Long-Term PDE-5A Inhibition Improves Myofilament Function in Left and Right Ventricular Cardiomyocytes through Partially Different Mechanisms in Diabetic Rat Hearts

Heart failure with preserved ejection fraction (HFpEF) and right ventricular (RV) dysfunction are frequent complications of diabetic cardiomyopathy. Here we aimed to characterize RV and left ventricular (LV) remodeling and its prevention by vardenafil (a long-acting phosphodiesterase-5A (PDE-5A) inhibitor) administration in a diabetic HFpEF model. Zucker Diabetic Fatty (ZDF) and control, ZDF Lean (Lean) male rats received 10 mg/kg vardenafil (ZDF + Vard; Lean + Vard) per os, on a daily basis for a period of 25 weeks. In vitro force measurements, biochemical and histochemical assays were employed to assess cardiomyocyte function and signaling. Vardenafil treatment increased cyclic guanosine monophosphate (cGMP) levels and decreased 3-nitrotyrosine (3-NT) levels in the left and right ventricles of ZDF animals, but not in Lean animals. Cardiomyocyte passive tension (Fpassive) was higher in LV and RV cardiomyocytes of ZDF rats than in those receiving preventive vardenafil treatment. Levels of overall titin phosphorylation did not differ in the four experimental groups. Maximal Ca2+-activated force (Fmax) of LV and RV cardiomyocytes were preserved in ZDF animals. Ca2+-sensitivity of isometric force production (pCa50) was significantly higher in LV (but not in RV) cardiomyocytes of ZDF rats than in their counterparts in the Lean or Lean + Vard groups. In accordance, the phosphorylation levels of cardiac troponin I (cTnI) and myosin binding protein-C (cMyBP-C) were lower in LV (but not in RV) cardiomyocytes of ZDF animals than in their counterparts of the Lean or Lean + Vard groups. Vardenafil treatment normalized pCa50 values in LV cardiomyocytes, and it decreased pCa50 below control levels in RV cardiomyocytes in the ZDF + Vard group. Our data illustrate partially overlapping myofilament protein alterations for LV and RV cardiomyocytes in diabetic rat hearts upon long-term PDE-5A inhibition. While uniform patterns in cGMP, 3-NT and Fpassive levels predict identical effects of vardenafil therapy for the diastolic function in both ventricles, the uneven cTnI, cMyBP-C phosphorylation levels and pCa50 values implicate different responses for the systolic function.

Melatonin Improves Levels of Zn and Cu in the Muscle of Diabetic Obese Rats

Melatonin improves metabolic alterations associated with obesity and its diabetes (diabesity). We intend to determine whether this improvement is exerted by changing Zn and/or Cu tissue levels in liver, muscle, pancreas, and brain, and in internal (perirenal, perigonadal, and omentum) and subcutaneous lumbar white adipose tissues (IWAT and SWAT, respectively). Male Zücker diabetic fatty (ZDF) rats and lean littermates (ZL) were orally supplemented either with melatonin (10 mg/kg body weight/day) or vehicle for 6 weeks. Zn and Cu concentrations were not significantly influenced by diabesity in the analyzed tissues (p > 0.05), with the exception of Zn in liver. In skeletal muscle Zn and Cu, and in perirenal WAT, only Zn levels increased significantly with melatonin supplementation in ZDF rats (p < 0.05). This cytoplasmic Zn enhancement would be probably associated with the upregulation of several Zn influx membrane transporters (Zips) and could explain the amelioration in the glycaemia and insulinaemia by upregulating the Akt and downregulating the inhibitor PTP1B, in obese and diabetic conditions. Enhanced Zn and Cu levels in muscle cells could be related to the reported antioxidant melatonin activity exerted by increasing the Zn, Cu-SOD, and extracellular Cu-SOD activity. In conclusion, melatonin, by increasing the muscle levels of Zn and Cu, joined with our previously reported findings improves glycaemia, insulinaemia, and oxidative stress in this diabesity animal model.

Melatonin Enhances the Mitochondrial Functionality of Brown Adipose Tissue in Obese—Diabetic Rats

Developing novel drugs/targets remains a major effort toward controlling obesity-related type 2 diabetes (diabesity). Melatonin controls obesity and improves glucose homeostasis in rodents, mainly via the thermogenic effects of increasing the amount of brown adipose tissue (BAT) and increases in mitochondrial mass, amount of UCP1 protein, and thermogenic capacity. Importantly, mitochondria are widely known as a therapeutic target of melatonin; however, direct evidence of melatonin on the function of mitochondria from BAT and the mechanistic pathways underlying these effects remains lacking. This study investigated the effects of melatonin on mitochondrial functions in BAT of Zücker diabetic fatty (ZDF) rats, which are considered a model of obesity-related type 2 diabetes mellitus (T2DM). At five weeks of age, Zücker lean (ZL) and ZDF rats were subdivided into two groups, consisting of control and treated with oral melatonin for six weeks. Mitochondria were isolated from BAT of animals from both groups, using subcellular fractionation techniques, followed by measurement of several mitochondrial parameters, including respiratory control ratio (RCR), phosphorylation coefficient (ADP/O ratio), ATP production, level of mitochondrial nitrites, superoxide dismutase activity, and alteration in the mitochondrial permeability transition pore (mPTP). Interestingly, melatonin increased RCR in mitochondria from brown fat of both ZL and ZDF rats through the reduction of the proton leak component of respiration (state 4). In addition, melatonin improved the ADP/O ratio in obese rats and augmented ATP production in lean rats. Further, melatonin reduced mitochondrial nitrosative and oxidative status by decreasing nitrite levels and increasing superoxide dismutase activity in both groups, as well as inhibited mPTP in mitochondria isolated from brown fat. Taken together, the present data revealed that chronic oral administration of melatonin improved mitochondrial respiration in brown adipocytes, while decreasing oxidative and nitrosative stress and susceptibility of adipocytes to apoptosis in ZDF rats, suggesting a beneficial use in the treatment of diabesity. Further research regarding the molecular mechanisms underlying the effects of melatonin on diabesity is warranted.

ZiBuPiYin Recipe Prevented and Treated Cognitive Decline in ZDF Rats With Diabetes-Associated Cognitive Decline via Microbiota–Gut–Brain Axis Dialogue

Gut microbiota is becoming one of the key determinants in human health and disease. Shifts in gut microbiota composition affect cognitive function and provide new insights for the prevention and treatment of neurological diseases. Diabetes-associated cognitive decline (DACD) is one of the central nervous system complications of type 2 diabetes mellitus (T2DM). ZiBuPiYin recipe (ZBPYR), a traditional Chinese medicine (TCM) formula, has long been used for the treatment of T2DM and prevention of DACD. However, the contribution of ZBPYR treatment to the interaction between the gut microbiota and metabolism for preventing and treating DACD remains to be clarified. Here, we investigate whether the gut microbiota plays a key role in ZBPYR-mediated prevention of DACD and treatment of T2DM via incorporating microbiomics and metabolomics, and investigate the links between the microbiota–gut–brain axis interaction and the efficacy of ZBPYR in ZDF rats. In the current study, we found that ZBPYR treatment produced lasting changes in gut microbiota community and metabolites and remotely affected hippocampus metabolic changes, thereby improving memory deficits and reversing β-amyloid deposition and insulin resistance in the brain of ZDF rats from T2DM to DACD. This may be related to a series of metabolic changes affected by gut microbiota, including alanine, aspartic acid, and glutamic acid metabolism; branched-chain amino acid metabolism; short-chain fatty acid metabolism; and linoleic acid/unsaturated fatty acid metabolism. In summary, this study demonstrates that prevention and treatment of DACD by ZBPYR partly depends on the gut microbiota, and the regulatory effects of bacteria-derived metabolites and microbiota–gut–brain axis are important protective mechanisms of ZBPYR.

Beneficial Effect of Quercetin on Erythrocyte Properties in Type 2 Diabetic Rats

Diabetes mellitus is characterized by tissue oxidative damage and impaired microcirculation, as well as worsened erythrocyte properties. Measurements of erythrocyte deformability together with determination of nitric oxide (NO) production and osmotic resistance were used for the characterization of erythrocyte functionality in lean (control) and obese Zucker diabetic fatty (ZDF) rats of two age categories. Obese ZDF rats correspond to prediabetic (younger) and diabetic (older) animals. As antioxidants were suggested to protect erythrocytes, we also investigated the potential effect of quercetin (20 mg/kg/day for 6 weeks). Erythrocyte deformability was determined by the filtration method and NO production using DAF-2DA fluorescence. For erythrocyte osmotic resistance, we used hemolytic assay. Erythrocyte deformability and NO production deteriorated during aging—both were lower in older ZDF rats than in younger ones. Three-way ANOVA indicates improved erythrocyte deformability after quercetin treatment in older obese ZDF rats only, as it was not modified or deteriorated in both (lean and obese) younger and older lean animals. NO production by erythrocytes increased post treatment in all experimental groups. Our study indicates the potential benefit of quercetin treatment on erythrocyte properties in condition of diabetes mellitus. In addition, our results suggest potential age-dependency of quercetin effects in diabetes that deserve additional research.

The Effects of Erchen Decoction on Gut Microbiota and Lipid Metabolism Disorders in Zucker Diabetic Fatty Rats

Obesity is a chronic metabolic disease caused by genetic and environmental factors that has become a serious global health problem. There is evidence that gut microbiota is closely related to the occurrence and development of obesity. Erchen Decoction (ECD), a traditional Chinese medicine, has been widely used for clinical treatment and basic research of obesity and related metabolic diseases in recent years. It can significantly improve insulin resistance (IR) and lipid metabolism disorders. However, there is no microbiological study on its metabolic regulation. In this study, we investigated the effects of ECD on obesity, especially lipid metabolism and the composition and function of gut microbiota in Zucker diabetic fatty (ZDF) rats, and explored the correlation between the biomarkers of gut microbiota and metabolite and host phenotype. The results showed that ECD could reduce body weight, improve IR and lipid metabolism, and reduce the concentration of free fatty acids (FFA) released from white adipose tissue (WAT) due to excessive lipolysis by interfering with the insulin receptor substrate 1 (IRS1)/protein kinase B (AKT)/protein kinase A (PKA)/hormone-sensitive triglyceride lipase (HSL) signaling pathway in ZDF rats. Additionally, ECD gradually adjusted the overall structure of changed gut microbiota, reversed the relative abundance of six genera, and changed the function of gut microbiota by reducing the content of propionic acid, a metabolite of gut microbiota, in ZDF rats. A potentially close relationship between biomarkers, especially Prevotella, Blautia, and Holdemania, propionic acid and host phenotypes were demonstrated through correlation analysis. The results suggested that the beneficial effects of ECD on obesity, especially lipid metabolism disorders, are related to the regulation of gut microbiota in ZDF rats. This provides a basis for further research on the mechanism and clinical application of ECD to improve obesity via gut microbiota.

Effects of bee bread, Cornelian cherries treatment on the femoral bone structure using Zucker diabetic fatty rats as an animal model

: This is a pilot study dealing with the reduced femoral bone structure in Zucker diabetic fatty (ZDF) rats following a 10 week treatment with bee bread and Cornelian cherries. The adult ZDF rats were divided into 4 groups: the E1 group received bee bread (500 mg/kg b.w. daily), the E2 group received Cornelian cherries (500 mg/kg b.w. per day), the E3 group was simultaneously supplemented by bee bread and Cornelian cherries (500 + 500 mg/kg b.w. daily), and the C group served as an untreated diabetic control. A non-significant decrease in the blood glucose, total cholesterol, triglyceride concentrations, ALP activity and a non-significant increase in the insulin, total body weight of the ZDF rats, femoral length and weight were observed in the E1, E2, and E3 groups. The treatments had no impact on the relative volume of the cortical bone, bone mineral density, bone surface and cortical bone thickness. On the contrary, the relative volume of the trabecular bone, trabecular thickness and trabecular bone surface were significantly increased in the E1 group. The relative volume of the trabecular bone and trabecular thickness were significantly elevated in the E2 group. In the E3 group, the trabecular thickness was found to have significantly increased. Single administrations of either bee bread or Cornelian cherries had more positive effects on the trabecular bone microarchitecture in the ZDF rats than the simultaneous supplementation.

Vitamin A Status Regulates the Development of Obesity and Type 2 Diabetes in Zucker Diabetic Fatty Rats

Objectives Here, we studied the effects of VA status on the development of obesity and type 2 diabetes in Zucker diabetic fat (ZDF) rats. Methods Zucker Lean (ZL) and ZDF rats at weaning were divided into 6 groups, VA deficient with basal fat (VAD-BF, 0 mg retinyl palmitate (RP)/kg and 22.1% fat energy), VA marginal with BF (VAM-BF, 0.35 mg RP/kg), VA sufficient with BF (VAS-BF, 4.0 mg RP/kg), VAD with high-fat (VAD-HF, 60% fat energy), VAM-HF and VAS-HF diets, and fed for 8 weeks (w). The body mass (BM), and peripheral blood glucose (PBG) were measured weekly. An oral glucose tolerance test (OGTT) were done at 6.5 and 7.5w, respectively. Plasma levels of glucose, insulin, triacylglycerol and cholesterol l were determined using commercially available kits. The expression levels of genes and proteins in the liver of rats were analyzed using PCR and Western blot. Results VAS-BF ZL and ZDF rats from 6w had respectively higher BM than VAD/VAM-BF ZL and ZDF rats. VAS-HF ZL and ZDF rats from 4w had respectively higher BM than VAD/VAM-HF ZL and ZDF rats. VAS-BF/HF ZDF rats from 6w had respectively higher PBG levels than VAD/VAM-BF/HF ZDF rats. The OGTT AUC values of VAS-BF/HF ZL/ZDF rats were respectively higher than that of VAD/VAM-BF/HF ZL/ZDF rats. The levels of glucose, insulin, triacylglycerol and cholesterol in VAD/VAM/VAS-BF ZDF and VAD/VAM/VAS-HF ZDF rats were higher than that in BF ZL rats (except for the glucose level) and HF ZL rats, respectively. The hepatic Gck mRNA and its protein levels in VAD-BF ZL rats were lower than that in VAS-BF ZL rats. The hepatic levels of Fas, and Acl mRNA and FAS, and ACL proteins in VAM/VAS-HF ZF rats were higher than that in VAM/VAS-HF ZL rats. The hepatic retinol content of VAD-BF/HF ZL/ZDF rats were lower than that of VAM groups, which are lower than that of VAS-BF/HF ZL/ZDF rats. Conclusions VA statuses affect BM gain in ZL and ZDF rats fed a BF or a HF diet. The expression levels of mRNAs and proteins in the fatty acid biosynthesis pathways were reduced in VAD-HF ZDF rats. The effects of VA on fatty acid biosynthesis in ZDF rats were masked in a HF diet setting. Reduced VA intake prevents obesity, and type 2 diabetes in ZDF rats. Funding Sources Diabetes Action Research and Education Foundation

Dapagliflozin Alleviates Hepatic Steatosis by Restoring Autophagy via the AMPK-mTOR Pathway

As a newly approved oral hypoglycaemic agent, the sodium-glucose cotransporter 2 (SGLT2) inhibitor dapagliflozin, which is derived from the natural product phlorizin can effectively reduce blood glucose. Recent clinical studies have found that dapagliflozin alleviates non-alcoholic fatty liver disease (NAFLD), but the specific mechanism remains to be explored. This study aimed to investigate the underlying mechanism of dapagliflozin in alleviating hepatocyte steatosis in vitro and in vivo. We fed the spontaneous type 2 diabetes mellitus rats with high-fat diets and cultured human normal liver LO2 cells and human hepatocellular carcinoma HepG2 cells with palmitic acid (PA) to induce hepatocellular steatosis. Dapagliflozin attenuated hepatic lipid accumulation both in vitro and in vivo. In Zucker diabetic fatty (ZDF) rats, dapagliflozin reduced hepatic lipid accumulation via promoting phosphorylation of acetyl-CoA carboxylase 1 (ACC1), and upregulating lipid β-oxidation enzyme acyl-CoA oxidase 1 (ACOX1). Furthermore, dapagliflozin increased the expression of the autophagy-related markers LC3B and Beclin1, in parallel with a drop in p62 level. Similar effects were observed in PA-stimulated LO2 cells and HepG2 cells. Dapagliflozin treatment could also significantly activated AMPK and reduced the phosphorylation of mTOR in ZDF rats and PA-stimulated LO2 cells and HepG2 cells. We demonstrated that dapagliflozin ameliorates hepatic steatosis by decreasing lipogenic enzyme, while inducing fatty acid oxidation enzyme and autophagy, which could be associated with AMPK activation. Moreover, our results indicate that dapagliflozin induces autophagy via the AMPK-mTOR pathway. These findings reveal a novel clinical application and functional mechanism of dapagliflozin in the treatment of NAFLD.

Berberine Slows the Progression of Prediabetes to Diabetes in Zucker Diabetic Fatty Rats by Enhancing Intestinal Secretion of Glucagon-Like Peptide-2 and Improving the Gut Microbiota

BackgroundBerberine is a plant alkaloid that has multiple beneficial effects against intestine inflammation. In our previous study, we have found that berberine also possesses an antidiabetic effect. However, whether berberine is useful in the prevention of type 2 diabetes mellitus (T2DM) through its effect on intestine endocrine function and gut microbiota is unclear.AimTo investigate the effects of berberine in the prevention of T2DM, as well as its effects on intestine GLP-2 secretion and gut microbiota in ZDF rats.MethodsTwenty Zucker Diabetic Fatty (ZDF) rats were fed a high-energy diet until they exhibited impaired glucose tolerance (IGT). The rats were then divided into two groups to receive berberine (100 mg/kg/d; berberine group) or vehicle (IGT group) by gavage for 3 weeks. Five Zucker Lean (ZL) rats were used as controls. Fasting blood glucose (FBG) was measured, an oral glucose tolerance test was performed, and the Homeostatic Model Assessment of Insulin Resistance (HOMA-IR) was calculated. Intestinal expression of TLR-4, NF-κB, TNF-α, mucin, zona occludens-1 (ZO-1) and occludin were assessed (immunohistochemistry). Plasma levels and glutamine-induced intestinal secretion of glucagon-like peptide-1 (GLP-1) and GLP-2 were measured (enzyme-linked immunosorbent assay). The plasma lipopolysaccharide (LPS) level was measured. Fecal DNA extraction, pyrosequencing, and bioinformatics analysis were performed.ResultsAfter 3 weeks of intervention, diabetes developed in all rats in the IGT group, but only 30% of rats in the berberine group. Treatment with berberine was associated with reductions in food intake, FBG level, insulin resistance, and plasma LPS level, as well as increases in fasting plasma GLP-2 level and glutamine-induced intestinal GLP-2 secretion. Berberine could increase the goblet cell number and villi length, and also reverse the suppressed expressions of mucin, occludin, ZO-1 and the upregulated expressions of TLR-4, NF-κB and TNF-α induced in IGT rats (P&lt;0.05). Berberine also improved the structure of the gut microbiota and restored species diversity.ConclusionBerberine may slow the progression of prediabetes to T2DM in ZDF rats by improving GLP-2 secretion, intestinal permeability, and the structure of the gut microbiota.