Mouse Model Of Insulin Resistance Research Articles

Exercise activates AMPK in mouse and human pancreatic islets to decrease senescence.

Beta (β)-cell senescence contributes to type 2 diabetes mellitus (T2DM). While exercise is vital for T2DM management and significantly affects cellular ageing markers, its effect on β-cell senescence remains unexplored. Here, we show that short-term endurance exercise training (treadmill running, 1 h per day for 10 days) in two male and female mouse models of insulin resistance decreases β-cell senescence. In vivo and in vitro experiments revealed that this effect is mediated, at least in part, by training-induced increases in serum glucagon, leading to activation of 5'-AMP-activated protein kinase (AMPK) signalling in β-cells. AMPK activation resulted in the nuclear translocation of NRF2 and decreased expression of senescence markers and effectors. Remarkably, human islets from male and female donors with T2DM treated with serum collected after a 10-week endurance exercise training programme showed a significant decrease in the levels of senescence markers. These findings indicate that exercise training decreases senescence in pancreatic islets, offering promising therapeutic implications for T2DM.

Relationship between Serum Concentrations and Muscular Expressions of Selenoproteins on Selenium-Supplemented Insulin Resistance Mouse Model.

Previously, insulin resistance and hepatic oxidative stress with increased expressions of glutathione peroxidase (GPx) 1 and selenoprotein P (SelP) were induced in NSY mice, a diabetic mouse model, by administrating a high fat diet (HFD) and seleno-L-methionine (SeMet) for 12 weeks. In this study we developed an analysis method for serum selenoproteins using LC-tandem mass spectrometry (LC-MS/MS) and investigated the effects of supplementary selenium on serum concentrations of selenoproteins as well as protein expression in skeletal muscle as a major insulin target tissue under the same experimental condition. The glucose area under the curves for oral glucose tolerance and insulin tolerance tests indicated that the HFD induced insulin resistance, whereas the treatment of SeMet + HFD showed insignificant promotion compared with the HFD-induced insulin resistance. Although the expressions of GPx1 in gastrocnemius and soleus were not significantly induced by supplementary SeMet nor HFD administration, the expressions of SelP in both skeletal muscles were significantly induced by the treatment of SeMet + HFD. There were also significant increases in serum concentrations of SelP by supplementary SeMet + HFD administration, whereas GPx3 was augmented by supplementary SeMet only. These results indicated that the HFD intake under the sufficient selenium status augmented the blood secretion of SelP, which may participate in the reduction of insulin sensitivity in skeletal muscles as well as liver or adipose tissues, and it is a better indicator of deterioration than GPx3 as it is a major selenoprotein in serum.

Systemic administration of miR-451 improves autophagy response in an accelerated mouse model of diabetic kidney disease

Diabetic kidney disease (DKD) is a common and severe microvascular complication of both type 1 and type 2 diabetes mellitus. It is the most common cause of end stage renal disease in the United States. Autophagy is a highly conserved pathway that recycles damaged organelles and macromolecules to maintain intracellular homeostasis. There is mounting evidence highlighting the role of autophagy in diabetic kidney disease. MicroRNAs (miRNA) affect the transcription of several genes involved in the development and progression of DKD, regulating key pathways and processes, including fibrotic signaling and autophagy. Thus, the dysregulation of miRs exacerbates kidney injury in the diabetic setting. Previous work from our group suggests that chronic systemic inhibition of miR-451 exacerbates fibrosis in the kidney of an obese insulin resistant mouse model (tallyHo/Jng), indicating a potential renoprotective role for miR-451 in the diabetic kidney. However, few studies have investigated this protective role of miR-451 in the diabetic kidney. This study aimed to understand the renoprotective effects of miR-451 in an accelerated mouse model of DKD. BTBR ob/ob mice rapidly develop morphological renal lesions characteristic of both early and advanced human DKD, with proteinuria typically beginning at 4-weeks of age. 12-week old male black and tan, brachyuric (BTBR) ob/ob mice and BTBR wild-type mice were treated with commercially available miR-451 mimics (2mg/kg/bw; n=3/treatment group; treatment groups: WT, BTBR ob/ob, WT+miR-451, and BTBR ob/ob+miR-451) 1X per week for 3-consective weeks. Spot urine was collected weekly until the end of study. Random blood glucose was measured by tail prick weekly. Mice were humanely euthanized, and kidneys and blood harvested at the end of the treatment period. Western blotting of whole kidney homogenate was performed following a standard protocol. Data was analyzed using students t-test or ANOVA, where appropriate, p<0.05 considered significant. Albumin: creatinine ratio was four times higher (p=0.01) in BTBR ob/ob mice beginning at 5 weeks. Treatment with miR-451 mimics had no significant effect on body weight in either group. Blood glucose was significantly higher in miR-451 treated and untreated BTBR ob/ob mice at 12-weeks (425± 33.1 mg/dL; p=0.04) and 13-weeks (383± 25.3 mg/dL; p=0.007). However, blood glucose decreased significantly from week 13 (554.7± 10.8 mg/dL) to week 14(289± 13.3 mg/dL; p=0.0002) in BTBR ob/ob miR-451 treated mice. Western blot analysis of MiR-451 mimic treated BTBR ob/ob and WT mice whole kidney homogenate revealed a 91% (p=0.02) and 95% (p=0.01) reduction, respectively, in YWHAZ (a predicted miR-451 target). Conversely, western blot analysis of autophagy related proteins revealed a 68% (p=0.01) increase in ATG101 and a 44% increase in Beclin-1 in miR-451 mimic treated BTBR ob/ob mice. Collectively, the findings of this study suggest that miR-451 may offer protection in the diabetic kidney via autophagic signaling and improved blood glucose control. GHUCCTS KL2 Scholar Program (Award Number: KL2TR001432) The Charles and Mary Latham Fund Howard University Department of Medicine Start-Up Funds; Howard University Bridge Fund and Pilot Study Awards. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Conjugated Linoleic Acid Ameliorates High Fat-Induced Insulin Resistance via Regulating Gut Microbiota-Host Metabolic and Immunomodulatory Interactions.

Interaction between gut microbiota, host immunity and metabolism has been suggested to crucially affect the development of insulin resistance (IR). This study aims to investigate how gut microbiota, inflammatory responses and metabolism in individuals with IR are affected by the supplementation of conjugated linoleic acid (CLA) and how this subsequently affects the pathophysiology of IR by using a high-fat diet-induced IR mouse model. Serum biochemical indices showed that 400 mg/kg body weight of CLA effectively attenuated hyperglycemia, hyperlipidemia, glucose intolerance and IR, while also promoting antioxidant capacities. Histomorphology, gene and protein expression analysis revealed that CLA reduced fat deposition and inflammation, and enhanced fatty acid oxidation, insulin signaling and glucose transport in adipose tissue or liver. Hepatic transcriptome analysis confirmed that CLA inhibited inflammatory signaling pathways and promoted insulin, PI3K-Akt and AMPK signaling pathways, as well as linoleic acid, arachidonic acid, arginine and proline metabolism. Gut microbiome analysis further revealed that these effects were highly associated with the enriched bacteria that showed positive correlation with the production of short-chain fatty acids (SCFAs), as well as the improved SCFAs production simultaneously. This study highlights the therapeutic actions of CLA on ameliorating IR via regulating microbiota-host metabolic and immunomodulatory interactions, which have important implications for IR control.

Sulforaphane Ameliorates High-Fat-Diet-Induced Metabolic Abnormalities in Young and Middle-Aged Obese Male Mice.

Type 2 diabetes (T2D) is still a fast-growing health problem globally. It is evident that chronic insulin resistance (IR) and progressive loss of β-cell mass and function are key features of T2D etiology. Obesity is a leading pathogenic factor for developing IR. The aim of the present study was to determine whether sulforaphane (SFN), a natural compound derived from cruciferous vegetables, can prevent (prevention approach) or treat (treatment approach) obesity and IR in mouse models. We show that dietary intake of SFN (0.5 g/kg of HFD) for 20 weeks suppressed high-fat diet (HFD)-induced fat accumulation by 6.04% and improved insulin sensitivity by 23.66% in young male mice. Similarly, dietary provision of SFN (0.25 g/kg) significantly improved blood lipid profile, glucose tolerance, and insulin sensitivity of the middle-aged male mice while it had little effects on body composition as compared with the HFD group. In the treatment study, oral administration of SFN (40 mg/kg) induced weight loss and improved insulin sensitivity and plasma lipid profile in the diet-induced-obesity (DIO) male mice. In all three studies, the metabolic effects of SFN administration were not associated with changes in food intake. In vitro, SFN increased glucose uptake in C2C12 myotubes and increased fatty acid and pyruvate oxidation in primary human skeletal muscle cells. Our results suggest that SFN may be a naturally occurring insulin-sensitizing agent that is capable of improving the metabolic processes in HFD-induced obesity and IR and thereby may be a promising compound for T2D prevention.

Comparison of Sitagliptin with Vildagliptin's effects on Mitochondrial Activity, Heart Rate Variability, and Cardiac Performance in Obese Insulin-Resistant Rats

Context and Objective: It has been demonstrated that a prolonged high-fat diet (HFD) can lead to insulin resistance, which is typified by hyperinsulinemia and metabolic inflexibility. Heart failure, cardiac mitochondrial dysfunction, and cardiac sympathovagal imbalance are all linked to insulin resistance. Oral anti-diabetic medications called sitagliptin and vildagliptin, which block dipeptidyl peptidase-4 (DPP-4), are frequently administered to individuals suffering from cardiovascular disease. Thus, using a mouse model of insulin resistance, we aimed to investigate the effects of sitagliptin and vildagliptin in this work. Method of Experimentation: Male For a period of 12 weeks, 180–200 g Wistar rats were fed either an HFD (59% energy from fat) or a regular diet (20% energy from fat). After that, these rats were split up into three subgroups and given either vehicle for an additional 21 days, sitagliptin (30 mg/kg/day-1), vildagliptin (3 mg/kg−1 day−1), or both days. Heart rate variability (HRV), cardiac function, oxidative stress, metabolic parameters, and cardiac mitochondrial function were all measured Important Findings: In rats fed a high-fat diet (HFD), they developed insulin resistance, which was manifested as a decrease in high-density lipoprotein (HDL) and an increase in body weight, plasma insulin, total cholesterol, and oxidative stress levels. Furthermore, HFD rats showed evidence of cardiac dysfunction, decreased HRV, cardiac mitochondrial dysfunction, and alterations in cardiac mitochondrial morphology. Vildagliptin and sitagliptin both raised HDL levels while lowering plasma insulin, total cholesterol, and oxidative stress. Additionally, sitagliptin and vildagliptin fully restored HRV and reduced cardiac dysfunction as well as cardiac mitochondrial dysfunction Conclusions and Significance: In obese insulin-resistant rats, vildagliptin and sitagliptin both had comparable cardioprotective effects.

TGF-β1 Signaling Impairs Metformin Action on Glycemic Control.

Hyperglycemia is a hallmark of type 2 diabetes (T2D). Metformin, the first-line drug used to treat T2D, maintains blood glucose within a normal range by suppressing hepatic glucose production (HGP). However, resistance to metformin treatment is developed in most T2D patients over time. Transforming growth factor beta 1 (TGF-β1) levels are elevated both in the liver and serum of T2D humans and mice. Here, we found that TGF-β1 treatment impairs metformin action on suppressing HGP via inhibiting AMPK phosphorylation at Threonine 172 (T172). Hepatic TGF-β1 deficiency improves metformin action on glycemic control in high fat diet (HFD)-induced obese mice. In our hepatic insulin resistant mouse model (hepatic insulin receptor substrate 1 (IRS1) and IRS2 double knockout (DKO)), metformin action on glycemic control was impaired, which is largely improved by further deletion of hepatic TGF-β1 (TKObeta1) or hepatic Foxo1 (TKOfoxo1). Moreover, blockade of TGF-β1 signaling by chemical inhibitor of TGF-β1 type I receptor LY2157299 improves to metformin sensitivity in mice. Taken together, our current study suggests that hepatic TGF-β1 signaling impairs metformin action on glycemic control, and suppression of TGF-β1 signaling could serve as part of combination therapy with metformin for T2D treatment.

Betaine for the prevention and treatment of insulin resistance and fatty liver in a high-fat dietary model of insulin resistance in C57BL mice.

The aim was to investigate mechanisms by which betaine improves hepatic insulin signaling in a dietary mouse model of insulin resistance and fatty liver. C57BL 6J mice were fed a standard diet (SF), a standard diet with betaine (SFB), a nutritionally complete high fat (HF) diet, or a high fat diet with betaine (HFB) for 14 weeks. In a separate experiment, mice were fed high fat diet for 18 weeks, half of whom received betaine for the final 4 weeks. Activation of insulin signaling in the liver was assessed by western blot. Insulin signaling was also assessed in insulin resistant primary human hepatocytes treated with betaine. As compared with SF, mice receiving HF diet were heavier, had more hepatic steatosis, and abnormal glucose tolerance test (GTT). Betaine content in liver and serum was 50% lower in HF than in SF; betaine supplementation restored serum and liver betaine content. Betaine treatment of HF reduced whole body insulin resistance as measured by GTT. Betaine treatment of HF increased tyrosine phosphorylation of insulin receptor substrate-1 and phosphorylation (activation) of Akt, and increased hepatic glycogen content. In vitro, betaine reversed insulin resistance in primary human hepatocytes by increasing insulin-stimulated tyrosine phosphorylation of IRS1 and of Akt. Betaine supplementation reduced whole body insulin resistance and increased activation of insulin signaling pathways in the liver in a mouse model of insulin resistance and fatty liver created by feeding a nutritionally complete high fat diet for 14 weeks. Betaine also reduced liver injury as assessed by ALT and by liver histology. In vitro, betaine reversed insulin resistance by increasing insulin-stimulated tyrosine phosphorylation of IRS1 and activation of downstream proteins in the insulin signaling cascade in insulin resistant primary human hepatocytes.

The Role of G0S2 in Lipid Metabolism Regulation in Chronic Myeloid Leukemia

Introduction: Chronic myeloid leukemia (CML) can be effectively treated with tyrosine kinase inhibitors (TKIs) targeting BCR::ABL1, but resistance remains a clinical problem. TKIs do not target the quiescent CML leukemic stem cell (LSC), and many patients are treated for life at a high economic burden and sometimes with significant side effects. We recently published a tumor suppressor role for G0/G1 switch gene 2 (G0S2) in CML. Our data showed that loss of G0S2 expression in CML promoted disease progression and drug resistance by disrupting glycerophospholipid metabolism (Gonzalezet al. Clin Transl Med, 2022). Interestingly, G0S2 is an estrogen-response gene that demonstrated anti-estrogenic and pro-migratory effects in ER+ versus ER- breast cancer. Additionally, deletion of G0S2 prevented obesity and insulin resistance in mouse models. Since sex-based differences in lipid metabolism and obesity are well documented, we hypothesized that the tumor suppressor role of G0S2 in CML would differ based on sex. Methods: We used peripheral blood from wild-type and G0s2 knockout ( G0s2 -/-) mice (both male and female) who were fed with a normal versus a high-fat diet. Resulting lineage-negative (Lin-) cells were cultured in recombinant murine cytokines ± granulocyte-colony stimulating factor (mG-CSF, 25 ng/ml, 7-10 days) in in vitro differentiation assays. Flow cytometry analysis was performed to measure CD11b+ neutrophils. Additionally, we transduced Lin- cells from wild-type or G0s2 knockout mice with BCR::ABL1, and assessed their ability to form a leukemia-like disease process in male versus female recipient mice. We also performed G0S2 ectopic expression and knockdown experiments in the K562 CML cell line and assessed the resulting cells by liquid chromatography (LC)/mass spectrometry (MS)-based lipidomics and proteomics analyses. Immunoblots were performed to assess the resulting cells for changes in the autophagy markers, LC3A/B I-II. Results: For the experiments conducted with mouse peripheral blood, RT-qPCR data showed high G0S2 mRNA expression upon neutrophil differentiation in wild-type male mice (n=5), but not in the wild-type female mice (n=5). Flow cytometry measuring CD11b markers showed that a high-fat diet-induced neutrophil production in wild-type male mice, but not in wild-type female mice or G0s2 -/- mice, suggesting hormonal regulation involving G0S2 and female mice. Consistent with this observation, forced BCR::ABL1 expression in G0s2 -/- bone marrow reduced overall survival in female (n=4/group) but not male (n=5/group) mice compared with wild-type controls. In mass spectrometry-based lipidomics analyses, G0S2 knockdown resulted in a substantial decrease of di- and triglycerides. G0S2 knockdown also resulted in substantial changes in phosphatidylethanolamine and phosphatidylcholine expression, implicating G0S2 in the production of lipid bilayer components. Conversely, ectopic G0S2 expression promoted the accumulation of long- and very long-chain triglycerides and species of phosphatidylcholine and phosphatidylethanolamine in K562 cells. Proteomics analyses showed autophagy as one of the top pathways affected by altered G0S2 expression. When we measured LC3A/B I-II by immunoblot, we observed opposing effects comparing G0S2 ectopic expression versus knockdown. Conclusion: Altogether, these findings suggest sex-based differences in the role of G0S2 and lipid metabolism in CML stem and progenitor cell differentiation, survival, and autophagy, both in vitro and in vivo. Restoring G0S2 expression combined with BCR::ABL1 inhibition may be a novel clinical strategy to induce treatment-free remission in CML. However, further studies should assess whether this strategy will have similar effects in male versus female patients.

Rap1 in the Context of PCSK9, Atherosclerosis, and Diabetes.

The focus of this article is to highlight the importance of the small GTPase, Ras-associated protein 1 (Rap1), in proprotein convertase subtilisin/kexin type 9 (PCSK9) regulation and atherosclerosis and type 2 diabetes etiology and discuss the potential therapeutic implications of targeting Rap1 in these disease areas. Cardiometabolic disease characterized by obesity, glucose intolerance, dyslipidemia, and atherosclerotic cardiovascular disease remain an important cause of mortality. Evidence using mouse models of obesity and insulin resistance indicates that Rap1 deficiency increases proatherogenic PCSK9 and low-density lipoprotein cholesterol levels and predisposes these mice to develop obesity- and statin-induced hyperglycemia, which highlights Rap1's role in cardiometabolic dysfunction. Rap1 may also contribute to cardiovascular disease through its effects on vascular wall cells involved in the atherosclerosis progression. Rap1 activation, specifically in the liver, could be beneficial in the prevention of cardiometabolic perturbations, including type 2 diabetes, hypercholesterolemia, and atherosclerosis.

Genetic Deletion of DNAJB3 Using CRISPR-Cas9, Produced Discordant Phenotypes.

Several pathways and/or genes have been shown to be dysregulated in obesity-induced insulin resistance (IR) and type 2 diabetes (T2D). We previously showed, for the first time, impaired expression of DNAJB3 mRNA and protein in subjects with obesity, which was concomitant with increased metabolic stress. Restoring the normal expression of DNAJB3 attenuated metabolic stress and improved insulin signaling both in vivo and in vitro, suggesting a protective role of DNAJB3 against obesity and T2D. The precise underlying mechanisms remained, however, unclear. This study was designed to confirm the human studies in a mouse model of dietary obesity-induced insulin resistance, and, if validated, to understand the underlying mechanisms. We hypothesized that mice lacking DNAJB3 would be more prone to high-fat (HF)-diet-induced increase in body weight and body fat, inflammation, glucose intolerance and insulin resistance as compared with wild-type (WT) littermates. Three DNAJB3 knockout (KO) lines were generated (KO 30, 44 and 47), using CRISPR-Cas9. Male and female KO and WT mice were fed a HF diet (45% kcal fat) for 16 weeks. Body weight was measured biweekly, and a glucose tolerance test (GTT) and insulin tolerance test (ITT) were conducted at week 13 and 14, respectively. Body composition was determined monthly by nuclear magnetic resonance (NMR). Following euthanasia, white adipose tissue (WAT) and skeletal muscle were harvested for further analyses. Compared with WT mice, male and female KO 47 mice demonstrated higher body weight and fat mass. Similarly, KO 47 mice also showed a slower rate of glucose clearance in GTT that was consistent with decreased mRNA expression of the GLUT4 gene in WAT but not in the muscle. Both male and female KO 47 mice exhibited higher mRNA levels of the pro-inflammatory marker TNF-a in WAT only, whereas increased mRNA levels of MCP1 chemokine and the ER stress marker BiP/Grp78 were observed in male but not in female KO 47 mice. However, we did not observe the same changes in the other KO lines. Taken together, the phenotype of the DNAJB3 KO 47 mice was consistent with the metabolic changes and low levels of DNAJB3 reported in human subjects. These findings suggest that DNAJB3 may play an important role in metabolic functions and glucose homeostasis, which warrants further phenotyping and intervention studies in other KO 47 and other KO mice, as well as investigating this protein as a potential therapeutic target for obesity and T2D.

Metformin targets intestinal immune system signaling pathways in a high-fat diet-induced mouse model of obesity and insulin resistance.

Research findings of the past decade have highlighted the gut as the main site of action of the oral antihyperglycemic agent metformin despite its pharmacological role in the liver. Extensive evidence supports metformin's modulatory effect on the composition and function of gut microbiota, nevertheless, the underlying mechanisms of the host responses remain elusive. Our study aimed to evaluate metformin-induced alterations in the intestinal transcriptome profiles at different metabolic states. The high-fat diet-induced mouse model of obesity and insulin resistance of both sexes was developed in a randomized block experiment and bulk RNA-Seq of the ileum tissue was the method of choice for comparative transcriptional profiling after metformin intervention for ten weeks. We found a prominent transcriptional effect of the diet itself with comparatively fewer genes responding to metformin intervention. The overrepresentation of immune-related genes was observed, including pronounced metformin-induced upregulation of immunoglobulin heavy-chain variable region coding Ighv1-7 gene in both high-fat diet and control diet-fed animals. Moreover, we provide evidence of the downregulation NF-kappa B signaling pathway in the small intestine of both obese and insulin-resistant animals as well as control animals after metformin treatment. Finally, our data pinpoint the gut microbiota as a crucial component in the metformin-mediated downregulation of NF-kappa B signaling evidenced by a positive correlation between the Rel and Rela gene expression levels and abundances of Parabacteroides distasonis, Bacteroides spp., and Lactobacillus spp. in the gut microbiota of the same animals. Our study supports the immunomodulatory effect of metformin in the ileum of obese and insulin-resistant C57BL/6N mice contributed by intestinal immunoglobulin responses, with a prominent emphasis on the downregulation of NF-kappa B signaling pathway, associated with alterations in the composition of the gut microbiome.

Intranasal insulin helps overcome brain insulin deficiency and improves survival and post-stroke cognitive impairment in male mice.

Obesity increases the risk for stroke and is associated with worse post-stroke outcomes; however, the mechanisms are poorly understood. Diet-induced obesity leads to insulin resistance and subsequently, brain insulin deficiency. The purpose of this study was to investigate the potential impact of brain insulin deficiency on post-stroke outcomes. To accomplish this, brain insulin levels were assessed in male C57BL/6J (B6) mice placed on either a standard diet or 54% kcal high-fat diet, a known model of insulin resistance. Mice were subjected to either a sham surgery (control) or 30-min middle cerebral artery occlusion to induce an ischemic stroke and administered either intranasal saline (0.9%) or intranasal insulin (1.75 U) twice daily for 5 days beginning on day 1 post-stroke. High-fat diet-induced brain insulin deficiency was associated with increased mortality, neurological and cognitive deficits. On the other hand, increasing brain insulin levels via intranasal insulin improved survival, neurological and cognitive function in high-fat diet mice. Our data suggests that brain insulin deficiency correlates with worse post-stroke outcomes in a diet-induced mouse model of insulin resistance and increasing brain insulin levels may be a therapeutic target to improve stroke recovery.

Adipose tissue-derived FABP4 mediates glucagon-stimulated hepatic glucose production in gestational diabetes.

One of the most common complications of pregnancy is gestational diabetes mellitus (GDM), which may result in significant health threats of the mother, fetus and the newborn. Fatty acid-binding protein 4 (FABP4) is an adipokine that regulates glucose homeostasis by promoting glucose production and liver insulin resistance in mouse models. FABP4 levels are increased in GDM and correlates with maternal indices of insulin resistance, with a rapid decline post-partum. We therefore aimed to determine the tissue origin of elevated circulating FABP4 levels in GDM and to assess its potential contribution in promoting glucagon-induced hepatic glucose production. FABP4 protein and gene expression was determined in biopsies from placenta, subcutaneous (sWAT) and visceral (vWAT) white adipose tissues from GDM and normoglycaemic pregnant women. FABP4 differential contribution in glucagon-stimulated hepatic glucose production was tested in conditioned media before and after its immune clearance. We showed that FABP4 is expressed in placenta, sWAT and vWAT of pregnant women at term, with a significant increase in its secretion from vWAT of women with GDM compared with normoglycaemic pregnant women. Neutralizing FABP4 from both normoglycaemic pregnant women and GDM vWAT secretome, resulted in a decrease in glucagon-stimulated hepatic glucose production. This study provides new insights into the role of adipose tissue-derived FABP4 in GDM, highlighting this adipokine, as a potential co-activator of glucagon-stimulated hepatic glucose production during pregnancy.

1738-P: Instrument-Free Single-Cell Resolution of Transcriptome in Mouse Pancreatic Islet Cells

Type 1 diabetes is characterized by an almost complete depletion of insulin-producing beta cells. Thus, regeneration of beta cells through beta-cell neogenesis represents a major therapeutic strategy to cure the disease. We previously reported a mouse model of severe insulin resistance that leads to high insulin secretion and massive beta-cell neogenesis. To understand the origins of the newly formed beta cells and the genic programs involved in high insulin secretion, we aimed at defining the pancreatic islet cells transcriptome at high resolution cell by cell. Here, we tested the compatibility of the novel instrument-free 3’ single-cell RNA-sequencing benchtop kit Asteria™, recently released by Scipio bioscience (Paris, France), on pancreatic islet cells isolated from adult mice, which underwent cell dissociation by trypsin treatment. In the Asteria™ procedure, differently from the microfluidic-based single-cell one, coupling of solid polymer-barcoded beads and cells occurs in homogenous phase in a reversible hydrogel named RevGel™. Such conditions are claimed by the vendor to be causing less mechanical stress to cells for an expected more preserved transcriptome. Successive steps were classic molecular biology and bioinformatic analysis using Scipio bioscience’s data analysis cloud-based software Cytonaut™. We profiled the transcriptome of 938 single cells from healthy mice and identified a total of 12 cell subtypes. SingleR annotation based on reference data revealed all the major cell types commonly described in the literature, including a well-represented beta cell population and some rare cell types such as ductal and stellate cells. We demonstrate 3’ single-cell RNA-sequencing using Scipio bioscience’s kit Asteria™ as an effective strategy to dissect the heterogeneity and the complexity of mouse pancreatic islets and identify the gene expression signatures of each cell. Disclosure D.G.Dimitrova: None. A.Liboz: None. S.Lipin: None. C.Beaupere: None. N.Roblot: None. J.Komatsu: None. G.Guillemain: None. B.Blondeau: None.

Tripeptide IRW Improves AMPK/eNOS Signaling Pathway via Activating ACE2 in the Aorta of High-Fat-Diet-Fed C57BL/6 Mice.

This study aims to investigate the effect of tripeptide IRW on the local renin-angiotensin system (RAS), particularly angiotensin-converting enzyme 2 (ACE2), and their association with signaling pathways in the aorta of a high-fat-diet (HFD)-induced insulin-resistant mouse model. C57BL/6 mice were fed HFD (45% of the total calories) for six weeks, and then IRW was added to the diet (45 mg/kg body weight (BW)) for another eight weeks. ACE2 mRNA expression and protein level(s) were increased (p < 0.05), while angiotensin II receptor (AT1R) and angiotensin-converting enzyme (ACE) protein abundance was significantly reduced (p < 0.05) in the aorta of HFD mice treated by IRW. IRW supplementation also improved glucose transporter 4 (GLUT4) abundance (p < 0.05) alongside AMP-activated protein kinase (AMPK) (p < 0.05), Sirtuin 1 (SIRT1) (p < 0.05), and endothelial nitric oxide synthase (eNOS) (p < 0.05) expression. IRW downregulated the levels of endothelin 1 (ET-1) and p38 mitogen-activated protein kinases (p38 MAPK, p < 0.05). Furthermore, the levels of AMPK and eNOS in vascular smooth muscle cells (VSMCs) were significantly reduced in ACE2 knockdown cells treated with or without IRW (p < 0.01). In conclusion, this study provided new evidence of the regulatory role of IRW on the aortic ACE2 against metabolic syndrome (MetS) in an HFD-induced insulin-resistant model.

Betahistine prevents development of endolymphatic hydrops in a mouse model of insulin resistance and diabetes

Background Diabetes is associated with inner ear dysfunction. Furthermore, C57BL/6J mice fed high fat diet (HFD), a model for insulin resistance and diabetes, develop endolymphatic hydrops (EH). Aim Evaluate if betahistine, spironolactone (aldosterone antagonist) and empagliflozin (sodium –glucose cotransporter2 inhibitor) can prevent EH induced by HFD and explore potential mechanisms. Methods C57BL/6J mice fed HFD were treated with respective drug. The size of the endolymphatic fluid compartment was measured using contrast enhanced MRI. Secondarily, mice treated with cilostamide, a phosphodiesterase3 inhibitor, to induce EH and HEI-OC1 auditory cells were used to study potential cellular mechanisms of betahistine. Results HFD-induced EH was prevented by betahistine but not by spironolactone and empagliflozin. Betahistine induced phosphorylation of protein kinaseA substrates but did not prevent cilostamide-induced EH. Conclusions Betahistine prevents the development of EH in mice fed HFD, most likely not involving pathways downstream of phosphodiesterase3, an enzyme with implications for dysfunction in diabetes. The finding that spironolactone did not prevent HFD-induced EH suggests different mechanisms for EH induction/treatment since spironolactone prevents EH induced by vasopressin, as previously observed. Significance This further demonstrates that independent mechanisms can cause hydropic inner ear diseases which suggests different therapeutic approaches and emphazises the need for personalized medicine.

Alternate-day fat diet and exenatide modulate the brain leptin JAK2/STAT3/SOCS3 pathway in a fat diet-induced obesity and insulin resistance mouse model.

Obesity is one of the most burdensome health problems and is closely linked to leptin resistance. The study examined whether an alternate-day high-fat diet (ADF) and/or GLP-1 agonist (exenatide) modulate brain leptin resistance caused by a high-fat diet (HFD). Sixty adult male mice were divided into 6 groups: (i) normal palatable diet (NPD), (ii) exenatide control (NPD received exenatide) (iii) HFD, (iv) ADF treated, (v) exenatide treated, (vi) ADF and exenatide treated. All animal groups were fed a HFD for 8 weeks, before they received treatment (ADF and/or exenatide) for 8 additional weeks. Body weight was assessed at the start and at the end of the experiment. Lipid profile, brain leptin and its receptor expression with the leptin-sensitive pathway, JAK2/STAT3/SOCS3/PTP1B, fasting blood glucose (FBG), serum insulin, liver metabolic handling via its regulators IRS1/PI3K/GLUT4 for hyperinsulinemia/obesity-induced PDK3/NAFLD2 modification, and liver enzymes were determined at the end of the experiment. ADF and exenatide reduced body weight and FBG in HFD-obese mice (p < 0.05). The combined ADF and exenatide regimen enhanced the brain anorexic leptin/JAK2/STAT3 and attenuated the SOCS3/PTP1B pathway (p < 0.05). The ADF/exenatide anorexigenic brain effect also modulated liver glucose via IRS1/PI3K/GLUT4 expression (p < 0.05), attenuating NAFLD2 and PDK3 expression (p < 0.05). Liver enzymes and the histopathological profile confirmed the improvement. In HFD caloric consumption, a combination of ADF and GLP-1 agonist enhances the brain leptin anorexigenic effect with the improvement of the metabolic sequelae of hyperinsulinemia, hyperlipidemia and liver steatosis.

Raphanus sativus L. var. caudatus Extract Alleviates Impairment of Lipid and Glucose Homeostasis in Liver of High-Fat Diet-Induced Obesity and Insulin Resistance in Mice.

The present study investigated the activities of Raphanus sativus L. var. caudatus extract (RS) on abnormal lipid and glucose homeostasis in a high-fat diet (HFD)-induced obesity and insulin resistance in a mouse model. Institute of Cancer Research mice were rendered obese by 16-week HFD feeding. Obese mice were administered with 100 or 200 mg/kg/d RS orally during the last 8 weeks of diet feeding. Then, the biochemical parameters were determined. The gene and protein expressions regulating lipid and glucose homeostasis in the liver were measured. This study revealed that the state of hyperglycemia, hyperleptinemia, hyperinsulinemia, and hyperlipidemia was reduced after 8 weeks of RS treatment (100 or 200 mg/kg). Administration of RS also improved insulin sensitivity and increased serum adiponectin. The liver total cholesterol and triglyceride concentrations were decreased by both doses of RS. Notably, a decrease in the expression of liver-specific genes, including sterol regulatory element-binding protein 1c, fatty acid synthase, and acetyl-CoA carboxylase, was found in the RS-treated groups. Moreover, administration of RS showed a significant increase in the expression of adenosine monophosphate-activated protein kinase (AMPK) phosphorylation and sirtuin1 (Sirt1) proteins. These findings indicated that RS improved abnormal lipid and glucose homeostasis in the liver of obesity-associated insulin resistance mouse model, possibly through the stimulation of the AMPK/Sirt1 pathway.

Liver-Secreted Hexosaminidase A Regulates Insulin-Like Growth Factor Signaling and Glucose Transport in Skeletal Muscle.

Nonalcoholic fatty liver disease (NAFLD) and impaired glycemic control are closely linked; however, the pathophysiological mechanisms underpinning this bidirectional relationship remain unresolved. The high secretory capacity of the liver and impairments in protein secretion in NAFLD suggest that endocrine changes in the liver are likely to contribute to glycemic defects. We identify hexosaminidase A (HEXA) as an NAFLD-induced hepatokine in both mice and humans. HEXA regulates sphingolipid metabolism, converting GM2 to GM3 gangliosides-sphingolipids that are primarily localized to cell-surface lipid rafts. Using recombinant murine HEXA protein, an enzymatically inactive HEXA(R178H) mutant, or adeno-associated virus vectors to induce hepatocyte-specific overexpression of HEXA, we show that HEXA improves blood glucose control by increasing skeletal muscle glucose uptake in mouse models of insulin resistance and type 2 diabetes, with these effects being dependent on HEXA's enzymatic action. Mechanistically, HEXA remodels muscle lipid raft ganglioside composition, thereby increasing IGF-1 signaling and GLUT4 localization to the cell surface. Disrupting lipid rafts reverses these HEXA-mediated effects. In this study, we identify a pathway for intertissue communication between liver and skeletal muscle in the regulation of systemic glycemic control.