysis. VR: methodology and validation. AAM: methodology and validation. AP: resources, supervision. NRG: Supervision, writing. VKR: Project administration, conceptualization, supervision writing and editing Acknowledgement We thank Dr. Jyotsna Dhawan, CCMB for providing C2C12 mouse myoblasts cell line (originally from Helen Blua), Dr. Anujith Kumar, MIRM for his critical comments on the manuscript. Vinay Kumar Rao is supported by Core Research Grant CRG/2022/001969, Anusandhan National Research Foundation, Govt. of India. Vision group on science and technology, Govt. of Karnataka (GRD1120), Intramural Research Grant, JSS Academy of Higher Education and Research, India. Parika Kala is supported by fellowships from Manipal Academy of Higher Education, Manipal. Nikhil R Gandasi’s (NRG) lab was funded by the Indian Institute of Science—seed grants, Department of Biotechnology (DBT)-Ramalingaswami fellowship, Indian Council of Medical Research (ICMR) – Grants in Aid Scheme, Science and Engineering Research (SERB) Board – Start-up grant, Infosys Young Investigator fellowship, Rajiv Gandhi University of Health Sciences (RGUHS) extramural grant and NovoNordisk Foundation awarded to NRG lab. Aishwarya A Makam’s salary was supported by Prime Ministers Research Fellowship (PMRF). Data availability RNA-Seq data has been deposited in GEO database under accession number GSE313392. For reviewer access - uvgbscwefjqfzol References Nellaiappan K, Preeti K, Khatri DK, Singh SB (2022) Diabetic Complications: An Update on Pathobiology and TherapeuticStrategies. 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Additional Declarations There is NO conflict of interest to disclose Supplementary Files DEGAAvsCTsig.xlsx RNA SEQ Gene expression data DEGHSDvsCTsig.xlsx RNA SEQ Gene expression data Supplementaryfile10122025.pdf Supplementary Figures DEGAAvsHSDsig.xlsx RNA SEQ Gene expression data Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: revise 07 Apr, 2026 Review # 1 received at journal 23 Mar, 2026 Review # 2 received at journal 04 Mar, 2026 Reviewer # 2 agreed at journal 27 Feb, 2026 Reviewer # 1 agreed at journal 27 Feb, 2026 Reviewers invited by journal 08 Feb, 2026 Submission checks completed at journal 15 Dec, 2025 Editor assigned by journal 12 Dec, 2025 First submitted to journal 12 Dec, 2025 You are reading this latest preprint version 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. 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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-8342512","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":587782971,"identity":"6e7d4ee2-1256-4577-b0f1-c4841a6f7dcd","order_by":0,"name":"Vinay Kumar Rao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6ElEQVRIiWNgGAWjYDACZgiVwA8mGRhkgJQBcVokGyBaeAhrgYIEgwMQBmEtBsd5D37mYdiWZ3zt8LMHD3fY8TCwN2+TwKvlMF+yNA/D7WKz22nmBolnknkYeI6V4dUi2cxjANKSuO12gplEYhszD4NEjhkhLca/QVo2z07/BtRSz8Mg/wa/Fn5mHjOwLRukc0C2HAbawkNYi+Ucg9vFErdzyiQSzxznYeNJK7bAp4WN/4zxjTcVt/P4Z6dvk/y5o1qOn/3wxhv4tIAAEw8sIhgbgIYQUg5W+APOaiBG/SgYBaNgFIw0AACoIkAYZrbQNAAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0009-0004-5577-0763","institution":"Manipal Institute of Regenerative Medicine, MAHE","correspondingAuthor":true,"prefix":"","firstName":"Vinay","middleName":"Kumar","lastName":"Rao","suffix":""},{"id":587782972,"identity":"7fb7d958-a28b-42a8-b19f-69ace2e0534b","order_by":1,"name":"Supriya Bevinakoppamath","email":"","orcid":"","institution":"JSS Medical College, JSS AHER","correspondingAuthor":false,"prefix":"","firstName":"Supriya","middleName":"","lastName":"Bevinakoppamath","suffix":""},{"id":587782973,"identity":"53a333a2-2895-48af-994d-50a9e2a8336a","order_by":2,"name":"Parika Kala","email":"","orcid":"","institution":"MIRM, Bengaluru","correspondingAuthor":false,"prefix":"","firstName":"Parika","middleName":"","lastName":"Kala","suffix":""},{"id":587782974,"identity":"e9395945-a16f-499f-96c7-bce6cec8cf53","order_by":3,"name":"Swarnaseetha Adusumalli","email":"","orcid":"","institution":"Nanyang Technological University","correspondingAuthor":false,"prefix":"","firstName":"Swarnaseetha","middleName":"","lastName":"Adusumalli","suffix":""},{"id":587782975,"identity":"27aeed82-64f8-40fe-a80b-a6a6e0e8005a","order_by":4,"name":"Vishnu Ramadas","email":"","orcid":"","institution":"Indian Institute of Science","correspondingAuthor":false,"prefix":"","firstName":"Vishnu","middleName":"","lastName":"Ramadas","suffix":""},{"id":587782976,"identity":"64bd74a5-d0a7-4579-8927-d13b2f6f4c12","order_by":5,"name":"Aishwarya Makam","email":"","orcid":"","institution":"Indian Institute of Science","correspondingAuthor":false,"prefix":"","firstName":"Aishwarya","middleName":"","lastName":"Makam","suffix":""},{"id":587782977,"identity":"c7706416-6986-44e9-b8d0-4332cf64dc74","order_by":6,"name":"Akila Prashant","email":"","orcid":"","institution":"JSS Medical College JSS AHER","correspondingAuthor":false,"prefix":"","firstName":"Akila","middleName":"","lastName":"Prashant","suffix":""},{"id":587782978,"identity":"07e11a65-82b5-4611-9764-4ecb0c672d1e","order_by":7,"name":"Nikhil Gandasi","email":"","orcid":"","institution":"Indian institute of Science","correspondingAuthor":false,"prefix":"","firstName":"Nikhil","middleName":"","lastName":"Gandasi","suffix":""}],"badges":[],"createdAt":"2025-12-12 06:45:41","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8342512/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8342512/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":102532759,"identity":"2e6fcf01-3662-48c5-805b-143aa87486d6","added_by":"auto","created_at":"2026-02-12 16:44:21","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":74009,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHigh sugar diet induce hyperglycemia and diabetic like phenotypes in wandering Drosophila 3\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003erd\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e instar larvae\u003c/strong\u003e. (A) Representative image of larvae fed with CT and HSD diets. (B) Quantitation of size of larvae fed with respective diets. n=9 larvae, Scale bars, 200µm. (C) Graph showing percentage of pupae formed, n=100 larvae. (D) Representative image showing larvae fed with bromophenol blue with respective diets. (E) Quantitation of amount of bromophenol blue consumed by larvae on both the diets at 595nm, n=25 larvae, (three independent experiments), Scale bar, 200µm. (F) Estimation of hemolymph glucose concentrations. n=50-100 larvae (three independent experiments). (G) Estimation of triglyceride levels, and n=25 larvae. (three independent experiments). Error bars indicate mean ± SEM. Unpaired student t-test with Welch’s correction was performed to derive all\u0026nbsp;p-values (*\u003cem\u003ep\u003c/em\u003e-value \u0026lt;0.05; **\u003cem\u003ep\u003c/em\u003e-value \u0026lt;0.01; ***\u003cem\u003ep\u003c/em\u003e-value \u0026lt;0.001)\u003c/p\u003e","description":"","filename":"OnlineFigure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8342512/v1/b7799c5fca619dddeabd40e4.png"},{"id":102532760,"identity":"96a9ef47-e202-4f9e-b95d-3b98fb46f2de","added_by":"auto","created_at":"2026-02-12 16:44:21","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":112094,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHigh sugar diet induces muscle loss in wandering 3\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003erd\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e instar larvae.\u003c/strong\u003e (A) Confocal images of larval body wall muscles stained with Phalloidin, and nuclei counter stained with DAPI. (B) VL3+VL4 muscle area. n=3 larval muscles, Scale bars, 100µm. (C) Quantitation of locomotory activity of 3\u003csup\u003erd\u003c/sup\u003e instar larvae n=9 larvae. (D\u0026amp;E) qRT PCR relative gene expression of muscle catabolic genes InR and Thor. n=30 larval muscles, Error bars indicate mean ±SEM. (F\u0026amp;G) Relative expression of muscle anabolic genes mef2 and Mhc, n=20 larval muscles, Error bars indicate ±SD (Representative graph of two biological replicates). Unpaired student t-test with Welch’s correction was performed to derive all\u0026nbsp;p-values (*\u003cem\u003ep\u003c/em\u003e-value \u0026lt;0.05; **\u003cem\u003ep\u003c/em\u003e-value \u0026lt;0.01;****\u003cem\u003ep\u003c/em\u003e-value \u0026lt;0.0001)\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"OnlineFigure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8342512/v1/266842d5aea70c043eb4508f.png"},{"id":102532762,"identity":"36a4fc35-1f96-49c2-bfb5-0fb712ccc529","added_by":"auto","created_at":"2026-02-12 16:44:21","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":136379,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVitamin C rescues the diabetic like phenotype and improves metabolism in 3\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003erd\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e instar larvae.\u003c/strong\u003e (A) Representative image showing 3\u003csup\u003erd\u003c/sup\u003e instar larvae fed with CT, HSD and HSD+AA diets. Scale bars 100µm. (B) Quantitation of locomotary activity of the larvae fed with all the three diets. n=10 larvae. (C) Representative image showing larvae fed with bromophenol blue along with respective diets. (D) Quantitation of bromophenol blue at 595nm to assess the amount of food consumed by the 3\u003csup\u003erd\u003c/sup\u003e instar larvae on all the three diets. Scale bar, 200µm, n=15 larvae (three independent experiments). (E) Estimation of hemolymph glucose levels. n=50-100 larvae (three independent experiments) (F) Estimation of triglyceride levels, n=15-30 larvae (three independent experiments). Error bars indicate mean ± SEM. (G) Relative expression of dilp2 n=20 larvae, Error bars indicate ±SD (Representative graph of two biological replicates). (H) Relative expression of akh n=30 larvae, Error bars indicate mean ±SEM. (I\u0026amp;J) Volcano plots showing differentially expressed genes. (K, L\u0026amp;M) Differentially regulated pathways (Combined RNA-seq data from three replicates). Ordinary one-way ANOVA was performed to derive all\u0026nbsp;p-values (*\u003cem\u003ep\u003c/em\u003e-value \u0026lt;0.05; **\u003cem\u003ep\u003c/em\u003e-value \u0026lt;0.01; ***\u003cem\u003ep\u003c/em\u003e-value \u0026lt;0.001; ****\u003cem\u003ep\u003c/em\u003e-value \u0026lt;0.0001)\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"OnlineFigure3.png","url":"https://assets-eu.researchsquare.com/files/rs-8342512/v1/289644190c0e822757d3b0cc.png"},{"id":102746844,"identity":"e1fa87e0-8b98-470b-bb4e-9397ae7f2fb8","added_by":"auto","created_at":"2026-02-16 09:02:03","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":74086,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVitamin C reduces oxidative stress and enhances dTet expression in HSD fed larvae. \u003c/strong\u003e(A)Total antioxidant capacity, n=30 larval muscles. (C) Relative expression of antioxidant gene SOD1, n=20 larval muscles. Error bars indicate mean ± SD (Representative graph of two biological replicates). (D\u0026amp;F) Relative expression of antioxidant genes SOD2 and CAT, n=30 larval muscles, (E) Relative expression of dTET, n=15-30 larval muscles. (F) Representative image showing larvae fed with CT and CT+AA diet. (G) Relative expression of dTET of larvae fed with CT and CT+AA diets. n=15-30 larval muscles, Error bars indicate mean ± SEM. (H) Relative enrichment of H3K9me2 on dTET promoter in the larval muscles. n=300-600 larval muscles, Error bars indicate ±SD. Ordinary one-way ANOVA was performed to derive all p-values (*\u003cem\u003ep\u003c/em\u003e-value \u0026lt;0.05; **\u003cem\u003ep\u003c/em\u003e-value \u0026lt;0.01; ***\u003cem\u003ep\u003c/em\u003e-value \u0026lt;0.001; ****\u003cem\u003ep\u003c/em\u003e-value \u0026lt;0.0001)\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"OnlineFigure4.png","url":"https://assets-eu.researchsquare.com/files/rs-8342512/v1/0b95dcefb8b5ba1eb67ab1f7.png"},{"id":102747327,"identity":"ab80175f-1d8e-4a45-a839-3b6c8f4f1cfa","added_by":"auto","created_at":"2026-02-16 09:04:31","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":152695,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAscorbic acid rescues the HSD induced muscle phenotype and increases muscle anabolism\u003c/strong\u003e. (A) Confocal images of larval body wall muscles stained with Phalloidin, and nuclei counter stained with DAPI. (B) VL3+VL4 muscle area. n=7 larval muscles, Scale bars, 100µm. (C) Quantitation of locomotory activity of 3\u003csup\u003erd\u003c/sup\u003e instar larvae. n=10 larvae. (D) Relative gene expression of muscle anabolic genes mef2 and Mhc, n=30 larval muscles. Error bars indicate mean ± SEM. Ordinary one-way ANOVA was performed to derive all\u0026nbsp;p-values (*\u003cem\u003ep\u003c/em\u003e-value \u0026lt;0.05; **\u003cem\u003ep\u003c/em\u003e-value \u0026lt;0.01; ***\u003cem\u003ep\u003c/em\u003e-value \u0026lt;0.001; ****\u003cem\u003ep\u003c/em\u003e-value \u0026lt;0.0001)\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"OnlineFigure5.png","url":"https://assets-eu.researchsquare.com/files/rs-8342512/v1/1c2415e6509bf3b9b523a24d.png"},{"id":102747049,"identity":"2f9d1719-2514-4b26-b6c7-a6a61d55a8dd","added_by":"auto","created_at":"2026-02-16 09:03:39","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":169064,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAscorbic acid (AA) increases muscle regeneration in diabetic mice with muscle injury.\u003c/strong\u003e (A) Schematic representation of the work plan. (B) Estimation of blood glucose levels in control, STZ and STZ+AA mice groups. n= 6-7 mice each group. (C) Representative images of injured TA muscle cross sections stained with haematoxylin and eosin of Wildtype uninjured, and injured muscles at day 7 after injury from all three groups. Scale bars 0.05mm. (D\u0026amp;E) Quantification of muscle regeneration is determined by the percentage of muscle fibers with centrally located nuclei. n=6-7 mice each group, 100-500 muscle fibers were analyzed. Error bars indicate mean ± SEM. (F) Western blots from C2C12 cells probed with myosin heavy chain, myogenin and β-actin antibodies (three independent replicates were performed). One-way ANOVA was performed to derive all p-values (*\u003cem\u003ep\u003c/em\u003e-value \u0026lt;0.05; **\u003cem\u003ep\u003c/em\u003e-value \u0026lt;0.01; ***\u003cem\u003ep\u003c/em\u003e-value \u0026lt;0.001; ****\u003cem\u003ep\u003c/em\u003e-value \u0026lt;0.0001)\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"OnlineFigure6.png","url":"https://assets-eu.researchsquare.com/files/rs-8342512/v1/a8dec068180442df1c45e0a6.png"},{"id":102750772,"identity":"0b757677-6919-4200-83a1-1efab8bc0a78","added_by":"auto","created_at":"2026-02-16 09:22:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2214940,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8342512/v1/ce145f86-3537-472c-a3db-4c594ec08430.pdf"},{"id":102532763,"identity":"bf86f4df-528b-4785-af56-7a829d666a8a","added_by":"auto","created_at":"2026-02-12 16:44:21","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":193427,"visible":true,"origin":"","legend":"RNA SEQ Gene expression data","description":"","filename":"DEGAAvsCTsig.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8342512/v1/e379e088cd23bcb197f5214f.xlsx"},{"id":102532764,"identity":"32f470e7-5fb8-4ae8-84f5-f527e0bf15d6","added_by":"auto","created_at":"2026-02-12 16:44:21","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":131115,"visible":true,"origin":"","legend":"RNA SEQ Gene expression data","description":"","filename":"DEGHSDvsCTsig.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8342512/v1/971fbf179242ec9f0b1b83de.xlsx"},{"id":102532768,"identity":"b3e32c72-471e-48d6-b76f-fbe60d222a64","added_by":"auto","created_at":"2026-02-12 16:44:21","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":622071,"visible":true,"origin":"","legend":"Supplementary Figures","description":"","filename":"Supplementaryfile10122025.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8342512/v1/7ccb41745907099450ae7e44.pdf"},{"id":102532766,"identity":"d733dab8-ba89-43b5-b1d2-1eabb0718495","added_by":"auto","created_at":"2026-02-12 16:44:21","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":340480,"visible":true,"origin":"","legend":"RNA SEQ Gene expression data","description":"","filename":"DEGAAvsHSDsig.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8342512/v1/4e37351e01ba01160e7d6843.xlsx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e conflict of interest to disclose","formattedTitle":"Vitamin C Supplementation Mitigates Diabetes-Associated Skeletal Muscle Atrophy","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDiabetes is generally accompanied with complications of various organs including heart, kidney, eye, skeletal muscles and tend to worsen over time [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Skeletal muscle is a vital metabolic organ that helps coordinate the body\u0026rsquo;s energy and protein balance primarily through glucose uptake, storage of amino acids making it a key tissue in maintaining insulin sensitivity and protein synthesis for tissue repair. Both type 1 and type 2 diabetes cause atrophy of skeletal muscles, characterised by shrinking of muscle fibres, altered metabolic activity, and significant loss of muscle mass and function ultimately resulting in reduced quality of life of diabetic patients [\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMechanistically, insulin resistance in diabetic muscle alters PI3K\u0026ndash;AKT\u0026ndash;mTOR signalling, which is critical for muscle anabolism [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. In addition, increased expression MuRF1 and Atrogin-1, which promote protein degradation, are activated concurrently with insulin resistance [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. When combined, these changes tilt the balance towards catabolism gradually leading to the loss of skeletal muscle mass and function. Additionally oxidative stress and mitochondrial dysfunction, contributes to energy imbalance and decreased muscle regeneration[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Although the current therapeutics for diabetes focuses on controlling blood glucose, they fall short in addressing the gradual loss of skeletal muscle mass and strength in diabetic patients. Nutritional supplements that not only restore normal glycemia but also counteract muscle atrophy linked to diabetes are therefore becoming increasingly necessary.\u003c/p\u003e \u003cp\u003eInterestingly, diabetic patients tend to have lower levels of antioxidant vitamins such as vitamin C, E and A [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Vitamin C is an essential nutrient critical for maintaining various metabolic and physiological functions. It serves as a cofactor for many enzymes notably for Fe(II) and 2-oxoglutarate-dependent dioxygenases including Tet enzymes, which catalyze the oxidation of DNA 5-methylcytosine into 5-hydroxymethylcytosine and regulate gene expression[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Increasing evidence indicates a critical role for vitamin C in skeletal muscle regeneration [\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Vitamin C uptake reduced oxidative stress and improved insulin sensitivity in diabetic patients [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Deficiency of vitamin C causes increased ROS and muscle wasting with concomitant increase in catabolic genes [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Recent mechanistic insights have shown that the vitamin C increases myoblast differentiation marked by reduction in global H3K9 methylation [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. While these studies demonstrate the role of vitamin C in skeletal muscle homeostasis, whether vitamin C counteracts muscle atrophy in diabetes and the underlying mechanisms remains to be further investigated.\u003c/p\u003e \u003cp\u003eIn this study, using a \u003cem\u003eDrosophila melanogaster\u003c/em\u003e (Here after referred as Drosophila) model of HSD-induced diabetes, which showed characteristics of diabetic-like phenotypes, we examined the effect of vitamin C on the reversal of diabetic-like phenotypes and skeletal muscle atrophy. Vitamin C supplementation led to rescue in larval myofiber size compared to the HSD group. Vitamin C increased food uptake by HSD-fed larvae, indicating reversal of food aversion. Transcriptomic analysis of VC-HSD larval muscles revealed enhanced expression of metabolic genes, and increased expression of genes linked to muscle development and differentiation. Vitamin C supplementation reduced oxidative stress and, interestingly, dTet expression is rescued in diabetic muscles. We extend our findings to the diabetic mouse model and find that vitamin C helps improve skeletal muscle regeneration in diabetic mice. Altogether, the data suggests that vitamin C could be beneficial in mitigating muscle atrophy in diabetes condition.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eFly stock\u003c/h2\u003e \u003cp\u003e \u003cem\u003eCanton-S\u003c/em\u003e wild type flies were obtained from the National Drosophila Stock Centre facility, University of Mysore. 100ml of media contained Semolina (10g), Agar (1g), propionic acid (0.75ml), Sucrose (5.13 g = 0.15 M for CT, 34.2g = 1.0 M for HSD) [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], Ascorbic acid (AA, 0.05g\u0026thinsp;=\u0026thinsp;10mM). Experimental larvae were reared on one of the following diets: control (CT), High-Sugar Diet (HSD), or HSD supplemented with either vitamin C (ascorbic acid) (HSD\u0026thinsp;+\u0026thinsp;AA), or CT diet supplemented with ascorbic acid (CT\u0026thinsp;+\u0026thinsp;AA).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eGlucose estimation\u003c/h3\u003e\n\u003cp\u003eHemolymph was pooled from 50\u0026ndash;100 third instar larvae to obtain 2\u0026micro;l for the assay. Glucose was measured by adding 2\u0026micro;l of the hemolymph to 198\u0026micro;l of Erba Glucose Reagent (# 120235, Erba Mannheim, Germany) in a 96-well plate. The plate was incubated at 37\u003csup\u003e0\u003c/sup\u003eC for 15min. The absorbance of standards and the samples were measured against blank at 505nm.\u003c/p\u003e\n\u003ch3\u003eTriglyceride (TAG) estimation\u003c/h3\u003e\n\u003cp\u003eFor whole body TAG, 5 larvae were homogenized in PBS\u0026thinsp;+\u0026thinsp;0.1% Tween and centrifuged at 5000rpm for 5 min at 4\u003csup\u003e0\u003c/sup\u003eC. The supernatant was heated for 5 min at 65\u0026deg;C to inactivate lipases [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. 2\u0026micro;l of the homogenate was mixed with 198\u0026micro;l of triglyceride reagent (# 120237, Erba Mannheim, Germany) and analysed as per the manufacturer's instructions.\u003c/p\u003e\n\u003ch3\u003eLarval feeding assay\u003c/h3\u003e\n\u003cp\u003eLarval feeding assay was performed according to published protocols [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Third instar larvae were starved for 2h and transferred into the 6 well plates containing the specific diets with 0.05% (wt/vol) bromophenol blue dye (#93676, SRL, India). After 10min of feeding, the larvae were washed with PBS and homogenized in Milli-Q water. The lysates were centrifuged and 100\u0026micro;l of supernatant was transferred to 96-well plate and absorbance was measured at 595 nm.\u003c/p\u003e\n\u003ch3\u003eSize assessment and crawling assay\u003c/h3\u003e\n\u003cp\u003eWandering third instar larvae were heat fixed and imaged using Trinocular stereo zoom (Model: SZX7, Olympus). Area was calculated using Gryphax software. For crawling assay, larvae were allowed to crawl on a 90mm petri dish containing 2% agarose over graph paper with a 0.2 cm\u003csup\u003e2\u003c/sup\u003e grid. Number of grid lines crossed in 90s were recorded with the camera attached to stereo zoom microscope [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ePhalloidin staining\u003c/h2\u003e \u003cp\u003eLarval muscle dissection and staining were performed as described previously [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Mid third instar larvae were dissected PBS. The muscle fillets were fixed in 4% formaldehyde for 20min at room temperature. The fillets were washed using 0.3% PBS with Triton X-100 stained with Phalloidin (#P1951, Sigma, USA) for 1h at room temperature. The fillets were washed with PBST, and the nuclei were counterstained with DAPI (#TC229, Himedia, India) for 5min. The fillets were washed and mounted onto a glass slide in mounting media. High-resolution images of VL3 and VL4 muscles were captured at 64\u0026times; magnification using a Leica Stellaris confocal microscope. Muscle area quantification was performed using ImageJ (NIH).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eQuantitative real-time PCR (qPCR)\u003c/h3\u003e\n\u003cp\u003eTotal RNA was isolated from 10 third instar larvae or larval body wall muscles using Qiazol reagent (#79306, Qiagen, USA). First-strand cDNA was synthesized from 2\u0026micro;g of total RNA with high-capacity cDNA synthesis kit (#4368814, ThermoFisher, USA). qPCRs were performed in Quant studio 5Dx System (Applied Bio systems, USA). Each 20\u0026micro;l reaction mixture contained 2\u0026micro;l of diluted cDNA, 10\u0026micro;M each of the forward and reverse primers, and 10\u0026micro;l 2 \u0026times; DyNamo color flash SYBR green (Thermo Fisher, USA). Reactions for each sample were carried out in triplicates. PCR amplification was performed with standard machine settings. Melting curves were generated for testing single product after amplification. Relative gene expression was quantified using the 2\u003csup\u003e\u0026minus;ΔCt\u003c/sup\u003e method. Data was normalized to endogenous control RPL32 [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. All the primers used are listed in the Supplementary Table\u0026nbsp;1.\u003c/p\u003e\n\u003ch3\u003eChromatin Immunoprecipitation\u003c/h3\u003e\n\u003cp\u003eChromatin Immunoprecipitation was performed using SimpleChIP\u0026reg; Enzymatic Chromatin (#9003S, CST, USA) with slight modifications. Briefly, 300\u0026ndash;600 larval body wall muscles were dissected and fixed in 1.8% formaldehyde for 15min at room temperature. The muscles were incubated in Buffer A for 5min followed by centrifugation at 4000rpm for 5min at 4\u003csup\u003e0\u003c/sup\u003eC. The muscles were then homogenized in Buffer B using micro pestles and subjected to micrococcal nuclease digestion (3ul for 100 larval body muscles) for 8min at 37\u003csup\u003e0\u003c/sup\u003eC. The samples were then sonicated using 4mm probe with 30% Amplitude with 20sec ON and 30sec OFF for 10 cycles (Sonics Vibracell 500). The lysates were centrifuged at 10000rpm for 10min at 4\u003csup\u003e0\u003c/sup\u003eC, the supernatant was subjected to chromatin immunoprecipitation. 10% of the lysate was kept as input. To the rest of the lysate H3K9me2 (1:600) (#9753S, CST, USA) or IgG antibody (1:1200) were added and incubated overnight at 4\u003csup\u003e0\u003c/sup\u003eC. 30\u0026micro;l of Protein G Magnetic Beads were added to each IP reaction and incubated for 2h at 4\u0026deg;C. After the elution of chromatin from antibody/protein G magnetic beads, the chromatin samples were subjected to reverse crosslinking, and the DNA was purified from both input and ChIP samples. The relative enrichment was calculated by normalizing values of ChIP-qPCR with the input. Primers used for ChIP-qPCR and the promoter region details are listed in Table\u0026nbsp;1.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eMuscle injury and ascorbic acid administration in diabetic mice\u003c/h2\u003e \u003cp\u003e All mice experiments were approved by the institutional animal ethical committee (IAEC). C57BL/6 male mice (6\u0026ndash;8-week-old) were housed at 24\u0026deg;C\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C and 55% \u0026plusmn; 5% humidity, with a 12hr light-dark cycle and free access to food and freshwater. Hyperglycemia was induced by administration of low doses of Streptozotocin (STZ) (#S0130 Sigma, USA) dissolved in sodium citrate buffer (50mM, pH 4.5) via intra-peritoneal route (50 mg/kg body weight per day) with a 27G needle for 5 consecutive days [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Control group mice were injected with an equal volume of citrate buffer. Fasting blood glucose was estimated seven days after the last STZ injection. Mice with \u0026gt;\u0026thinsp;150mg/dL glucose levels were considered to be diabetic.\u003c/p\u003e \u003cp\u003eTA muscles of hind limbs were unilaterally injected with 50\u0026micro;l of either 1.2% BaCl2 in 0.9% NaCl or 0.9%NaCl (vehicle control). Post-injury hyperglycemic and injured mice were randomly divided into 3 groups. One group received ascorbic acid (AA) prepared as detailed in previous reports [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Mice were administered with AA at 4g/kg body weight concentration per day via intra-peritoneal route for the next 7 days [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Other two groups received only saline as vehicle control. After 7 days, mice from all the groups were euthanized and TA muscles were collected, fixed in paraformaldehyde for further analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eHaematoxylin and Eosin staining\u003c/h2\u003e \u003cp\u003eMuscles were fixed in 4% paraformaldehyde. Fixed tissues were processed overnight using a Leica automated tissue processor. Tissues were embedded in paraffin, and 4\u0026micro;m sections were cut and mounted onto glass slides, followed by heat fixation. Sections were stained with haematoxylin for 5min, differentiated in acid alcohol, and rinsed with water. Eosin staining (1%) was performed for 30s, followed by dehydration in 100% ethanol. Bright-field images were acquired using a Leica light microscope with LAS EZ software. Quantitative image analysis was performed using ImageJ (NIH).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eCell Culture\u003c/h2\u003e \u003cp\u003eC2C12 murine myoblast cells were maintained in growth medium consisting of DMEM (#11960044, Gibco, USA) supplemented with 20% fetal bovine serum (#10270106, Gibco, USA) and 1% anti-anti (#15240062, Gibco, USA). Cells were cultured at 37\u0026deg;C in a humidified atmosphere with 5% CO₂.\u003c/p\u003e \u003cp\u003eDifferentiation was induced by switching to differentiation medium composed of DMEM supplemented with 2% horse serum. For treatment groups, AA was added to the differentiation medium at a final concentration of 100\u0026micro;M.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eWestern Blot\u003c/h2\u003e \u003cp\u003eSamples were resolved on 10% SDS-PAGE gels and subsequently transferred onto PVDF membrane. Membrane was blocked in 5% non-fat dry milk for 1h at room temperature and incubated overnight at 4\u0026deg;C with anti-myosin heavy chain (#M4276, Sigma, USA), anti-myogenin (#sc-52903, Santa Cruz, USA) 1:1000 dilution and anti- β-actin (#CAB340Mi22, Cloud clone, USA) 1:10000 dilution. After washing, membranes were incubated with HRP-conjugated secondary antibodies for 1h at room temperature. Bands were visualized using Clarity\u0026trade; Western ECL Substrate (#1705060, Bio-Rad, USA), and images were captured using ChemiDoc Imaging System (Bio-Rad, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error (SEM) or standard deviation (SD) unless otherwise mentioned. Means of different groups were compared and analysed using unpaired student\u0026rsquo;s t-test with Welch\u0026rsquo;s corrections. Statistical analysis for multiple groups was compared using ANOVA. Differences were reported as statistically significant when p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. GraphPad Prism 10 software was used for statistical analysis and generation of graphs. Asterisks indicate degrees of significance: *\u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05; **\u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.01; ***\u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.001; ****\u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.0001.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eHSD induces type 2 diabetes in Drosophila larvae\u003c/h2\u003e \u003cp\u003eDrosophila larvae reared on HSD display similar pathophysiology of type 2 diabetes in humans [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. To examine whether high sugar feeding to wild type \u003cem\u003eCanton- S\u003c/em\u003e larvae induce hyperglycemia and displays characteristics of diabetes, larvae in the HSD condition were fed media with a final concentration of 1 M sucrose. In contrast, control larvae (CT) were fed media containing 0.1 M sucrose. HSD-fed larvae displayed a significant reduction in body size and delayed development \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B\u003cb\u003e\u0026amp;C)\u003c/b\u003e. Our observations are consistent with the phenotypes observed in IPC ablated flies or insulin receptor mutant flies [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. We next tested the larval feeding behaviour in control and HSD diet by larval feeding assay. We found that HSD-fed larvae had reduced food intake as evidenced by lower blue stains, which were also quantified \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC\u003cb\u003e\u0026amp;D)\u003c/b\u003e. Despite eating less, HSD-fed larvae developed hyperglycemia and elevated triglyceride levels in the hemolymph suggesting a higher calorie intake \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE\u003cb\u003e\u0026amp;F)\u003c/b\u003e. These results indicate that HSD-fed larvae are hyperglycemic and display characteristics of type 2 diabetes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eHSD-fed diabetic larvae exhibit muscle atrophy\u003c/h2\u003e \u003cp\u003eWe examined whether HSD-fed diabetic larvae exhibited some of the characteristics of muscle atrophy. We analyzed myofiber size of ventral longitudinal VL3 and VL4 body wall muscles, which are each comprised of single myofibers. A significant reduction in the size of VL3 and VL4 muscles was evident from the phalloidin staining \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA\u003cb\u003e\u0026amp;B)\u003c/b\u003e. In addition, locomotory function was significantly impaired in HSD-fed larvae compared to the controls \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e. We investigated the impact of HSD on the expression of FOXO target genes in the body wall muscles. HSD larval muscles had significantly higher expression of InR and Thor, indicating insulin resistance and increased catabolism \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD\u003cb\u003e\u0026amp;E)\u003c/b\u003e. While the expression of muscle anabolic genes mef2 and mhc were reduced \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF\u003cb\u003e\u0026amp;G)\u003c/b\u003e. These results suggest that HSD-fed diabetic larvae had significant impact on the muscles and display muscle atrophy phenotype.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eVitamin C supplementation rescues diabetic-like phenotypes in HSD-fed larvae\u003c/h2\u003e \u003cp\u003eNext, we tested the beneficial effect of vitamin C on diabetic-like phenotypes. Consistent with the earlier data, HSD-fed larvae had reduced body size and growth inhibition. Interestingly, vitamin C-supplementation to HSD-fed larvae (HSD\u0026thinsp;+\u0026thinsp;AA) showed a rescue from the diabetic-like phenotype \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. A significant rescue in body size was observed in HSD\u0026thinsp;+\u0026thinsp;AA larvae \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. Vitamin C supplementation led to increased food consumption, as evidenced by increased blue stain and dye absorbance \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC\u003cb\u003e\u0026amp;D)\u003c/b\u003e. Despite higher calorie in HSD, a reduction in hemolymph glucose levels was seen; however, the triglyceride levels remained unchanged in the HSD\u0026thinsp;+\u0026thinsp;AA group \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE\u003cb\u003e\u0026amp;F)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eHSD causes peripheral insulin resistance in Drosophila, marked by changes in the expression of circulating Drosophila insulin-like peptides (DILPs). DILPs are released from insulin-producing cells (IPCs) in the central nervous system (CNS) and regulate glucose homeostasis [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Similarly, Adipokinetic hormones (akh), expressed in the fat body, helps mobilize stored fat and is increased in diabetic flies [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. We observed a significant increase in the expression of dilp2 and akh in HSD-fed larvae, indicating insulin resistance \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG\u003cb\u003e\u0026amp;H)\u003c/b\u003e. A substantial reduction in both dilp2 and akh expression was evident in HSD\u0026thinsp;+\u0026thinsp;AA larvae, indicating maintenance of glucose metabolism homeostasis.\u003c/p\u003e \u003cp\u003eNext, we performed RNA-seq to understand the transcriptional changes in control, HSD, and HSD\u0026thinsp;+\u0026thinsp;AA larval body wall muscles. Compared to controls, 1,144 genes were differentially regulated in HSD larval muscles. In comparison, 2881 genes were differentially regulated in HSD\u0026thinsp;+\u0026thinsp;AA larval muscle compared to HSD \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI\u003cb\u003e\u0026amp;J Supplementary files)\u003c/b\u003e. KEGG pathways and biological process analysis showed enrichment for genes involved in various metabolic pathways in HSD\u0026thinsp;+\u0026thinsp;AA muscles \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eK\u003cb\u003e\u0026amp;L)\u003c/b\u003e. In contrast, the HSD group had enrichment for genes involved in cuticle development, suggesting that HSD-fed larvae had a delayed developmental program and were metabolically less active. In addition, genes linked to muscle development were significantly enriched in HSD\u0026thinsp;+\u0026thinsp;AA compared to HSD \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eM\u003cb\u003e)\u003c/b\u003e. Furthermore, the expression of mef2 target genes, Mhc, Arp8, Act87E, Act57B were enriched in the HSD\u0026thinsp;+\u0026thinsp;AA compared to the HSD, indicating enhanced muscle anabolism with vitamin C (\u003cb\u003eSupplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e)\u003c/b\u003e. Overall, the data suggest vitamin C supplementation improved metabolism and promoted muscle gene expression in diabetic larvae.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eVitamin C reduces oxidative stress and rescues Tet expression in HSD-fed larvae\u003c/h2\u003e \u003cp\u003eVitamin C is a potent antioxidant. Since vitamin C supplementation rescued diabetic- like phenotypes, we measured the total antioxidant capacity (TAC). Compared to HSD, HSD\u0026thinsp;+\u0026thinsp;AA larvae showed increased TAC indicating reduced oxidative stress upon vitamin C supplementation \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. Consistent with this the expression of antioxidant genes SOD1, SOD2 and Catalase were higher in HSD\u0026thinsp;+\u0026thinsp;AA fed larvae \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, C\u003cb\u003e\u0026amp;D)\u003c/b\u003e. In addition to its role as an antioxidant, vitamin C functions as a co-factor for several enzymes including epigenetic modifier Tet whose expression and activity are critical for muscle differentiation and development [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. To this end, we examined the expression of Drosophila Tet (dTet) in the muscles of control, HSD, and HSD\u0026thinsp;+\u0026thinsp;AA larvae. dTet was significantly downregulated in HSD larval muscles compared to controls. In contrast, dTet expression was rescued upon vitamin C supplementation \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE\u003cb\u003e)\u003c/b\u003e. Alternatively, we also checked if dTet is altered in controls upon vitamin C supplementation. We did not see changes in dTet expression or in the larval size \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF\u003cb\u003e)\u003c/b\u003e. Consistent with its reduced expression, we found higher repressive H3K9me2 on the dTet promoter in HSD larval muscles, while it was reversed in HSD\u0026thinsp;+\u0026thinsp;AA \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG\u003cb\u003e)\u003c/b\u003e. These results confirm that dTet is transcriptionally downregulated in HSD larval muscles.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eVitamin C mitigates muscle atrophy in diabetic larvae\u003c/h2\u003e \u003cp\u003eSince muscle genes were enriched in RNA-seq analysis in the HSD\u0026thinsp;+\u0026thinsp;AA group, we analyzed the myofiber size in all three groups. Phalloidin staining showed increased size of VL3 and VL4 muscles in the HSD\u0026thinsp;+\u0026thinsp;AA group compared to HSD (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA\u003cb\u003e\u0026amp;B\u003c/b\u003e). Improved locomotion was evident in the HSD\u0026thinsp;+\u0026thinsp;AA larvae compared to the HSD larvae \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e. Consistently, expression of mef2 and mhc was significantly higher in the HSD\u0026thinsp;+\u0026thinsp;AA compared to HSD group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC\u003cb\u003e\u0026amp;D)\u003c/b\u003e. These findings indicate that vitamin C supplementation increases muscle size and function and alleviates muscle atrophy in diabetic larvae.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eVitamin C improves skeletal muscle regeneration in diabetic mice\u003c/h2\u003e \u003cp\u003eWe furthered our findings on a diabetic mouse model to examine the effect of vitamin C on skeletal muscle regeneration. Five low doses of Streptozotocin (STZ) were injected intraperitoneally to induce a type 2 diabetes in C57BL/6 mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA) [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Mice were divided into three groups: controls (No STZ), STZ, and STZ with vitamin C (4g/kg body weight) (STZ\u0026thinsp;+\u0026thinsp;AA). The STZ group had significantly higher blood glucose, indicating that the mice were hyperglycemic \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Surprisingly, STZ\u0026thinsp;+\u0026thinsp;AA mice did not exhibit changes in glucose levels, compared to STZ mice. Consistently, we did not observe changes in insulin levels in the pancreatic β-cells between the STZ and STZ\u0026thinsp;+\u0026thinsp;AA groups, indicating that vitamin C did not improve the pancreatic β-cells function in the diabetic mice \u003cb\u003e(Supplementary Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further assess the effect of vitamin C on skeletal muscle regeneration, we injured the Tibialis Anterior (TA) muscle with barium chloride [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Interestingly, seven days after the injury, mice treated with AA intraperitoneally had improved muscle regeneration compared to STZ diabetic mice \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e. Increased myofiber size and enhanced fusion index were evident in the STZ\u0026thinsp;+\u0026thinsp;AA diabetic mice \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD\u003cb\u003e\u0026amp;E)\u003c/b\u003e. In addition, presence of vitamin C led to enhanced \u003cem\u003ein vitro\u003c/em\u003e myogenic differentiation of C2C12 mouse myoblasts as evidenced by increased expression myogenin and myosin heavy chain \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF\u003cb\u003e).\u003c/b\u003e Overall, these results suggest a beneficial role of vitamin C in skeletal muscle differentiation and regeneration.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study we demonstrate the beneficial effect of vitamin C in mitigating muscle atrophy and improving skeletal muscle regeneration in diabetic larvae and mouse respectively. These results highlight the critical role of essential micronutrient vitamin C, in maintaining muscle homeostasis in diabetes. Importantly, in addition to its role as an antioxidant, our findings highlight a role for vitamin C in modulating epigenetic regulators in diabetic skeletal muscles.\u003c/p\u003e \u003cp\u003eWe used Drosophila larvae for several reasons: 1. In Drosophila, the conserved insulin signaling regulates constant feeding behaviour and energy storage mechanisms, making it an effective model to study metabolic dysfunction [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. 2. Hyperglycemia in Drosophila causes insulin resistance, leading to developmental delay and reduced body size, which mimics a key feature of diabetes pathology in mammals [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. 3. It eliminates sex specific variabilities due to hyperglycemia which otherwise could act as a confounding factor [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. 4. Larval body wall muscle undergoes a 50-fold increase in muscle mass during larval development, which provides an excellent window to study muscle atrophy and hypertrophy \u003cem\u003ein vivo\u003c/em\u003e [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHSD-fed larvae exhibited increased expression of FOXO target genes, decreased mef2 and mhc, and reduced myofiber size, all of which reflect increased catabolic signaling. Such changes are evident in diabetic mammals where FOXO activity drives proteasomal and autophagy degradation pathways [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Vitamin C supplementation alleviated these changes in diabetic larvae. In a randomized, crossover study involving individuals with type 2 diabetes, vitamin C supplementation resulted in improved glucose uptake and reduced oxidative stress in skeletal muscles (Mason et al., 2016). Another trial with diabetic patients on metformin showed that supplementation with vitamin C (and/or vitamin E) improved fasting blood glucose, HbA1c, and insulin resistance [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Our findings support a beneficial role for vitamin C in mitigating muscle atrophy in diabetes.\u003c/p\u003e \u003cp\u003eA rescue in dTet expression in HSD\u0026thinsp;+\u0026thinsp;AA larvae indicates that in addition to its antioxidant and metabolic effects, vitamin C may also induce epigenetic changes in diabetic muscle. Vitamin C\u0026rsquo;s ability to modulate the epigenome through Tet dioxygenases is emerging as an important gene regulatory mechanism. Tet catalyze oxidation of 5-methylcytosine (5mc) to 5-hydroxymethylcytosine (5hmc), facilitating DNA demethylation and activation of gene expression. Vitamin C functions as a cofactor for Tet and enhances their catalytic activity [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. In the context of myogenesis, vitamin C induces expression of myogenic genes through Tet-dependent epigenetic mechanisms [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. During development, dTet has been shown to co-localize with mef2 in muscle precursor cells and depletion of dTet in flies causes defects in locomotion and pupal lethality [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Since vitamin C alleviated the diabetic phenotype in HSD-fed larvae, our findings suggest that the expression and activity of dTet may be crucial in the larvals muscle.\u003c/p\u003e \u003cp\u003eA significant limitation of our study is that, although vitamin C restored dTet expression in HSD-fed larvae, we did not assess dTet enzymatic activity or its downstream epigenetic effects. Notably, DNA methylation is not evident in Drosophila; however, dTet is implicated in RNA hydroxymethylation [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. A further understanding of the consequences of dTet-mediated RNA hydroxymethylation, particularly on muscle specific mRNA, will help determine whether increased dTet expression upon vitamin C translates into functional epigenetic regulation in diabetic muscle.Furthermore, Tet loss-of-function studies under diabetic conditions would help clarify the muscle-protective effects of vitamin C. The observed effect of vitamin C on muscle regeneration in injured diabetic mice may be due to its direct interaction with PAX7 thereby by activating myogenic genes or through the transport of vitamin C to the injured site, which enhances cell adhesion and migration, both of which are essential for regeneration [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. However, the vitamin C-mediated epigenetic mechanisms underlying skeletal muscle regeneration warrant further investigation.\u003c/p\u003e \u003cp\u003eOverall, this study demonstrates that vitamin C mitigates muscle atrophy, and supplementation of vitamin C with anti-diabetic medications may be beneficial in controlling diabetic complications.\u003c/p\u003e "},{"header":"Declarations","content":"\u003ch2\u003e \u003cb\u003eAuthor information\u003c/b\u003e \u003c/h2\u003e \u003cp\u003e \u003cstrong\u003eManipal Institute of Regenerative Medicine, Manipal Academy of Higher Education, Manipal, India.\u003c/strong\u003e \u003cp\u003eParika Kala, Vinay Kumar Rao\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eDepartment of Medical Genetics, JSS Medical College, JSS Academy of Higher Education and Research, Mysuru, India.\u003c/strong\u003e \u003cp\u003eSupriya Bevinakoppamath, Akila Prashant\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eLee Kong Chian School of Medicine, Nanyang Technological University, Singapore.\u003c/strong\u003e \u003cp\u003eSwarnaseetha Adusumalli\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eDepartment of Developmental Biology and Genetics (DBG), Indian Institute of Science (IISc), India\u003c/strong\u003e \u003cp\u003eVishnu Ramada, Aishwarya A Makam, Nikhil R Gandasi\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eConflict of Interest\u003c/h2\u003e \u003cp\u003eThe authors declare no conflict of interest\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eEthical declaration\u003c/h2\u003e \u003cp\u003e All animal experiments were conducted after the approval of Institutional animal ethical committee clearance (JSS AHER, Mysore).\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor contribution\u003c/h2\u003e \u003cp\u003eSB: conceptualization, methodology, validation, writing. PK: methodology and validation. SA: RNA-seq analysis. VR: methodology and validation. AAM: methodology and validation. AP: resources, supervision. NRG: Supervision, writing. VKR: Project administration, conceptualization, supervision writing and editing\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e \u003cp\u003eWe thank Dr. Jyotsna Dhawan, CCMB for providing C2C12 mouse myoblasts cell line (originally from Helen Blua), Dr. Anujith Kumar, MIRM for his critical comments on the manuscript. Vinay Kumar Rao is supported by Core Research Grant CRG/2022/001969, Anusandhan National Research Foundation, Govt. of India. Vision group on science and technology, Govt. of Karnataka (GRD1120), Intramural Research Grant, JSS Academy of Higher Education and Research, India. Parika Kala is supported by fellowships from Manipal Academy of Higher Education, Manipal. Nikhil R Gandasi\u0026rsquo;s (NRG) lab was funded by the Indian Institute of Science\u0026mdash;seed grants, Department of Biotechnology (DBT)-Ramalingaswami fellowship, Indian Council of Medical Research (ICMR) \u0026ndash; Grants in Aid Scheme, Science and Engineering Research (SERB) Board \u0026ndash; Start-up grant, Infosys Young Investigator fellowship, Rajiv Gandhi University of Health Sciences (RGUHS) extramural grant and NovoNordisk Foundation awarded to NRG lab. Aishwarya A Makam\u0026rsquo;s salary was supported by Prime Ministers Research Fellowship (PMRF).\u003c/p\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eRNA-Seq data has been deposited in GEO database under accession number GSE313392. For reviewer access - uvgbscwefjqfzol\u003c/p\u003e \u003c/div\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eNellaiappan K, Preeti K, Khatri DK, Singh SB (2022) Diabetic Complications: An Update on Pathobiology and TherapeuticStrategies. Curr Diabetes Rev 18(1):e030821192146\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKalyani RR, Tra Y, Yeh HC, Egan JM, Ferrucci L, Brancati FL (2013) Quadriceps strength, quadriceps power, and gait speed in older U.S. adults with diabetes mellitus: results from the National Health and Nutrition Examination Survey, 1999\u0026ndash;2002. J Am Geriatr Soc 61(5):769\u0026ndash;775\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePark SW, Goodpaster BH, Strotmeyer ES, de Rekeneire N, Harris TB, Schwartz AV, Tylavsky FA, Newman AB (2006 June) Decreased muscle strength and quality in older adults with type 2 diabetes: the health, aging, and body composition study. Diabetes 55(6):1813\u0026ndash;1818\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVolpato S, Bianchi L, Lauretani F, Lauretani F, Bandinelli S, Guralnik JM, Zuliani G, Ferrucci L (2012) Role of muscle mass and muscle quality in the association between diabetes and gait speed. Diabetes Care 35(8):1672\u0026ndash;1679\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eda Silva Rosa SC, Nayak N, Caymo AM, Gordon JW (2020) Mechanisms of muscle insulin resistance and the cross-talk with liver and adipose tissue. Physiol Rep 8(19):e14607\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eO\u0026rsquo;Neill BT, Lee KY, Klaus K, Softic S, Krumpoch MT, Fentz J, Stanford KI, Robinson MM, Cai W, Kleinridders A, Pereira RO, Hirshman MF, Abel ED, Accili D, Goodyear LJ, Nair KS, Kahn CR Insulin and IGF-1 receptors regulate FoxO-mediated signaling in muscle proteostasis. 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Stem Cell Res 12(2):354\u0026ndash;363\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 is not available with this version.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nutrition-and-diabetes","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"nutd","sideBox":"Learn more about [Nutrition \u0026 Diabetes](http://www.nature.com/nutd/)","snPcode":"41387","submissionUrl":"https://mts-nutd.nature.com/cgi-bin/main.plex","title":"Nutrition \u0026 Diabetes","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-8342512/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8342512/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eSkeletal muscle atrophy is a major complication of diabetes linked with poor prognosis and reduced quality of life. Antioxidant vitamin C has shown promise in alleviating diabetic complications in rodents and mammals. Whether vitamin C is effective in mitigating diabetic muscle atrophy and the underlying molecular mechanisms remains unclear. The Drosophila larval body wall muscle offers a powerful system to identify interventions that target muscle atrophy and hypertrophy.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eTo induce diabetic conditions, wild-type \u003cem\u003eCanton-S\u003c/em\u003e Drosophila larvae were fed with High Sugar Diet (HSD) with a final concentration of 1 M sucrose. Control larvae (CT) were fed a diet containing 0.1 M sucrose. Body wall muscles of mid-third instar larvae were used for molecular analysis. The effect of vitamin C on skeletal muscle regeneration was assessed using a streptozotocin-induced diabetic mouse model.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eHSD-fed larvae exhibited severe growth inhibition and developed hyperglycemia, accompanied by increased triglycerides in the hemolymph. Ventral longitudinal VL3 and VL4 body wall myofibers were reduced in size, exhibited decreased expression of mef2 and mhc, along with increased expression of FOXO target genes, indicating muscle atrophy. Supplementation of Vitamin C (Ascorbic acid) to HSD (HSD\u0026thinsp;+\u0026thinsp;AA) rescued growth inhibition, reduced food aversion, and alleviated diabetic phenotypes. Transcriptomic analysis of HSD\u0026thinsp;+\u0026thinsp;AA larval muscles revealed enhanced expression of genes linked to metabolism, muscle development, and differentiation. Vitamin C reduced oxidative stress and, interestingly, rescued the expression of epigenetic regulator Ten-eleven-translocation (Tet), which utilizes vitamin C as a cofactor for its activity. Furthermore, vitamin C improved skeletal muscle regeneration in the injured diabetic mice.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eCollectively, our data demonstrate that vitamin C mitigates muscle atrophy and enhances skeletal muscle regeneration in diabetic muscles. These results suggest that vitamin C intake, in combination with anti-diabetic medications, may offer promising strategy to mitigate long-term diabetic complications.\u003c/p\u003e","manuscriptTitle":"Vitamin C Supplementation Mitigates Diabetes-Associated Skeletal Muscle Atrophy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-12 16:44:16","doi":"10.21203/rs.3.rs-8342512/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2026-04-07T10:50:36+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-03-23T21:38:56+00:00","index":1,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-03-04T19:16:28+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-02-27T13:49:12+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-02-27T13:36:11+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2026-02-08T17:55:54+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-15T16:39:02+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-12T06:42:23+00:00","index":"","fulltext":""},{"type":"submitted","content":"Nutrition \u0026 Diabetes","date":"2025-12-12T06:42:22+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nutrition-and-diabetes","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"nutd","sideBox":"Learn more about [Nutrition \u0026 Diabetes](http://www.nature.com/nutd/)","snPcode":"41387","submissionUrl":"https://mts-nutd.nature.com/cgi-bin/main.plex","title":"Nutrition \u0026 Diabetes","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0c8a1d6c-865d-479e-bf3f-9515fb8ed214","owner":[],"postedDate":"February 12th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[{"id":62534213,"name":"Health sciences/Diseases/Endocrine system and metabolic diseases/Diabetes/Diabetes complications"},{"id":62534214,"name":"Biological sciences/Physiology/Metabolism/Metabolic diseases/Diabetes/Diabetes complications"}],"tags":[],"updatedAt":"2026-04-07T10:57:28+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-12 16:44:16","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8342512","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8342512","identity":"rs-8342512","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}