Effects of Huanglian-Renshen-Decoction, a Fixed Mixture of Traditional Chinese Medicine, on the Improvement of Glucose Metabolism by Maintenance of Pancreatic ? Cell Identity in db/db Mice.
ABSTRACT: Huanglian-Renshen-Decoction (HRD) is widely used to treat type 2 diabetes mellitus (T2DM) in China. However, the underlying mechanism is unclear. We aimed to investigate the mechanism by which HRD regulates the glucose level. Forty 7-8-week-old db/db (BSK) mice were randomly assigned to the following four groups: model, low dose HRD (LHRD), high dose HRD (HHRD), and saxagliptin (SAX). Additionally, 10 db/m mice were assigned to control group. The experimental mice were administered 3.03g/kg/d and 6.06g/kg/d of HRD in the LHRD and HHRD groups, respectively, and 10mg/kg/d saxagliptin in the SAX group for 8 weeks. The control and model groups were supplied with distilled water. After the intervention, the pancreas and blood were collected and tested. Compared with that of model group, the fasting blood glucose (FBG) was significantly decreased in all intervention groups (p < 0.05 or 0.01), whereas fasting serum insulin (FINS) was increased significantly in both HHRD and SAX groups. The immunofluorescence images showed that the mass of insulin+ cells was increased and that of glucagon+ cells was reduced obviously in experimental groups compared to those of the model group. In addition, the coexpression of insulin, glucagon, and PDX1 was decreased in HHRD group, and the level of caspase 12 in islet was decreased significantly in all intervention groups. However, little difference was found in the number and morphology of islet, and the expression of ki67, bcl2, bax, caspase 3, and cleaved-caspase 3 in the pancreas among groups. Interestingly, the cleaved-Notch1 level was increased and the Ngn3 level in islet was decreased significantly in HHRD group. The HRD showed dose-dependent effects on glucose metabolism improvement through maintenance of ? cell identity via a mechanism that might involve the Notch1/Ngn3 signal pathway in db/db mice.
Project description:The p38 mitogen-activated protein kinase (MAPK) pathway is involved in endoplasmic reticulum stress (ERS) and inflammation, which may play an important role in the pathogenesis of type 2 diabetes (T2DM). This study aimed to investigate whether p38 MAPK contributes to the pathogenesis of T2DM. 6-week-old female db/db mice were randomly assigned to Dmo and Dmi groups, and C57 mice were assigned as controls. The Dmi group was gavaged with the p38 MAPK inhibitor SB203580 for 9?weeks, and the effects on ? cell dysfunction and apoptosis were investigated. db/db mice showed higher food intake, body mass, fasting glucose, and plasma insulin levels than C57 mice. After SB203580 administration, blood glucose was significantly lower. HOMA ? and HOMA IR were improved. Islet mRNA expression levels of the ERS markers were lower. P38 MAPK inhibition reduced blood glucose and improved ? cell function, at least in part by reducing ? cell apoptosis.
Project description:This study aimed to evaluate the validation of the leptin receptor-deficient mice model for secondary osteoporosis associated with type 2 diabetes mellitus (T2DM) at bone micro-architectural level. Thirty three 36-week old male mice were divided into four groups: normal control (db/m) (n = 7), leptin receptor-deficient T2DM (db/db) (n = 8), human C-reactive protein (CRP) transgenic normal control (crp/db/m) (n = 7), and human CRP transgenic T2DM (crp/db/db) (n = 11). Lumber vertebrae (L5) and bilateral lower limbs were scanned by micro-CT to analyze trabecular and cortical bone quality. Right femora were used for three-point bending to analyze the mechanical properties. Trabecular bone quality at L5 was better in db/db or crp/db/db group in terms of bone mineral density (BMD), bone volume fraction, connectivity density, trabecular number and separation (all p < 0.05). However the indices measured at proximal tibia showed comparable trabecular BMD and microarchitecture among the four groups. Femur length in crp/db/db group was significantly shorter than db/m group (p < 0.05) and cortices were thinner in db/db and crp/db/db groups (p > 0.05). Maximum loading and energy yield in mechanical test were similar among groups while the elastic modulus in db/db and crp/db/db significantly lower than db/m. The leptin-receptor mice is not a proper model for secondary osteoporosis associated with T2DM.
Project description:Continuous deficiency of leucine, a member of branched chain amino acids, is able to reduce obesity and improve insulin sensitivity in mice. Intermittent fasting has been shown to be effective in intervention of metabolic disorders including diabetes. However, it is unknown whether intermittent leucine deprivation can intervene in type 2 diabetes progression. We administered leucine-deprived food every other day in db/db mice, a type 2 diabetes model, for a total of eight weeks to investigate the interventional effect of intermittent leucine deprivation. Intermittent leucine deprivation significantly reduces hyperglycemia in db/db mice independent of body weight change, together with improvement in glucose tolerance and insulin sensitivity. The total area of pancreatic islets and ? cell number are increased by intermittent leucine deprivation, accompanied by elevated proliferation of ? cells. The expression level of Ngn3, a ? cell progenitor marker, is also increased by leucine-deleted diet. However, leucine deficiency engenders an increase in fat mass and a decrease in lean mass. Lipid accumulation in the liver is elevated and liver function is compromised by leucine deprivation. In addition, leucine deficiency alters the composition of gut microbiota. Leucine deprivation increases the genera of Bacteroides, Alloprevotella, Rikenellaceae while reduces Lachnospiraceae and these changes are correlated with fasting blood glucose levels of the mice. Collectively, our data demonstrated that intermittent leucine deprivation can intervene in the progression of type 2 diabetes in db/db mice. However, leucine deficiency reduces lean mass and aggravates hepatic steatosis in the mouse.
Project description:Recent data have indicated the emerging role of glomerular autophagy in diabetic kidney disease. We aimed to assess the effect of the SGLT2 inhibitor empagliflozin, the DPP4 inhibitor linagliptin, and their combination, on glomerular autophagy in a model of type 2 diabetes. Eight-week-old male db/db mice were randomly assigned to treatment with empagliflozin, linagliptin, empagliflozin-linagliptin or vehicle for 8 weeks. Age-matched non-diabetic db/+ mice acted as controls. To estimate glomerular autophagy, immunohistochemistry for beclin-1 and LAMP-1 was performed. Podocyte autophagy was assessed by counting the volume density (Vv) of autophagosomes, lysosomes and autolysosomes by transmission electron microscopy. LC3B and LAMP-1, autophagy markers, and caspase-3 and Bcl-2, apoptotic markers, were evaluated in renal cortex by western blot. Vehicle-treated db/db mice had weak glomerular staining for beclin-1 and LAMP-1 and reduced Vv of autophagosomes, autolysosomes and lysosomes in podocytes. Empagliflozin and linagliptin, both as monotherapy and in combination, enhanced the areas of glomerular staining for beclin-1 and LAMP-1 and increased Vv of autophagosomes and autolysosomes in podocytes. Renal LC3B and Bcl-2 were restored in actively treated animals. LAMP-1 expression was enhanced in the empagliflozin group; caspase-3 expression decreased in the empagliflozin-linagliptin group only. Mesangial expansion, podocyte foot process effacement and urinary albumin excretion were mitigated by both agents. The data provide further explanation for the mechanism of the renoprotective effect of SGLT2 inhibitors and DPP4 inhibitors in diabetes.
Project description:Background. This study was to explore the pharmacokinetics of saxagliptin (Sax) in Goto-Kakizaki (GK) rats complicated with depression induced by chronic unpredicted mild stress (CUMS). The comorbidity of diabetic patients with depression is becoming more and more epidemic. Whether depression mental disorder alters the pharmacokinetics of hypoglycemic drugs in diabetes patients is not clear. Methods. Five-week-old male GK rats were kept in the cage for 7 weeks in a specific pathogen free (SPF)-grade lab until the emergence of diabetes and were then divided into two groups: control group and depression model group. Rats in the CUMS-induced depression group were exposed to a series of stressors for 8 weeks. Plasma serotonin and dopamine levels and behavior of open-field test were used to confirm the establishment of the depression model. All rats were given 0.5 mg/kg Sax orally after 8 weeks and blood samples were collected at different time points. The Sax concentration was assayed by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). The CYP450 activity of the liver microsomes was determined by using cocktails of probe drugs in which the activities of CYP enzymes were assessed through the determination of the production of the probe drugs. Results. Statistically significant differences in Sax pharmacokinetics were observed for area under curve, clearance, peak concentration, peak time and mean residence time between the depression rats and the control rats, while no statistical differences were observed for half-time and distribution volume by HPLC-MS/MS analysis. The CYP450 activity had different changes in the depression group. Conclusions. These results indicated that CUMS-induced depression alters the drug metabolic process of Sax and CYP450 activity of the liver microsomal enzymes in GK rats.
Project description:Diabetes poses a substantial burden to society as it can lead to serious complications and premature death. The number of cases continues to increase worldwide. Two major causes of diabetes are insulin resistance and insulin insufficiency. Currently, there are few antidiabetic drugs available that can preserve or protect ?-cell function to overcome insulin insufficiency in diabetes. We describe a therapeutic strategy to preserve ?-cell function by overexpression of follistatin (FST) using an AAV vector (AAV8-Ins-FST) in diabetic mouse model. Overexpression of FST in the pancreas of db/db mouse increased ?-cell islet mass, decreased fasting glucose level, alleviated diabetic symptoms, and essentially doubled lifespan of the treated mice. The observed islet enlargement was attributed to ?-cell proliferation as a result of bioneutralization of myostatin and activin by FST. Overall, our study indicates overexpression of FST in the diabetic pancreas preserves ?-cell function by promoting ?-cell proliferation, opening up a new therapeutic avenue for the treatment of diabetes.
Project description:Aim/Introduction:? Preservation of ?-cell mass is crucial for maintaining long-term glucose homeostasis. Therapies based on incretin and its mimetics are expected to achieve this goal through various biological functions, particularly the restoration of ?-cell mass. Here we tested the effects of gastrin and exendin-4 in type 2 diabetic animals.? The effects of exendin-4 and gastrin on ?-cell function and mass were examined in 8-week-old db/db mice. INS-1 beta cells and AR42J cells were used to determine the molecular mechanism underlying the effects of the two agents. Immunohistochemistry, western blotting and RT-PCR assays were used to assess the biological effects of the two agents.? Two weeks of combination administration of exendin-4 plus gastrin resulted in a significant improvement of glucose tolerance associated with a marked preservation of ?-cell mass in db/db mice. Immunohistochemical analysis showed that such treatment resulted in the appearance of numerous irregularly-shaped small islets and single insulin-positive cells. While gastrin had little biological effect on INS-1 ?-cells consistent with low expression of its intrinsic receptor on these cells, it caused differentiation of AR42J cells into insulin-producing cells. Co-stimulation with exendin-4 significantly enhanced gastrin-induced endocrine differentiation of AR42J precursor cells. These findings were further supported by enhanced expression of key genes involved in ?-cell differentiation and maturation, such as neurogenin3 (Ngn3) and MafA.? These results suggest that combination treatment of db/db mice with exendin-4 and gastrin preserves ?-cell mass by stimulating ?-cell growth and differentiation. (J Diabetes Invest, doi: 10.1111/j.2040-1124.00044.x, 2010).
Project description:There are conflicting reports on the link between the micronutrient selenium and the prevalence of diabetes. To investigate the possibility that selenium acts as a "double-edged sword" in diabetes, cDNA microarray profiling and two-dimensional differential gel electrophoresis coupled with mass spectrometry were used to determine changes in mRNA and protein expression in pancreatic and liver tissues of diabetic db/db mice in response to dietary selenate supplementation. Fasting blood glucose levels increased continuously in db/db mice administered placebo (DMCtrl), but decreased gradually in selenate-supplemented db/db mice (DMSe) and approached normal levels after termination of the experiment. Pancreatic islet size was increased in DMSe mice compared with DMCtrl mice, resulting in a clear increase in insulin production and a doubling of plasma insulin concentration. Genes that encode proteins involved in key pancreatic ?-cell functions, including regulation of ?-cell proliferation and differentiation and insulin synthesis, were found to be specifically upregulated in DMSe mice. In contrast, apoptosis-associated genes were downregulated, indicating that islet function was protected by selenate treatment. Conversely, liver fat accumulation increased in DMSe mice together with significant upregulation of lipogenic and inflammatory genes. Genes related to detoxification were downregulated and antioxidant enzymatic activity was reduced, indicating an unexpected reduction in antioxidant defense capacity and exacerbation of fatty liver degeneration. Moreover, proteomic analysis of the liver showed differential expression of proteins involved in glucolipid metabolism and the endoplasmic reticulum assembly pathway. Taken together, these results suggest that dietary selenate supplementation in db/db mice decreased hyperglycemia by increasing insulin production and secretion; however, long-term hyperinsulinemia eventually led to reduced antioxidant defense capacity, which exacerbated fatty liver degeneration.
Project description:Background:Diabetic retinopathy (DR) is a severe complication of diabetes mellitus. DR is considered as a neurovascular disease. Retinal ganglion cell (RGC) loss plays an important role in the vision function disorder of diabetic patients. Histone deacetylase3 (HDAC3) is closely related to injury repair and nerve regeneration. The correlation between HDAC3 and retinal ganglion cells in diabetic retinopathy is still unclear yet. Methods:To investigate the chronological sequence of the abnormalities of retinal ganglion cells in diabetic retinopathy, we choose 15 male db/db mice (aged 8 weeks, 12 weeks, 16 weeks, 18 weeks, and 25 weeks; each group had 3 mice) as diabetic groups and 3 male db/m mice (aged 8 weeks) as the control group. In this study, we examined the morphological and immunohistochemical changes of HDAC3, Caspase3, and LC3B in a sequential manner by characterizing the process of retinal ganglion cell variation. Results:Blood glucose levels and body weights of db/db mice were significantly higher than that of the control group, P < 0.01. Compared with the control group, the number of retinal ganglion cells decreased with the duration of disease increasing. HDAC3 expression gradually increased in RGCs of db/db mice. Caspase3 expression gradually accelerated in RGCs of db/db mice. LC3B expression dynamically changed in RGCs of db/db mice. HDAC3 was positively correlated with Caspase3 expression (r = 0.7424), P < 0.01. Compared with the control group, the number of retinal ganglion cells decreased with the duration of disease increasing. HDAC3 expression gradually increased in RGCs of db/db mice. Caspase3 expression gradually accelerated in RGCs of db/db mice. LC3B expression dynamically changed in RGCs of db/db mice. HDAC3 was positively correlated with Caspase3 expression (r = 0.7424), P < 0.01. Compared with the control group, the number of retinal ganglion cells decreased with the duration of disease increasing. HDAC3 expression gradually increased in RGCs of db/db mice. Caspase3 expression gradually accelerated in RGCs of db/db mice. LC3B expression dynamically changed in RGCs of db/db mice. HDAC3 was positively correlated with Caspase3 expression (Discussion. We clarified the dynamic expression changes of HDAC3, Caspase3, and LC3B in retinal ganglion cells of db/db mice. Our results suggest the HDAC3 expression has a positive correlation with apoptosis and autophagy.
Project description:Chronic hyperglycemia causes oxidative stress, which contributes to damage in various tissues and cells, including pancreatic beta-cells. The expression levels of antioxidant enzymes in the islet are low compared with other tissues, rendering the beta-cell more susceptible to damage caused by hyperglycemia. The aim of this study was to investigate whether increasing levels of endogenous glutathione peroxidase-1 (GPx-1), specifically in beta-cells, can protect them against the adverse effects of chronic hyperglycemia and assess mechanisms that may be involved. C57BLKS/J mice overexpressing the antioxidant enzyme GPx-1 only in pancreatic beta-cells were generated. The biological effectiveness of the overexpressed GPx-1 transgene was documented when beta-cells of transgenic mice were protected from streptozotocin. The transgene was then introgressed into the beta-cells of db/db mice. Without use of hypoglycemic agents, hyperglycemia in db/db-GPx(+) mice was initially ameliorated compared with db/db-GPx(-) animals and then substantially reversed by 20 wk of age. beta-Cell volume and insulin granulation and immunostaining were greater in db/db-GPx(+) animals compared with db/db-GPx(-) animals. Importantly, the loss of intranuclear musculoaponeurotic fibrosarcoma oncogene homolog A (MafA) that was observed in nontransgenic db/db mice was prevented by GPx-1 overexpression, making this a likely mechanism for the improved glycemic control. These studies demonstrate that enhancement of intrinsic antioxidant defenses of the beta-cell protects it against deterioration during hyperglycemia.