Leptin Receptor-deficient Mice Research Articles

Sex-specific effect of AQP9 deficiency on hepatic triglyceride metabolism in mice with diet-induced obesity.

Studies in obese rats and human cell models of non-alcoholic fatty liver disease have indicated that knockdown of the hepatic glycerol channel aquaporin 9 (AQP9) leads to decreased hepatic steatosis. However, a study in leptin receptor-deficient mice did not find that knockout (KO) of AQP9 alleviated hepatic steatosis. The aim of this study was to investigate the effect of high-fat diet (HFD) on hepatic glycerol and triglyceride metabolism in male and female AQP9 KO mice. Male and female AQP9 KO mice and wild-type (WT) littermates were fed a HFD for 12weeks. Weight, food intake and blood glucose were monitored throughout the study and tissue analysis included determination of hepatic triglyceride content and triglyceride secretion. The expression of key molecules for hepatic glycerol and triglyceride metabolism was evaluated using qPCR and western blotting. AQP9 KO and WT mice demonstrated a similar weight gain throughout the study period, and we found no evidence for AQP9 deficiency being associated with a reduced hepatic accumulation of triglyceride or a reduced blood glucose level. Instead, we show that the effect of AQP9 deficiency on hepatic lipid metabolism is sex-specific, with only male AQP9 KO mice having a reduced hepatic secretion of triglycerides and an elevated expression of peroxisome proliferator-activated receptor α. Male AQP9 KO mice had an elevated blood glucose level after 12weeks of HFD when compared to baseline levels. Thus, we found no evidence for AQP9 inhibition being a target for alleviating the development of hepatic steatosis in mice with diet-induced obesity. KEY POINTS: This study investigates the effect of AQP9 deficiency on hepatic triglyceride metabolism in both male and female mice fed a high-fat diet (HFD) for 12weeks. No evidence was found for AQP9 deficiency being associated with a reduced hepatic accumulation of triglyceride or a reduced blood glucose level. The effect of AQP9 deficiency on hepatic triglyceride metabolism is sex-specific. Male AQP9 KO mice had a reduced hepatic secretion of triglycerides and an elevated expression of peroxisome proliferator-activated receptor α, which likely promotes an increased hepatic fatty acid oxidation. Male AQP9 KO had an elevated blood glucose level after 12weeks of HFD when compared to baseline levels.

Synchronized proinsulin trafficking reveals delayed Golgi export accompanies β-cell secretory dysfunction in rodent models of hyperglycemia

The pancreatic islet β-cell’s preference for release of newly synthesized insulin requires careful coordination of insulin exocytosis with sufficient insulin granule production to ensure that insulin stores exceed peripheral demands for glucose homeostasis. Thus, the cellular mechanisms regulating insulin granule production are critical to maintaining β-cell function. In this report, we utilized the synchronous protein trafficking system, RUSH, in primary β-cells to evaluate proinsulin transit through the secretory pathway leading to insulin granule formation. We demonstrate that the trafficking, processing, and secretion of the proinsulin RUSH reporter, proCpepRUSH, are consistent with current models of insulin maturation and release. Using both a rodent dietary and genetic model of hyperglycemia and β-cell dysfunction, we show that proinsulin trafficking is impeded at the Golgi and coincides with the decreased appearance of nascent insulin granules at the plasma membrane. Ultrastructural analysis of β-cells from diabetic leptin receptor deficient mice revealed gross morphological changes in Golgi structure, including shortened and swollen cisternae, and partial Golgi vesiculation, which are consistent with defects in secretory protein export. Collectively, this work highlights the utility of the proCpepRUSH reporter in studying proinsulin trafficking dynamics and suggests that altered Golgi export function contributes to β-cell secretory defects in the pathogenesis of Type 2 diabetes.

Kcnma1 is involved in mitochondrial homeostasis in diabetes-related skeletal muscle atrophy.

Uncontrolled diabetes causes a catabolic state with multi-organic complications, of which impairment on skeletal muscle contributes to the damaged mobility. Kcnma1 gene encodes the pore-forming α-subunit of Ca2+ - and voltage-gated K+ channels of large conductance (BK channels), and loss-of-function mutations in Kcnma1 are in regards to impaired myogenesis. Herein, we observed a time-course reduction of Kcnma1 expression in the tibialis anterior muscles of leptin receptor-deficient (db/db) diabetic mice. To investigate the role of Kcnma1 in diabetic muscle atrophy, muscle-specific knockdown of Kcnma1 was achieved by mice receiving intravenous injection of adeno-associated virus-9 (AAV9)-encoding shRNA against Kcnma1 under the muscle creatine kinase (MCK) promoter. Impairment on muscle mass and myogenesis were observed in m/m mice with AAV9-shKcnma1 intervention, while this impairment was more obvious in diabetic db/db mice. Simultaneously, damaged mitochondrial dynamics and biogenesis showed much severer in db/db mice with AAV9-shKcnma1 intervention. RNA sequencing revealed the large transcriptomic changes resulted by Kcnma1 knockdown, and changes in mitochondrial homeostasis-related genes were validated. Besides, the artificial alteration of Kcnma1 in mouse C2C12 myoblasts was achieved with an adenovirus vector. Consistent results were demonstrated by Kcnma1 knockdown in palmitate-treated cells, whereas opposite results were exhibited by Kcnma1 overexpression. Collectively, we document Kcnma1 as a potential keeper of mitochondrial homeostasis, and the loss of Kcnma1 is a critical event in priming skeletal muscle loss in diabetes.

Zinc finger transcription factor Egf1 promotes non-alcoholic fatty liver disease

Background & AimsNon-alcoholic fatty liver disease (NAFLD) contributes to the global epidemic of metabolic syndrome and is considered a prelude to end-stage liver diseases such as cirrhosis and hepatocellular carcinoma. During NAFLD pathogenesis, hepatic parenchymal cells (hepatocytes) undergo both morphological and functional changes owing to a rewired transcriptome. The underlying mechanism is not entirely clear. In the present study, we investigated the involvement of early growth response 1 (Egr1) in NAFLD. MethodsQuantitative PCR, Western blotting, and histochemical staining were used to assess gene expression levels. Chromatin immunoprecipitation was used to evaluate protein binding to DNA. NAFLD was evaluated in leptin receptor-deficient (db/db) mice. ResultsWe report here that Egr1 was upregulated by pro-NAFLD stimuli in vitro and in vivo. Further analysis revealed that serum response factor (SRF) was recruited to the Egr1 promoter and mediated Egr1 transactivation. Importantly, Egr1 depletion markedly mitigated NAFLD in db/db mice. RNA sequencing revealed that Egr1 knockdown in hepatocytes, on the one hand, boosted fatty acid oxidation (FAO) and, on the other hand, suppressed the synthesis of chemoattractants. Mechanistically, Egr1 interacted with peroxisome proliferator-activated receptor α (PPARα) to repress PPARα-dependent transcription of FAO genes by recruiting its co-repressor NGFI-A binding protein 1 (Nab1), which potentially led to promoter deacetylation of FAO genes. ConclusionsOur data identify Egr1 as a novel modulator of NAFLD and a potential target for NAFLD intervention. Impact and ImplicationsNon-alcoholic fatty liver disease (NAFLD) precedes cirrhosis and hepatocellular carcinoma. In this paper, we describe a novel mechanism whereby early growth response 1 (Egr1), a transcription factor, contributes to NAFLD pathogenesis by regulating fatty acid oxidation. Our data provide novel insights and translational potential for NAFLD intervention.

Cyclopia intermedia (Honeybush) Induces Uncoupling Protein 1 and Peroxisome Proliferator-Activated Receptor Alpha Expression in Obese Diabetic Female db/db Mice.

Previously, we reported that a crude polyphenol-enriched fraction of Cyclopia intermedia (CPEF), a plant consumed as the herbal tea, commonly known as honeybush, reduced lipid content in 3T3-L1 adipocytes and inhibited body weight gain in obese, diabetic female leptin receptor-deficient (db/db) mice. In the current study, the mechanisms underlying decreased body weight gain in db/db mice were further elucidated using western blot analysis and in silico approaches. CPEF induced uncoupling protein 1 (UCP1, 3.4-fold, p < 0.05) and peroxisome proliferator-activated receptor alpha (PPARα, 2.6-fold, p < 0.05) expression in brown adipose tissue. In the liver, CPEF induced PPARα expression (2.2-fold, p < 0.05), which was accompanied by a 31.9% decrease in fat droplets in Hematoxylin and Eosin (H&E)-stained liver sections (p < 0.001). Molecular docking analysis revealed that the CPEF compounds, hesperidin and neoponcirin, had the highest binding affinities for UCP1 and PPARα, respectively. This was validated with stabilising intermolecular interactions within the active sites of UCP1 and PPARα when complexed with these compounds. This study suggests that CPEF may exert its anti-obesity effects by promoting thermogenesis and fatty acid oxidation via inducing UCP1 and PPARα expression, and that hesperidin and neoponcirin may be responsible for these effects. Findings from this study could pave the way for designing target-specific anti-obesity therapeutics from C. intermedia.

Inhibitory Potential of α-Amylase, α-Glucosidase, and Pancreatic Lipase by a Formulation of Five Plant Extracts: TOTUM-63.

Controlling post-prandial hyperglycemia and hyperlipidemia, particularly by regulating the activity of digestive enzymes, allows managing type 2 diabetes and obesity. The aim of this study was to assess the effects of TOTUM-63, a formulation of five plant extracts (Olea europaea L., Cynara scolymus L., Chrysanthellum indicum subsp. afroamericanum B.L.Turner, Vaccinium myrtillus L., and Piper nigrum L.), on enzymes involved in carbohydrate and lipid absorption. First, in vitro inhibition assays were performed by targeting three enzymes: α-glucosidase, α-amylase, and lipase. Then, kinetic studies and binding affinity determinations by fluorescence spectrum changes and microscale thermophoresis were performed. The in vitro assays showed that TOTUM-63 inhibited all three digestive enzymes, particularly α-glucosidase (IC50 of 13.1 µg/mL). Mechanistic studies on α-glucosidase inhibition by TOTUM-63 and molecular interaction experiments indicated a mixed (full) inhibition mechanism, and higher affinity for α-glucosidase than acarbose, the reference α-glucosidase inhibitor. Lastly, in vivo data using leptin receptor-deficient (db/db) mice, a model of obesity and type 2 diabetes, indicated that TOTUM-63 might prevent the increase in fasting glycemia and glycated hemoglobin (HbA1c) levels over time, compared with the untreated group. These results show that TOTUM-63 is a promising new approach for type 2 diabetes management via α-glucosidase inhibition.

Modulation of Lymphangiogenesis in Incisional Murine Diabetic Wound Healing Using Negative Pressure Wound Therapy.

Objective: Despite the significant function of lymphatics in wound healing, and frequent clinical use of Negative Pressure Wound Therapy (NPWT), the effect of mechanical force application on lymphangiogenesis remains to be elucidated. We utilize a murine incisional wound healing model to assess the mechanisms of lymphangiogenesis following NPWT. Approach: Dorsal incisional skin wounds were created on diabetic mice (genetically obese leptin receptor-deficient mice [db/db]; n = 30) and covered with an occlusive dressing (Control, n = 15) or NPWT (-125 mmHg, continuous, 24 h for 7 days; NPWT, n = 15). The wounds were macroscopically assessed for 28 days. Tissue was harvested on day 10 for analysis. Qualitative functional analysis of lymphatic drainage was performed on day 28 using Evans Blue staining (n = 2). Results: NPWT increased lymphatic vessel density (40 ± 20 vs. 12 ± 6 podoplanin [PDPN]+ and 25 ± 9 vs. 14 ± 8 lymphatic vessel endothelial receptor 1 [LYVE-1]+) and vessel diameter (28 ± 9 vs. 12 ± 2 μm). Western blotting verified the upregulation of LYVE-1 with NPWT. Leukocyte presence was higher with NPWT (22% ± 3.7% vs. 9.1% ± 4.1% lymphocyte common antigen [CD45]+) and the leukocytes were predominately B cells clustered within vessels (8.8% ± 2.5% vs. 18% ± 3.6% B-lymphocyte antigen CD20 [CD20]+). Macrophage presence was lower in the NPWT group. Lymphatic drainage was increased in the NPWT group, which exhibited greater Evans Blue positivity. Innovation: The lymphangiogenic effects take place independent of macrophage infiltration, appearing to correlate with B cell presence. Conclusion: NPWT promotes lymphangiogenesis in incisional wounds, significantly increasing the lymph vessel density and diameter. This study highlights the potential of NPWT to stimulate lymphatic drainage and wound healing of surgical incisions.

Effects of continuous thiamine intake on onset and progression of type 2 diabetes in leptin-receptor deficient mice

Aside from the COVID-19 pandemic, the obesity and diabetes pandemics have threatened global health. Patients with diabetes are more likely to experience serious complications from COVID-19; thus, preventing obesity-associated diabetes is of paramount important. Furthermore, the development of a method to prevent diabetes and elucidation of its pathology is a currently urgent issue. We previously reported that thiamine plays a key role in suppressing abnormal glycolipid metabolism in Otsuka Long-Evans Tokushima fatty (OLETF) rats, an animal model of obesity-associated diabetes. However, whether thiamine affects only OLETF rats or other animal models including a type 2 diabetes model with a different pathology requires elucidation. In this study, leptin-receptor deficiency mice were used as a model of type 2 diabetes with a different pathology to evaluate the efficacy of thiamine. The mice had free access to water containing 0.2% thiamine for 9 weeks, and the results showed that food and water consumption decreased in db/db-homo mice. Urine output, body weight gains and blood glucose levels decreased in mice that received thiamine. There were 5 mice and 1 mouse with a fasting glucose level of ≥ 300 mg/dL in the db/db-homo control group (n = 10) and db/db-homo thiamine group (n = 10), respectively, suggesting that thiamine intake may suppress an increase in blood glucose levels. The results of the present study suggest that demand for thiamine may exceed the normal range in in vivo mouse models of diabetes and continuous thiamine intake affects diabetes onset and progression.

Positive feedback loop of miR-320 and CD36 regulates the hyperglycemic memory-induced diabetic diastolic cardiac dysfunction

Intensive glycemic control is insufficient for reducing the risk of heart failure among patients with diabetes mellitus (DM). While the "hyperglycemic memory" phenomenon is well documented, little is known about its underlying mechanisms. In this study, a type 1 DM model was established in C57BL/6 mice using streptozotocin (STZ). Leptin receptor-deficient (db/db) mice were used as a model of type 2 DM. A type 9 adeno-associated virus was used to overexpress or knock down miR-320 invivo. Diastolic dysfunction was observed in the type 1 DM mice with elevated miR-320 expression. However, glycemic control using insulin failed to reverse diastolic dysfunction. miR-320 knockdown protected against STZ-induced diastolic dysfunction. Similar results were observed in the type 2 DM mice. Invitro, we found that miR-320 promoted CD36 expression, which in turn induced further miR-320 expression. CD36 was rapidly induced by hyperglycemia at protein level compared with the much slower induction of miR-320, suggesting a positive feedback loop of CD36/miR-320 with CD36 protein induction as the initial triggering event. In conclusion, in DM-induced cardiac injury, miR-320 and CD36 mutually enhance each other's expression, leading to a positive feedback loop and a sustained hyperlipidemic state in the heart.

Praliciguat Promotes Ischemic Leg Reperfusion in Leptin Receptor-Deficient Mice.

Lower-limb peripheral artery disease is one of the major complications of diabetes. Peripheral artery disease is associated with poor limb and cardiovascular prognoses, along with a dramatic decrease in life expectancy. Despite major medical advances in the treatment of diabetes, a substantial therapeutic gap remains in the peripheral artery disease population. Praliciguat is an orally available sGC (soluble guanylate cyclase) stimulator that has been reported both preclinically and in early stage clinical trials to have favorable effects in metabolic and hemodynamic outcomes, suggesting that it may have a potential beneficial effect in peripheral artery disease. We evaluated the effect of praliciguat on hind limb ischemia recovery in a mouse model of type 2 diabetes. Hind limb ischemia was induced in leptin receptor-deficient (Leprdb/db) mice by ligation and excision of the left femoral artery. Praliciguat (10 mg/kg/day) was administered in the diet starting 3 days before surgery. Twenty-eight days after surgery, ischemic foot perfusion and function parameters were better in praliciguat-treated mice than in vehicle controls. Improved ischemic foot perfusion was not associated with either improved traditional cardiovascular risk factors (ie, weight, glycemia) or increased angiogenesis. However, treatment with praliciguat significantly increased arteriole diameter, decreased ICAM1 (intercellular adhesion molecule 1) expression, and prevented the accumulation of oxidative proangiogenic and proinflammatory muscle fibers. While investigating the mechanism underlying the beneficial effects of praliciguat therapy, we found that praliciguat significantly downregulated Myh2 and Cxcl12 mRNA expression in cultured myoblasts and that conditioned medium form praliciguat-treated myoblast decreased ICAM1 mRNA expression in endothelial cells. These results suggest that praliciguat therapy may decrease ICAM1 expression in endothelial cells by downregulating Cxcl12 in myocytes. Our results demonstrated that praliciguat promotes blood flow recovery in the ischemic muscle of mice with type 2 diabetes, at least in part by increasing arteriole diameter and by downregulating ICAM1 expression.

The natural product rotundic acid treats both aging and obesity by inhibiting PTP1B

AbstractThe occurrence of obesity is associated with age. But their interplay remains mysterious. Here, we discovered that rotundic acid (RA), a plant-derived pentacyclic triterpene, was a powerful agent for both anti-aging and treating obesity. Considering that obese individuals decrease the appetite-suppressing and energy-expenditure-enhancing functions of leptin leading to obesity, we found RA was a leptin sensitizer, evidenced by observations that RA enhanced the leptin sensitivity to normal diet-induced obese (DIO) mice, and had minimal or no use to normal lean mice, leptin receptor-deficient (db/db) mice, and leptin-deficient (ob/ob) mice. Simultaneously, RA significantly increased energy expenditure, BAT thermogenesis, and glucose metabolism in DIO mice, as the results of enhancing leptin sensitivity. Regarding mode of action, we demonstrated that RA is a noncompetitive inhibitor of leptin negative regulators protein tyrosine phosphatase 1B (PTP1B) and T-cell PTP through interaction with their C-terminus, thus leading to weight loss through enhancing leptin sensitivity. Besides, we showed that deletion of yPTP1 in yeast completely abolished the lifespan extension effect of RA, celstrol, and withaferin A, while these compounds exhibited PTP1B inhibition activity. Furthermore, PTP1B knockdown extend lifespan in yeast and human cells, indicating PTP1B is an important factor regulating cellular aging.

Cardioprotective Effects of 1-(3,6-Dibromo-carbazol-9-yl)-3-Phenylamino-Propan-2-Ol in Diabetic Hearts via Nicotinamide Phosphoribosyltransferase Activation.

Diabetes is associated with increased cardiac injury and sudden death. Nicotinamide phosphoribosyltransferase (Nampt) is an essential enzyme for the NAD+ salvage pathway and is dysregulated in diabetes. Nampt activation results in rescued NADH/NAD+ ratios and provides pharmacological changes necessary for diabetic cardioprotection. Computer docking shows that 1-(3,6-Dibromo-carbazol-9-yl)-3-phenylamino-propan-2-ol (P7C3) allows for enhanced Nampt dimerization and association. To test the pharmacological application, we used male leptin receptor-deficient (db/db) mice and treated them with Nampt activator P7C3. The effects of 4-week P7C3 treatment on cardiac function were evaluated along with molecular signaling changes for phosphorylated protein kinase B (p-AKT), phosphorylated endothelial nitric oxide synthase (p-eNOS), and sirtuin 1 (SIRT1). The cardiac function evaluated by ECG and echocardiography were significantly improved after 4 weeks of P7C3 treatment. Biochemically, higher NADH/NAD+ ratios in diabetic hearts were rescued by P7C3 treatment. Moreover, activities of Nampt and SIRT1 were significantly increased in P7C3-treated diabetic hearts. P7C3 treatment significantly decreased the blood glucose in diabetic mice with 4-week treatment as noted by glucose tolerance test and fasting blood glucose measurements compared with vehicle-treated mice. P7C3 activated Nampt enzymatic activity both in vitro and in the 4-week diabetic mouse hearts, demonstrating the specificity of the small molecule. P7C3 treatment significantly enhanced the expression of cardioprotective signaling of p-AKT, p-eNOS, and Beclin 1 in diabetic hearts. Nampt activator P7C3 allows for decreased infarct size with decreased Troponin I and lactose dehydrogenase (LDH) release, which is beneficial to the heart. Overall, the present study shows that P7C3 activates Nampt and SIRT1 activity and decreases NADH/NAD+ ratio, resulting in improved biochemical signaling providing cardioprotection. SIGNIFICANCE STATEMENT: This study shows that 1-(3,6-Dibromo-carbazol-9-yl)-3-phenylamino-propan-2-ol (P7C3) is effective in treating diabetes and cardiovascular diseases. The novel small molecule is antiarrhythmic and improves the ejection fraction in diabetic hearts. The study successfully demonstrated that P7C3 decreases the infarct size in hearts during myocardial infarction and ischemia-reperfusion injury. Biochemical and cellular signaling show increased NAD+ levels, along with Nampt activity involved in upregulating protective signaling in the diabetic heart. P7C3 has high therapeutic potential for rescuing heart disease.

Single-Cell Transcriptomic Atlas of Gingival Mucosa in Type 2 Diabetes

The oral gingival barrier is a constantly stimulated and dynamic environment where homeostasis is often disrupted, resulting in inflammatory periodontal diseases. Type 2 diabetes (T2D) has been reported to be associated with gingival barrier dysfunction, but the effect and underlying mechanism are inconclusive. Herein, we performed single-cell RNA sequencing (scRNA-seq) of gingiva from leptin receptor-deficient mice (db/db) to examine the gingival heterogeneity in the context of T2D. Periodontal health of control mice is characterized by populations of Krt14+-expressing epithelial cells and Col1a1+-fibroblasts mediating immune homeostasis primarily through the enrichment of innate lymphoid cells. The db/db gingiva exhibited decreased epithelial/stromal ratio and dysfunctional barrier. We further observed stromal, particularly fibroblast immune hyperresponsiveness, linked to the recruitment of myeloid-derived cells at the db/db gingiva. Both scRNA-seq and histological analysis suggested the inflammatory signaling between fibroblasts and neutrophils as a potential driver of diabetes-induced periodontal damage. Notably, the “immune-like” stromal cells were wired toward the induction of gingival IL-17A hyperresponsiveness in db/db mice. Our work reveals that the “immune-like” fibroblasts with transcriptional diversity are involved in the innate immune homeostasis at the diabetic gingiva. It highlights a potentially significant role of these cell types in its pathogenesis.

Caveolin-1 Alleviates Diabetes-Associated Cognitive Dysfunction Through Modulating Neuronal Ferroptosis-Mediated Mitochondrial Homeostasis.

Aims: Iron metabolism is involved in many biological processes in the brain. Alterations in iron homeostasis have been associated with several neurodegenerative disorders. Instead of stroke and ischemic heart disease, dementia has become the second leading cause of mortality among the type 2 diabetes mellitus (T2DM) population. Therefore, we attempted to investigate the role of ferroptosis in diabetes-associated cognitive dysfunction (DACD). Results: We evaluated ferroptosis hallmarks in the hippocampus of T2DM (high-fat diet/streptozotocin, HFD/STZ) mice, primary hippocampal neurons, as well as in the blood of patients. The results of Gene Set Enrichment Analysis showed significantly differentially expressed genes related to ferroptosis-related pathways between normal control (db/m) and leptin receptor-deficient (db/db) mice. Here, ferroptosis, mitochondrial dysfunction and cognitive impairment were revealed, and caveolin-1 (cav-1) was significantly downregulated in the hippocampus of T2DM (HFD/STZ) mice. In addition, ferrostatin-1 and cav-1 restoration neutralized ferroptosis-related symbolic changes, mitochondrial dysfunction, and improved cognitive dysfunction. Notably, the plasma levels of Fe2+ and 4-hydroxynonenal (4-HNE) in T2DM patients showed a tendency to increase compared with those in nondiabetic subjects, and the Fe2+ level was negatively correlated with the cognitive ability in T2DM subjects. Innovation: For the first time, this study suggested that ferroptosis promoted the progression of DACD induced by T2DM both in vivo and in vitro, and supported the clinical evidence for the correlation between ferroptosis and T2DM-related DACD, which provided new insights into the potential antioxidant effects of ferroptosis inhibitors and cav-1 on DACD. Conclusions: The overexpression of cav-1 may attenuate DACD by modulating neuronal ferroptosis-mediated mitochondrial homeostasis. We put cav-1 on the spotlight as a promising candidate to prevent DACD. Antioxid. Redox Signal. 37, 867-886.

Human Amniotic Membrane Promotes Angiogenesis in an Oxidative Stress Chronic Diabetic Murine Wound Model.

Objective: The development of animal models, which adequately replicate the pathophysiology of chronic wounds, has been challenging. In this study, we utilized an oxidative stress (OS) murine model, which was previously developed by our group, to study the effect of a human amniotic membrane (AM) on chronic wound healing. Approach: Forty-five diabetic (genetically obese leptin receptor-deficient mice [db/db]) mice were separated into three groups. Thirty mice received an OS regimen and a 1 - × 1 cm2 full-thickness excisional dorsal wound. The wounds were either covered with AM and occlusive dressing (db/dbOS-AM) or occlusive dressing only (db/dbOS). Fifteen mice did not receive the OS regimen, and were covered with AM and occlusive dressing (db/db-AM). The wounds were photographed, and tissue was harvested at various time points. Results: Vascular density was higher in the AM-treated groups (db/dbOS-AM: 34 ± 12; db/db-AM: 37 ± 14; vs. db/dbOS: 19 ± 9 cluster of differentiation 31 [CD31+]/high power field [HPF] photograph; p = 0.04 and p = 0.003). Vessel maturity was lowest in the db/dbOS group (21% ± 4%; vs. db/dbOS-AM: 38% ± 10%, p = 0.004; db/db-AM: 40% ± 11%, p = 0.0005). Leukocyte infiltration was higher in the AM groups (db/dbOS-AM: 15 ± 4; db/db-AM: 16 ± 4 vs. db/dbOS: 8 ± 3 lymphocyte common antigen [CD45+]/HPF; p = 0.005 and p = 0.06). AM upregulated various proangiogenic factors, including vascular endothelial growth factor (VEGF), and downregulated genes involved in chronicity, such as osteopontin, as visualized through proteome analysis and western blotting. Cell death was lower in the AM groups (db/dbOS-AM: 28 ± 10, db/db-AM: 7 ± 5 vs. db/dbOS: 17% ± 9% Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling [TUNEL+]; p = 0.03 and p < 0.0001). Innovation: This study offers new insight on the mechanisms of action of human AM in chronic wound healing. Conclusion: AM treatment promoted healing in mice with complex chronic wounds. The AM stimulated angiogenesis through upregulation of proangiogenic factors, improving the wound milieu by increasing leukocyte and growth factor delivery and decreasing cell death.

Aerobic Exercise Improves Type 2 Diabetes Mellitus-Related Cognitive Impairment by Inhibiting JAK2/STAT3 and Enhancing AMPK/SIRT1 Pathways in Mice.

Type 2 diabetes mellitus (T2DM) is a prevalent risk factor for cognitive impairment. Aerobic exercise can improve T2DM-related cognitive impairment; however, the possible mechanisms remain elusive. Thus, we assessed db/m mice and leptin receptor-deficient (db/db) mice that did or did not perform aerobic exercise (8 m/min, 60 min/day, and 5 days/week for 12 weeks). In this study, cognitive function was significantly impaired in the T2DM mice; aerobic exercise improved cognitive impairment through activating the AMPK/SIRT1 signalling pathway and inhibiting the JAK2/STAT3 signalling pathway in T2DM mice. However, after the application of RO8191 (JAK2 activator) or Compound C (AMPK inhibitor), the positive improvement of the exercise was evidently suppressed. Taken together, our data indicated that long-term aerobic exercise improves type 2 diabetes mellitus-related cognitive impairment by inhibiting JAK2/STAT3 and enhancing AMPK/SIRT1 pathways in mice.

Soluble Epoxide Hydrolase Inhibition Protected against Diabetic Cardiomyopathy through Inducing Autophagy and Reducing Apoptosis Relying on Nrf2 Upregulation and Transcription Activation.

Background Many patients with diabetes die from diabetic cardiomyopathy (DCM); however, effective strategies for the prevention or treatment of DCM have not yet been clarified. Methods Leptin receptor-deficient (db/db) mice were treated with either the soluble epoxide hydrolase (sEH) inhibitor AUDA or vehicle alone. A virus carrying Nrf2 shRNA was used to manipulate Nrf2 expression in db/db mice. Cardiac structures and functions were analyzed using echocardiography and hemodynamic examinations. Primary cardiomyocytes cultured under high glucose and high fat (HGHF) conditions were used to conduct in vitro loss-of-function assays after culture in the presence or absence of AUDA (1 μM). Fluorescence microscopy-based detection of mCherry-GFP-LC3 was performed to assess autophagic flux. Results The sEH inhibitor AUDA significantly attenuated ventricular remodeling and ameliorated cardiac dysfunction in db/db mice. Interestingly, AUDA upregulated Nrf2 expression and promoted its nuclear translocation in db/db mice and the HGHF-treated cardiomyocytes. Additionally, AUDA increased autophagy and decreased apoptosis in db/db mice heart. Furthermore, the administration of AUDA promoted autophagic flux and elevated LC3-II protein level in the presence of bafilomycin A1. However, AUDA-induced autophagy was abolished, and the antiapoptotic effect was partially inhibited upon Nrf2 knockdown. Conclusion Our findings suggest that the sEH inhibitor AUDA attenuates cardiac remodeling and dysfunction in DCM via increasing autophagy and reducing apoptosis, which is relevant to activate Nrf2 signaling pathway.

Dysbiotic oral microbiota contributes to alveolar bone loss associated with obesity in mice.

Periodontal diseases (PD) are inflammatory conditions that affect the teeth supporting tissues. Increased body fat tissues may contribute to activation of the systemic inflammatory response, leading to comorbidities. Some studies have shown that individuals with obesity present higher incidence of PD than eutrophics. To investigate the impact of obesity on periodontal tissues and oral microbiota in mice. Two obesity mice models were performed, one using 12 weeks of the dietary protocol with a high-fat (HF) diet in C57BL/6 mice and the other using leptin receptor-deficient mice (db/db-/-), which became spontaneously obese. After euthanasia, a DNA-DNA hybridization technique was employed to evaluate the microbiota composition and topical application of chlorhexidine (CHX), an antiseptic, was used to investigate the impact of the oral microbiota on the alveolar bone regarding obesity. Increased adipose tissue may induce alveolar bone loss, neutrophil recruitment, and changes in the oral biofilm, similar to that observed in an experimental model of PD. Topical application of CHX impaired bone changes. Obesity may induce changes in the oral microbiota and neutrophil recruitment, which are associated with alveolar bone loss.

Analysis of N6-Methyladenosine Methylation Modification in Fructose-Induced Non-Alcoholic Fatty Liver Disease

Improvements in living standards have led to non-alcoholic fatty liver disease (NAFLD), one of the most common chronic liver diseases worldwide. Recent studies have shown that N6-methyladenosine (m6A), a type of RNA modification, is strongly associated with many important biological processes. However, the relationship between m6A methylation modifications and NAFLD remains poorly understood. In the present study, through methylated RNA immunoprecipitation sequencing and RNA transcriptome sequencing in high fructose diet-induced NAFLD mice, we found that hypermethylation-encoding genes were mainly enriched in lipid metabolism processes. We identified 266 overlapping and differentially expressed genes (DEGs) that changed at both the mRNA expression level and m6A modification level. Among them, 193 genes displayed increased expression and m6A modification, indicating that m6A RNA modifications tend to be positively correlated with NAFLD. We further compared the high fructose diet-induced NAFLD mouse model with leptin receptor-deficient mice and found that DEGs enriched in the lipid metabolism pathway were up-regulated in both groups. In contrast, DEGs associated with the immune inflammatory response were up-regulated in the high fructose diet group, but down-regulated in leptin receptor-deficient mice. Taken together, our results demonstrate that m6A methylation modifications may play an important role in the development of NAFLD.

Estradiol overcomes adiponectin-resistance in diabetic mice by regulating skeletal muscle adiponectin receptor 1 expression

Adiponectin and insulin resistance creates a vicious cycle that exacerbates type 2 diabetes. Earlier, we observed that female leptin receptor-deficient BLKS mice (BKS-db/db) were more sensitive to an adiponectin mimetic GTDF than males, which led us to explore if E2 plays a crucial role in modulation of adiponectin-sensitivity. Male but not female BKS-db/db mice were resistant to metabolic effects of globular adiponectin treatment. Male BKS-db/db displayed reduced skeletal muscle AdipoR1 protein expression, which was consequent to elevated polypyrimidine tract binding protein 1 (PTB) and miR-221. E2 treatment in male BKS-db/db, and ovariectomized BALB/c mice rescued AdipoR1 protein expression via downregulation of PTB and miR-221, and also directly increased AdipoR1 mRNA by its classical nuclear receptors. Estrogen receptor regulation via dietary or pharmacological interventions may improve adiponectin resistance and consequently ameliorate insulin resistance in type 2 diabetes.