Streptozotocin-induced Type-1 Diabetes Research Articles

Immunomodulation of streptozotocin induced Type 1 diabetes mellitus in mouse model by Macrophage migration inhibitory factor-2 (MIF-2) homologue of human lymphatic filarial parasite, Wuchereria bancrofti

Helminth parasites modulate the host immune system to ensure a long-lasting asymptomatic form of infection generally, mediated by the secretion of immunomodulatory molecules and one such molecule is a homologue of human host cytokine, Macrophage migratory Inhibitory Factor (hMIF). In this study, we sought to understand the role of homologue of hMIF from the lymphatic filarial parasite, Wuchereria bancrofti (Wba-MIF2), in the immunomodulation of the Streptozotocin (STZ)-induced Type1 Diabetes Mellitus (T1DM) animal model. Full-length recombinant Wba-MIF2 was expressed and found to have both oxidoreductase and tautomerase activities. Wba-MIF2 recombinant protein was treated to STZ induced T1DM animals, and after 5 weeks pro-inflammatory (IL-1, IL-2, IL-6, TNF-α, IFN-γ) and anti-inflammatory (IL-4, IL-10) cytokines and gene expressions were determined in sera samples and spleen respectively. Pro-inflammatory and anti-inflammatory cytokine levels were significantly (p<0.05) up-regulated and down-regulated respectively, in the STZ-T1DM animals, as compared to treated groups. Histopathology showed macrophage infiltration and greater damage of islets of beta cells in the pancreatic tissue of STZ-T1DM animals, than Wba-MIF2 treated STZ-T1DM animals. The present study clearly showed the potential of Wba-MIF2 as an immunomodulatory molecule, which could modulate the host immune system in the STZ-T1DM mice model from a pro-inflammatory to anti-inflammatory milieu.

Corn silk extract alleviate type 1 diabetes via regulating gut microbiota and inflammation

Type 1 diabetes (T1D) is a metabolic disease commonly affecting adolescents and causing various health complications. Corn silk (CS), a traditional Chinese medicine with medicinal and edible properties, has been extensively researched for its anti-diabetes and anti-inflammatory effects. This study aimed to investigate the hypoglycemic effect of CS extract on T1D mice by modulating the composition of gut microbiota. The results demonstrated that CS extract had a significant therapeutic effect on STZ-induced T1D by reducing the fasting blood glucose (FBG) levels and pancreas injury. Moreover, CS extract was found to modify the gut microbiota by altering the abundance of Firmicutes, Actinobacteria, Desulfobacterota, and Proteobacteria, which subsequently influenced the production of short-chain fatty acids (SCFAs). These SCFAs then mediated the expression of G protein-coupled receptor 41 (GPR41), interleukin-10 (IL-10), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α), ultimately resulting in a reduction in the inflammatory response.

Novel AHR ligand AGT-5 ameliorates type 1 diabetes in mice through regulatory cell activation in the early phase of the disease.

Type 1 diabetes (T1D) is an autoimmune disease with a strong chronic inflammatory component. One possible strategy for the treatment of T1D is to stimulate the regulatory arm of the immune response, i.e. to promote the function of tolerogenic dendritic cells (tolDC) and regulatory T cells (Treg). Since both cell types have been shown to be responsive to the aryl hydrocarbon receptor (AHR) activation, we used a recently characterized member of a new class of fluorescent AHR ligands, AGT-5, to modulate streptozotocin-induced T1D in C57BL/6 mice. Prophylactic oral administration of AGT-5 reduced hyperglycemia and insulitis in these mice. Phenotypic and functional analysis of cells in the pancreatic infiltrates of AGT-5-treated mice (at the early phase of T1D) revealed a predominantly anti-inflammatory environment, as evidenced by the upregulation of tolDC and Treg frequency, while CD8+ cell, Th1 and Th17 cells were significantly reduced. Similarly, AGT-5 enhanced the proportion of Treg and tolDC in small intestine lamina propria and suppressed the activation status of antigen-presenting cells through down-regulation of co-stimulatory molecules CD40, CD80 and CD86. The expression levels of Cyp1a1, controlled by the AHR, were increased in CD4+, CD8+ and Treg, confirming the AHR-mediated effect of AGT-5 in these cells. Finally, AGT-5 stimulated the function of regulatory cells in the pancreatic islets and lamina propria by upregulating indoleamine 2,3-dioxigenase 1 (IDO1) in tolDC. These findings were supported by the abrogation of AGT-5-mediated in vitro effects on DC in the presence of IDO1 inhibitor. AGT-5 also increased the expression of CD39 or CD73 ATP-degrading ectoenzymes by Treg. The increase in Treg is further supported by the upregulated frequency of IL-2-producing type 3 innate lymphoid cells (ILC3) in the lamina propria. Anti-inflammatory effects of AGT-5 were also validated on human tonsil cells, where in vitro exposure to AGT-5 increased the proportion of immunosuppressive dendritic cells and ILC3. These results suggest that AGT-5, by stimulating AHR, may promote a general immunosuppressive environment in the pancreas and small intestine lamina propria at the early phase of disease, and thereby inhibit the severity of T1D in mice.

The protective effects of icariin against testicular dysfunction in type 1 diabetic mice Via AMPK-mediated Nrf2 activation and NF-κB p65 inhibition

BackgroundOwing to the early suffering age and the rising incidence of type 1 diabetes (T1D), the resulting male reproductive dysfunction and fertility decline have become a disturbing reality worldwide, with no effective strategy being available. Icariin (ICA), a flavonoid extracted from Herba Epimedium, has been proved its promising application in improving diabetes-related complications including diabetic nephropathy, endothelial dysfunction and erectile dysfunction. Ensuring the future reproductive health of children and adolescents with T1D is crucial to improve global fertility. However, its roles in the treatment of T1D-induced testicular dysfunction and the potential mechanisms remain elusive. PurposeThe purpose of this present study was to investigate whether ICA ameliorates T1D-induced testicular dysfunction as well as its potential mechanisms. MethodsT1D murine model was established by intraperitoneal injection of STZ with or without treated with ICA for eleven weeks. Morphological, pathological and serological experiments were used to determine the efficacy of ICA on male reproductive function of T1D mice. Western blotting, Immunohistochemistry analysis, qRT-PCR and kit determination were performed to investigated the underlying mechanisms. ResultsWe found that replenishment of ICA alleviated testicular damage, promoted testosterone production and spermatogenesis, ameliorated apoptosis and blood testis barrier impairment in streptozotocin-induced T1D mice. Functionally, ICA treatment triggered adenosine monophosphate protein kinase (AMPK) activation, which in turn inhibited the nuclear translocation of nuclear factor kappa B p65 (NF-κB p65) to reduce inflammatory responses in the testis and activated nuclear factor erythroid 2-related factor 2(Nrf2), thereby enhancing testicular antioxidant capacity. Further studies revealed that supplementation with the AMPK antagonist Compound C or depletion of Nrf2 weakened the beneficial effects of ICA on testicular dysfunction of T1D mice. ConclusionCollectively, these results demonstrate the feasibility of ICA in the treatment of T1D-induced testicular dysfunction, and reveal the important role of AMPK-mediated Nrf2 activation and NF-κB p65 inhibition in ICA-associated testicular protection during T1D.

Therapeutic potential of a novel peripherally restricted CB1R inverse agonist on the progression of diabetic nephropathy.

This study assessed the efficacy of INV-202, a novel peripherally restricted cannabinoid type-1 receptor (CB1R) inverse agonist, in a streptozotocin-induced type-1 diabetes nephropathy mouse model. Diabetes was induced in 8-week-old C57BL6/J male mice via intraperitoneal injection of streptozotocin (45 mg/kg/day for 5 days); nondiabetic controls received citrate buffer. Diabetic mice were randomized to 3 groups based on blood glucose, polyuria, and albuminuria, and administered daily oral doses for 28-days of INV-202 at 0.3 or 3 mg/kg or vehicle. INV-202 did not affect body weight but decreased kidney weight compared with the vehicle group. While polyuria was unaffected by INV-202 treatment, urinary urea (control 30.77 ± 14.93; vehicle 189.81 ± 31.49; INV-202 (0.3 mg/kg) 127.76 ± 20; INV-202 (3 mg/kg) 93.70 ± 24.97 mg/24h) and albumin (control 3.06 ± 0.38; vehicle 850.08 ± 170.50; INV-202 (0.3 mg/kg) 290.65 ± 88.70; INV-202 (3 mg/kg) 111.29 ± 33.47 µg/24h) excretion both decreased compared with vehicle-treated diabetic mice. Compared with the vehicle group, there was a significant improvement in the urinary albumin to creatinine ratio across INV-202 groups. Regardless of the dose, INV-202 significantly reduced angiotensin II excretion in diabetic mice. The treatment also decreased Agtr1a renal expression in a dose-dependent manner. Compared with nondiabetic controls, the glomerular filtration rate was increased in the vehicle group and significantly decreased by INV-202 at 3 mg/kg. While the vehicle group showed a significant loss in the mean number of podocytes per glomerulus, INV-202 treatment limited podocyte loss in a dose-dependent manner. Moreover, in both INV-202 groups, expression of genes coding for podocyte structural proteins nephrin (Nphs1), podocin (Nphs2), and podocalyxin (Pdxl) were restored to levels similar to nondiabetic controls. INV-202 partially limited the proximal tubular epithelial cell (PTEC) hyperplasia and normalized genetic markers for PTEC lesions. INV-202 also reduced expression of genes contributing to oxidative stress (Nox2, Nox4, and P47phox) and inflammation (Tnf). In addition, diabetes-induced renal fibrosis was significantly reduced by INV-202. INV-202 reduced glomerular injury, preserved podocyte structure and function, reduced injury to PTECs, and ultimately reduced renal fibrosis in a streptozotocin-induced diabetic nephropathy mouse model. These results suggest that INV-202 may represent a new therapeutic option in the treatment of diabetic kidney disease.

Corinthian Currants Supplementation Restores Serum Polar Phenolic Compounds, Reduces IL-1beta, and Exerts Beneficial Effects on Gut Microbiota in the Streptozotocin-Induced Type-1 Diabetic Rat

The present study aimed at investigating the possible benefits of a dietary intervention with Corinthian currants, a rich source of phenolic compounds, on type 1 diabetes (T1D) using the animal model of the streptozotocin-(STZ)-induced diabetic rat. Male Wistar rats were randomly assigned into four groups: control animals, which received a control diet (CD) or a diet supplemented with 10% w/w Corinthian currants (CCD), and diabetic animals, which received a control diet (DCD) or a currant diet (DCCD) for 4 weeks. Plasma biochemical parameters, insulin, polar phenolic compounds, and inflammatory factors were determined. Microbiota populations in tissue and intestinal fluid of the caecum, as well as fecal microbiota populations and short-chain fatty acids (SCFAs), were measured. Fecal microbiota was further analyzed by 16S rRNA sequencing. The results of the study showed that a Corinthian currant-supplemented diet restored serum polar phenolic compounds and decreased interleukin-1b (IL-1b) (p < 0.05) both in control and diabetic animals. Increased caecal lactobacilli counts (p < 0.05) and maintenance of enterococci levels within normal range were observed in the intestinal fluid of the DCCD group (p < 0.05 compared to DCD). Higher acetic acid levels were detected in the feces of diabetic rats that received the currant diet compared to the animals that received the control diet (p < 0.05). Corinthian currant could serve as a beneficial dietary component in the condition of T1D based on the results coming from the animal model of the STZ-induced T1D rat.

Free and bound phenols from Cymbopogon citratus mitigated hepatocellular injury in streptozotocin-induced type 1 diabetic male rats via decrease in oxidative stress, inflammation, and other risk markers

Background/AimDiabetes mellitus is a debilitating disease still prevailing in the developing world. This study aimed to examine the protective potentials of Cymbopogon citratus extracts (crude (CCC), free (CFP), and bound phenol (CBP) fractions), in streptozotocin (STZ)-induced type 1 diabetic male rats. MethodsForty-five (45) animals of about 3–4 months were used for the study. About forty animals were induced with STZ. After induction and confirmation of diabetes, animals were grouped (five animals per group) into nine groups; group 1 (Control), group 2 received 50 mg/kg body weight (bw) (STZ). Groups 3, 4 and 5 were healthy animals and received C. citratus extracts (200 mg/kg bw CCC, 100 mg/kg bw CFP, and 100 mg/kg bw CBP) respectively; while groups 6 – 9 were diabetic group (induced with STZ) and later treated with C. citratus extracts (200 mg/kg bw CCC, 100 mg/kg bw CFP, 100 mg/kg bw CBP, and metformin (200 mg/kg bw) respectively for 14 days. Then, the animals were euthanized, blood/serum was collected, liver samples were collected and divided into two; for biochemical and histopathological studies. Liver biomarkers (ALT, AST, ALP, GGT), fructose-1,6-bisphosphatase, hexokinase, antioxidant enzymes (SOD, CAT, and GST) activities, albumin, bilirubin, hepatic glucose, GSH, MDA, pro-and anti-inflammatory cytokines concentrations, were estimated using standard protocols. ResultsOur results revealed that C. citratus extracts counteracted the deleterious effects of type 1 diabetes induction with STZ by optimizing the liver biomarkers, antioxidants enzymes, MDA and GSH levels, reducing the levels of pro-inflammatory cytokines and increasing anti-inflammatory. ConclusionOur results correlated with previous researches; the free phenols fraction revealed brilliant results above others. These suggested that C. citratus could serve as an alternative therapy in ameliorating liver complications linked to oxidative damage and inflammation in STZ-induced type-1 diabetes.

Ginseng-derived panaxadiol ameliorates STZ-induced type 1 diabetes through inhibiting RORγ/IL-17A axis.

Retinoic-acid-receptor-related orphan receptor γ (RORγ) is a major transcription factor for proinflammatory IL-17A production. Here, we revealed that the RORγ deficiency protects mice from STZ-induced Type 1 diabetes (T1D) through inhibiting IL-17A production, leading to improved pancreatic islet β cell function, thereby uncovering a potential novel therapeutic target for treating T1D. We further identified a novel RORγ inverse agonist, ginseng-derived panaxadiol, which selectively inhibits RORγ transcriptional activity with a distinct cofactor recruitment profile from known RORγ ligands. Structural and functional studies of receptor-ligand interactions reveal the molecular basis for a unique binding mode for panaxadiol in the RORγ ligand-binding pocket. Despite its inverse agonist activity, panaxadiol induced the C-terminal AF-2 helix of RORγ to adopt a canonical active conformation. Interestingly, panaxadiol ameliorates mice from STZ-induced T1D through inhibiting IL-17A production in a RORγ-dependent manner. This study demonstrates a novel regulatory function of RORγ with linkage of the IL-17A pathway in pancreatic β cells, and provides a valuable molecule for further investigating RORγ functions in treating T1D.

Justicia carnea extracts ameliorated hepatocellular damage in streptozotocin-induced type 1 diabetic male rats via decrease in oxidative stress, inflammation and increasing other risk markers

Introduction Diabetes mellitus is still a raging disease not fully subdued globally, especially in Africa. Our study aims to evaluate the anti-diabetic potentials of Justicia carnea extracts [crude (JCC), free (JFP) and bound phenol (JBP) fractions], in streptozotocin (STZ)-induced type-1 diabetes in male albino rats. Materials and Methods About thirty (30) animals were induced for type 1 diabetes with STZ; thereafter, treatment began for 14 days, after which the animals were euthanized, blood/serum was collected, the liver was removed and divided into two portions, for biochemical and histopathological analyses. Standard procedures were used to evaluate the liver biomarkers, like alanine transaminase (ALT), fructose-1,6-bisphosphatase, glucose-6- phosphatase, hexokinase activities, albumin, bilirubin, hepatic glucose concentrations; antioxidant status and pro- and anti-inflammatory cytokines were similarly assessed. Results These results revealed that the extracts ameliorated the harmful effects of STZ-induced diabetes in the liver by enhancing the activities of liver-based biomarkers, reducing the concentrations of pro-inflammatory cytokines and increasing the anti-inflammatory cytokine. Discussion The results agreed with previous research, and the free phenol fraction showed excellent results compared to others Conclusion These suggested that J. carnea could serve as an alternative remedy in ameliorating liver complications linked to oxidative damage and inflammation in STZ-induced type-1 diabetes.

Pre-transplant Exercise May Improve Glycemic Outcomes After Marginal Islet Mass Transplant in Type 1 Diabetes

Pancreatic islet transplantation (ITx) is a treatment option for restoring glucose balance in some individuals with type 1 diabetes (T1D). However, the need for multiple donors to achieve insulin independence is an obstacle preventing ITx from becoming the standard of care in T1D. Exercise improves insulin sensitivity, increases insulin-independent glucose uptake, shortens recovery from surgery and decreases surgery-related complication risk. We tested the hypothesis that pre-transplant exercise training (RUN, n=7) would improve blood glucose outcomes in an STZ-induced T1D rat model having undergone syngeneic ITx with marginal islet mass (7000 IEQ/kg) compared to similarly-treated sedentary controls (SIT, n=6).

γ-aminobutyric acid modulates α-cell hyperplasia but not β-cell regeneration induced by glucagon receptor antagonism in type 1 diabetic mice.

To investigate whether treatment with γ-aminobutyric acid (GABA) alone or in combination with glucagon receptor (GCGR) monoclonal antibody (mAb) exerted beneficial effects on β-cell mass and α-cell mass, and to explore the origins of the regenerated β-cells in mice with type 1 diabetes (T1D). Streptozotocin (STZ)-induced T1D mice were treated with intraperitoneal injection of GABA (250μg/kg per day) and/or REMD 2.59 (a GCGR mAb, 5mg/kg per week), or IgG dissolved in PBS for 8weeks. Plasma hormone levels and islet cell morphology were evaluated by ELISA and immunofluorescence, respectively. The origins of the regenerated β-cells were analyzed by double-immunostaining, α-cell lineage-tracing and BrdU-tracing studies. After the 8-week treatment, GABA or GCGR mAb alone or in combination ameliorated hyperglycemia in STZ-induced T1D mice. GCGR mAb upregulated plasma insulin level and increased β-cell mass, and GABA appeared to have similar effects in T1D mice. However, combination treatment did not reveal any additive or synergistic effect. Interestingly, the GCGR mAb-induced increment of plasma glucagon level and α-cell mass was attenuated by the combined treatment of GABA. In addition, duct-derived β-cell neogenesis and α-to-β cell conversion but not β-cell proliferation contributed to the increased β-cell mass in T1D mice. These results suggested that GABA attenuated α-cell hyperplasia but did not potentiates β-cell regeneration induced by GCGR mAb in T1D mice. Our findings provide novel insights into a combination treatment strategy for β-cell regeneration in T1D.

Fish Oil Intake During Gestation and Lactation Attenuated STZ-Induced Diabetes in Male Offspring via Activation of Brown Fat and Modulating Oxylipin Profile

ObjectivesFish oil (FO) has been demonstrated to activate brown thermogenesis and attenuate inflammation in the brown adipose tissue (BAT). Previously, we have reported that maternal FO supplementation promotes BAT activity of the weaned mice pups. However, whether maternal FO intake could confer sustainable metabolic benefits to offspring remains uncovered. Therefore, this study aimed to determine the differential impact of maternal FO during pregnancy versus lactation on BAT transcriptome and evaluate the role of bioactive lipid metabolites derived from maternal FO supplementation on the extended metabolic benefits of older pups in the context of type 1 diabetes (T1D).MethodsBAT samples were collected for RNA sequencing at birth and weaning in the pups with or without maternal FO supplementation. Transcriptomic data were analyzed for profiling of differential expression genes (DEGs) and pathway enrichment analysis. The separate set of 8-week-old male pups with or without maternal FO were injected with streptozotocin (STZ) to induce type 1 diabetes (T1D). In these animals, glucose tolerance, insulin secretion, and plasma levels of oxylipins were assessed. The expression levels of thermogenesis-related genes, inflammatory markers, and mitochondrial respiratory chain complexes were measured in the BATs.ResultsThe RNA-seq analysis revealed that FO intake throughout pregnancy and lactation, but not gestation only, significantly altered BAT transcriptome by upregulating glucose and lipid metabolism pathways and downregulating apoptosis-related pathways. The n-6/n-3 ratio maintained lowered in the 8-week-old pups with maternal fish oil. Consequently, the pups with maternal FO 1) provided the resistance to STZ-induced T1D, 2) promoted thermogenesis-related gene expression and mitochondrial respiratory protein complex in the BAT, 3) reduced proinflammatory oxylipin production compared with the control mice.ConclusionsOur results suggested that maternal FO intake in pregnancy and lactation, at least partly, protects against the risk of T1D of the offspring through augmented BAT function and anti-inflammatory oxylipin production.Funding SourcesNational Institute of Health (NIH), Project #7R21HD094273-02.

Specific Expression of KCC2 in the α Cells of Normal and Type 1 Diabetes Model Mouse Pancreatic Islets.

Gamma-aminobutyric acid (GABA) is an inhibitory neurotransmitter in the mature brain; however, it acts excitatory during development. This difference in action depends on the intracellular chloride ion concentration, primarily regulated by potassium chloride co-transporter2 (KCC2). Sufficient KCC2 expression results in its inhibitory action. GABA is also abundant in pancreatic islets, where it acts differentially on the islet cells, and is involved in carbohydrate metabolism. However, the mechanisms underlying the differential action remain unknown. We performed immunohistochemistry for glutamic acid decarboxylase (GAD), a synthetic enzyme for GABA, and KCC2 in normal adult islets. GAD was co-localized with insulin in β cells, whereas KCC2 was expressed in glucagon-positive α cells. These results are in line with previous observations that GABA decreases glucagon release but increases insulin release, and suggest that GABA and insulin may work together in reducing blood glucose levels under hyperglycemia. Next, we examined the streptozotocin-induced type1 diabetes mellitus mouse model. GAD and insulin expression levels were markedly decreased. KCC2 was expressed in glucagon-positive cells, whereas insulin- and somatostatin-positive cells were KCC2-negative. These findings suggest that in diabetes model, reduced GABA release may cause disinhibition of glucagon release, resulting in increased blood sugar levels and the maintenance of hyperglycemic state.

Therapeutic effects of menstrual blood-derived endometrial stem cells on mouse models of streptozotocin-induced type 1 diabetes.

BACKGROUNDType 1 diabetes (T1D), a chronic metabolic and autoimmune disease, seriously endangers human health. In recent years, mesenchymal stem cell (MSC) transplantation has become an effective treatment for diabetes. Menstrual blood-derived endometrial stem cells (MenSC), a novel MSC type derived from the decidual endometrium during menstruation, are expected to become promising seeding cells for diabetes treatment because of their noninvasive collection procedure, high proliferation rate and high immunomodulation capacity.AIMTo comprehensively compare the effects of MenSC and umbilical cord-derived MSC (UcMSC) transplantation on T1D treatment, to further explore the potential mechanism of MSC-based therapies in T1D, and to provide support for the clinical application of MSC in diabetes treatment.METHODSA conventional streptozotocin-induced T1D mouse model was established, and the effects of MenSC and UcMSC transplantation on their blood glucose and serum insulin levels were detected. The morphological and functional changes in the pancreas, liver, kidney, and spleen were analyzed by routine histological and immunohistochemical examinations. Changes in the serum cytokine levels in the model mice were assessed by protein arrays. The expression of target proteins related to pancreatic regeneration and apoptosis was examined by western blot.RESULTSMenSC and UcMSC transplantation significantly improved the blood glucose and serum insulin levels in T1D model mice. Immunofluorescence analysis revealed that the numbers of insulin+ and CD31+ cells in the pancreas were significantly increased in MSC-treated mice compared with control mice. Subsequent western blot analysis also showed that vascular endothelial growth factor (VEGF), Bcl2, Bcl-xL and Proliferating cell nuclear antigen in pancreatic tissue was significantly upregulated in MSC-treated mice compared with control mice. Additionally, protein arrays indicated that MenSC and UcMSC transplantation significantly downregulated the serum levels of interferon γ and tumor necrosis factor α and upregulated the serum levels of interleukin-6 and VEGF in the model mice. Additionally, histological and immunohistochemical analyses revealed that MSC transplantation systematically improved the morphologies and functions of the liver, kidney, and spleen in T1D model mice.CONCLUSIONMenSC transplantation significantly improves the symptoms in T1D model mice and exerts protective effects on their main organs. Moreover, MSC-mediated angiogenesis, antiapoptotic effects and immunomodulation likely contribute to the above improvements. Thus, MenSC are expected to become promising seeding cells for clinical diabetes treatment due to their advantages mentioned above.

POS-369 CHARACTERIZATION OF A NOVEL MOUSE MODEL OF METABOLIC SYNDROME INDUCED-CHRONIC KIDNEY DISEASE

Chronic kidney disease (CKD) induced in the context of metabolic syndrome (MS) involves underlying hypertension, hyperglycemia, excess body fat and dyslipidemia. A previously generated renin-dependent hypertensive/type-1 diabetic mouse model develops several signs associated human DKD, however the extent and impact of dyslipidemia in this model remains unknown. We hypothesized that combining a high-fat diet (HFD) regimen onto the hypertensive/ diabetic phenotype would mimic features of MS induced-CKD in mice. To this end, 8-week old male genetically hypertensive mice (Lin+) were subjected to streptozotocin (STZ) injections (i.p., 50 mg/kg, 5 days consecutive) to induce hyperglycemia. Four-week later hypertensive/ diabetic mice (LinSTZ) were fed a HFD for 8 weeks. Blood pressure, body weight and blood glucose (BG) were measured throughout the study, while urine albumin levels and histological assessment were performed at endpoint. A HFD-regimen led to significant weight gain and increased circulating triglycerides in WT and to lesser extent LinSTZ mice. Throughout the study, HFD-feeding seemed to reduce systolic blood pressure in LinSTZ mice, while BG remained elevated. Surprisingly, HFD-fed LinSTZ mice developed less glomerular hypertrophy, scarring and albuminuria at endpoint than regular-diet fed littermates. In contrast, histological assessment of liver injury revealed that both WT+HFD and LinSTZ mice developed steatosis (fat accumulation) lesions, while HFD-feeding in LinSTZ mice led to pronounced hepatocyte ballooning, liver inflammation and fibrosis. Moreover, circulating albumin levels, as a surrogate for liver function, were significantly albeit similarly reduced in both vehicle and HFD-fed LinSTZ mice. Taken together, our results show that HFD-feeding in LinSTZ did not lead to a more robust model of MS-induced CKD. In fact, several indices of renal injury were reduced by feeding LinSTZ mice a HFD. The combination of renin-driven hypertension and STZ-induced type-1 diabetes did however accelerate the development of liver injury in the context of MS. Subsequent studies will be undertaken to determine whether feeding LinSTZ a HFD may represent a novel model of accelerated non-alcoholic fatty liver disease.

Abstract 16563: FOXO1 Induces Cardiomyocyte-KLF5, Which Drives Oxidative Stress and Diabetic Cardiomyopathy

Introduction: Cardiomyopathy in type 1 diabetes (T1D) is accompanied by impaired mitochondrial function, oxidative stress and lipotoxicity. We showed that cardiomyocyte (CM) Krüppel-like factor 5 (KLF5) is increased in streptozotocin-induced T1D and induces Peroxisome Proliferator Activated Receptor (PPAR)α in mice. Hypothesis: KLF5 upregulation by FOXO1 induces diabetic cardiomyopathy (DbCM). Methods and Results: Analyses in CM from diabetic patients showed higher KLF5 mRNA levels compared to non-diabetic individuals. In vitro mechanistic and in vivo analyses in αMHC- Foxo1 -/- mice revealed that FOXO1 stimulates KLF5 expression via direct promoter binding. Genetic inhibition of CM FOXO1 alleviated DbCM. Additionally, AAV-mediated CM-specific KLF5 overexpression in C57Bl/6 (WT) mice induced cardiac dysfunction. Mice with CM-specific KLF5 constitutive expression (αMHC-rtTA- Klf5 ), which we generated, recapitulated cardiomyopathy without T1D. Moreover, Pparα -/- mice with T1D, had higher CM-KLF5 levels and developed DbCM, suggesting that KLF5-driven DbCM is PPARα-independent. Additionally, CM-KLF5 induced oxidative stress through increased NADPH oxidase (NOX)4 expression and lower mitochondria abundance. Conversely, KLF5 inhibition prevented NOX4 upregulation and superoxide formation. Furthermore, CM-KLF5 promoted NOX4 expression via direct promoter binding. Antioxidant treatment in diabetic WT and αMHC-rtTA- Klf5 mice alleviated cardiac dysfunction partially, suggesting other pathways that contribute in KLF5-induced DbCM. For that, we performed cardiac lipidome analysis where we found clustering of αMHC-rtTA- Klf5 with diabetic WT mice. Of note, KLF5 inhibition in diabetic mice resulted in similar lipidome with non-diabetic WT mice. Individual lipid species analysis showed increased ceramide accumulation in diabetic WT and αMHC-rtTA- Klf5 mice that was reversed upon KLF5 inhibition. Thus, CM-KLF5 activation correlates with cardiac ceramide accumulation, that has been associated with cardiac lipotoxicity. Conclusions: In conclusion, T1D stimulates FOXO1, which induces CM-KLF5 expression that leads to oxidative stress and DbCM in a non-PPARα-dependent manner, as well as to cardiac ceramide accumulation.

Differential Effects of Empagliflozin on Microvascular Complications in Murine Models of Type 1 and Type 2 Diabetes.

Simple SummaryType 1 and type 2 diabetes can lead to serious health problems that affect many organs including the nerve, the eye, and the kidney. These health problems can be disabling or even life-threatening, and to date, there are no effective therapies that can slow or prevent their development. We studied the effect of empagliflozin, a medication that reduces blood sugar levels and the risk of cardiovascular disease, on the nerve, eye, and kidney in a side-by-side comparison of type 1 and type 2 mouse models. Empagliflozin had no beneficial effects on disease progression in type 2 diabetic mice, but improved nerve function in the type 1 diabetic model, an effect that was accompanied by reduced markers of oxidative injury. These findings support the concept that different mechanisms contribute to nerve damage in type 1 and type 2 diabetes. Our results further support our view that the concept of one therapy will benefit all complications should be abandoned as we pursue more personalized, tissue-specific, diabetes type-specific treatments.Microvascular complications account for the significant morbidity associated with diabetes. Despite tight glycemic control, disease risk remains especially in type 2 diabetes (T2D) patients and no therapy fully prevents nerve, retinal, or renal damage in type 1 diabetes (T1D) or T2D. Therefore, new antidiabetic drug classes are being evaluated for the treatment of microvascular complications. We investigated the effect of empagliflozin (EMPA), an inhibitor of the sodium/glucose cotransporter 2 (SGLT2), on diabetic neuropathy (DPN), retinopathy (DR), and kidney disease (DKD) in streptozotocin-induced T1D and db/db T2D mouse models. EMPA lowered blood glycemia in T1D and T2D models. However, it did not ameliorate any microvascular complications in the T2D model, which was unexpected, given the protective effect of SGLT2 inhibitors on DKD progression in T2D subjects. Although EMPA did not improve DKD in the T1D model, it had a potential modest effect on DR measures and favorably impacted DPN as well as systemic oxidative stress. These results support the concept that glucose-centric treatments are more effective for DPN in T1D versus T2D. This is the first study that provides an evaluation of EMPA treatment on all microvascular complications in a side-by-side comparison in T1D and T2D models.

Recombinant ling zhi-8 enhances Tregs function to restore glycemic control in streptozocin-induced diabetic rats.

To explore the effect of recombinant LZ-8 (rLZ-8) on streptozocin (STZ)-induced diabetic rats and further illustrate its underlying mechanism. Rats were intraperitoneally injected with single-dose STZ 50mg/kg for induction of type 1 diabetes (T1D), and then, the diabetic rats were treated with rLZ-8 for 3months. The clinical symptoms, fasting blood glucose, insulin, cytokines, histopathology, flow cytometry and immunofluorescence were used to evaluate the therapeutic effect and underlying mechanism of rLZ-8 on alleviating diabetes mellitus (DM). Treatment with rLZ-8 obviously alleviated the clinical symptoms of T1D and dose-dependently reduced the levels of blood glucose, blood lipid and haemoglobin A1c (HbA1c) in diabetic rat model. Meanwhile, rLZ-8 markedly increased insulin secretion and protected against STZ-induced pancreatic tissue injury. Additionally, rLZ-8 dramatically inhibited the levels of TNF-α and IL-1β, and obviously increased the level of IL-10 in serum and pancreas. Further investigation indicated that rLZ-8 treatment significantly increased the number of regulatory T cells (Tregs) and up-regulated the expression of Foxp3 to restore balance between anti-inflammatory and inflammatory cytokines. These data suggest that rLZ-8 can antagonize STZ-induced T1D, and its mechanism may be related to inhibit inflammation and enhance Tregs generation.

Transient gain of function of cannabinoid CB1 receptors in the control of frontocortical glucose consumption in a rat model of Type-1 diabetes

Here we aimed to unify some previous controversial reports on changes in both cannabinoid CB1 receptor (CB1R) expression and glucose metabolism in the forebrain of rodent models of diabetes. We determined how glucose metabolism and its modulation by CB1R ligands evolve in the frontal cortex of young adult male Wistar rats, in the first 8 weeks of streptozotocin-induced type-1 diabetes (T1D). We report that frontocortical CB1R protein density was biphasically altered in the first month of T1D, which was accompanied with a reduction of resting glucose uptake ex vivo in acute frontocortical slices that was normalized after eight weeks in T1D. This early reduction of glucose uptake in slices was also restored by ex vivo treatment with both the non-selective CB1R agonists, WIN55212−2 (500 nM) and the CB1R-selective agonist, ACEA (3 μM) while it was exacerbated by the CB1R-selective antagonist, O-2050 (500 nM). These results suggest a gain-of-function for the cerebrocortical CB1Rs in the control of glucose uptake in diabetes. Although insulin and IGF-1 receptor protein densities remained unaffected, phosphorylated GSKα and GSKβ levels showed different profiles 2 and 8 weeks after T1D induction in the frontal cortex. Altogether, the biphasic response in frontocortical CB1R density within a month after T1D induction resolves previous controversial reports on forebrain CB1R levels in T1D rodent models. Furthermore, this study also hints that cannabinoids may be useful to alleviate impaired glucoregulation in the diabetic cortex.

The DNA Sensor AIM2 Protects against Streptozotocin-Induced Type 1 Diabetes by Regulating Intestinal Homeostasis via the IL-18 Pathway.

Pattern recognition receptors (PRRs), such as Nod2, Nlrp3, Tlr2, Trl4, and Tlr9, are directly involved in type 1 diabetes (T1D) susceptibility. However, the role of the cytosolic DNA sensor, AIM2, in T1D pathogenesis is still unknown. Here, we demonstrate that C57BL/6 mice lacking AIM2 (AIM2−/−) are prone to streptozotocin (STZ)-induced T1D, compared to WT C57BL/6 mice. The AIM2−/− mice phenotype is associated with a greater proinflammatory response in pancreatic tissues, alterations in gut microbiota and bacterial translocation to pancreatic lymph nodes (PLNs). These alterations are related to an increased intestinal permeability mediated by tight-junction disruption. Notably, AIM2−/− mice treated with broad-spectrum antibiotics (ABX) are protected from STZ-induced T1D and display a lower pancreatic proinflammatory response. Mechanistically, the AIM2 inflammasome is activated in vivo, leading to an IL-18 release in the ileum at 15 days after an STZ injection. IL-18 favors RegIIIγ production, thus mitigating gut microbiota alterations and reinforcing the intestinal barrier function. Together, our findings show a regulatory role of AIM2, mediated by IL-18, in shaping gut microbiota and reducing bacterial translocation and proinflammatory response against insulin-producing β cells, which ultimately results in protection against T1D onset in an STZ-induced diabetes model.