Overexpression of hexokinase 2 reduces mitochondrial calcium overload in coronary endothelial cells of type 2 diabetic mice.

Coronary microvascular rarefaction, due to endothelial cell (EC) dysfunction, is one of the causes of increased morbidity and mortality in diabetes. Coronary ECs in diabetes are more apoptotic due partly to mitochondrial calcium overload. This study was designed to investigate the role of hexokinase 2 (HK2, an endogenous inhibitor of voltage-dependent anion channel) in coronary endothelial dysfunction in type 2 diabetes. We used mouse coronary ECs (MCECs) isolated from type 2 diabetic mice and human coronary ECs (HCECs) from type 2 diabetic patients to examine protein levels and mitochondrial function. ECs were more apoptotic and capillary density was lower in the left ventricle of diabetic mice than the control. MCECs from diabetic mice exhibited significant increase in mitochondrial Ca2+ concentration ([Ca2+]mito) compared with the control. Among several regulatory proteins for [Ca2+]mito, hexokinase 1 (HK1) and HK2 were significantly lower in MCECs from diabetic mice than control MCECs. We also found that the level of HK2 ubiquitination was higher in MCECs from diabetic mice than in control MCECs. In line with the data from MCECs, HCECs from diabetic patients showed lower HK2 protein levels than HCECs from nondiabetic patients. High-glucose treatment, but not high-fat treatment, significantly decreased HK2 protein levels in MCECs. HK2 overexpression in MCECs of diabetic mice not only lowered the level of [Ca2+]mito, but also reduced mitochondrial reactive oxygen species production toward the level seen in control MCECs. These data suggest that HK2 is a potential therapeutic target for coronary microvascular disease in diabetes by restoring mitochondrial function in coronary ECs.

High fat diet-induced oxidative stress blocks hepatocyte nuclear factor 4α and leads to hepatic steatosis in mice.

Nonalcoholic fatty liver disease (NAFLD) is the most common form of chronic liver disease with manifestation of over-accumulation of fat in liver. Increasing evidences indicate that NAFLD may be in part caused by malfunction of very low density lipoprotein (VLDL) secretion. Hepatocyte nuclear factor 4α (HNF4α), a nuclear receptor protein, plays an important role in sustain hepatic lipid homeostasis via transcriptional regulation of genes involved in secretion of VLDL, such as apolipoprotein B (ApoB). However, the exact functional change of HNF4α in NAFLD remains to be elucidated. In the present study, we found that high fat diet (HFD) induced cytoplasmic retention of HNF4α in hepatocytes, which led to down-regulation of hepatic ApoB expression and its protein level in serum, as well as reduced secretion of VLDL. We further revealed that oxidative stress, elevated in fatty liver, was the key factor inducing the cytoplasmic retention of HNF4α in hepatocytes by activating protein kinase C (PKC)-mediated phosphorylation in HNF4α. Thus, our findings reveal a novel mechanism underlying HFD-induced fatty liver that oxidative stress impairs function of HNF4α on ApoB expression and VLDL secretion via PKC activation, eventually promoting fat accumulation in the liver. Therefore, oxidative stress/PKC/HNF4α pathway may be a novel target to treat diet-induced fatty liver.

High glucose induces the apoptosis of HUVECs in mitochondria dependent manner by enhancing VDAC1 expression.

Mellithemia are diabetic patients' emblematic syndrome, which would induce vasculopathy resulting from apoptosis of vascular endothelial cells. The pathological mechanism of high glucose-induced apoptosis of vascular endothelial cells is investigated in the present study utilizing HUVEC cells. As high glucose-induced apoptosis is caused by elevated mitochondrial permeability-mediated release of mitochondrial cytochrome c, voltage-dependent anion channel (VDAC1), the controller of mitochondrial permeability, and its regulator Bax were investigated. Our results suggest that upregulation of VDAC1 is the central event in high glucose-induced cell apoptosis, since silencing VDAC1 reduced high glucose-induced upregulation of mitochondrial/cellular Bax; thus silencing VDAC1 recovered the high glucose-reduced binding of Bax to VDAC1, which finally reduced the high mitochondrial permeability. Besides, high glucose increased VDAC1 expression by elevating the expression of SREBP1 and SREBP2, the tanscriptional factor of VDAC1. Those findings indicate that SREBP1 or SREBP2/VDAC1 could be novel targets for the prevention of diabetic vasculopathy.

SGLT inhibitors attenuate NO-dependent vascular relaxation in the pulmonary artery but not in the coronary artery.

Inhibitors of sodium-glucose cotransporter (SGLT)2 are a new class of oral drugs for type 2 diabetic patients that reduce plasma glucose levels by inhibiting renal glucose reabsorption. There is increasing evidence showing the beneficial effect of SGLT2 inhibitors on glucose control; however, less information is available regarding the impact of SGLT2 inhibitors on cardiovascular outcomes. The present study was designed to determine whether SGLT inhibitors regulate vascular relaxation in mouse pulmonary and coronary arteries. Phlorizin (a nonspecific SGLT inhibitor) and canagliflozin (a SGLT2-specific inhibitor) relaxed pulmonary arteries in a dose-dependent manner, but they had little or no effect on coronary arteries. Pretreatment with phlorizin or canagliflozin significantly inhibited sodium nitroprusside (SNP; a nitric oxide donor)-induced vascular relaxation in pulmonary arteries but not in coronary arteries. Phlorizin had no effect on cGMP-dependent relaxation in pulmonary arteries. SNP induced membrane hyperpolarization in human pulmonary artery smooth muscle cells, and pretreatment of cells with phlorizin and canagliflozin attenuated SNP-induced membrane hyperpolarization by decreasing K(+) activities induced by SNP. Contrary to the result observed in ex vivo experiments with SGLT inhibitors, SNP-dependent relaxation in pulmonary arteries was not altered by chronic administration of canagliflozin. On the other hand, canagliflozin administration significantly enhanced SNP-dependent relaxation in coronary arteries in diabetic mice. These data suggest that SGLT inhibitors differentially regulate vascular relaxation depending on the type of arteries, duration of the treatment, and health condition, such as diabetes.

Serum bile acid and unsaturated fatty acid profiles of non-alcoholic fatty liver disease in type 2 diabetic patients.

BACKGROUND: The understanding of bile acid (BA) and unsaturated fatty acid (UFA) profiles, as well as their dysregulation, remains elusive in individuals with type 2 diabetes mellitus (T2DM) coexisting with non-alcoholic fatty liver disease (NAFLD). Investigating these metabolites could offer valuable insights into the pathophy-siology of NAFLD in T2DM. AIM: To identify potential metabolite biomarkers capable of distinguishing between NAFLD and T2DM. METHODS: A training model was developed involving 399 participants, comprising 113 healthy controls (HCs), 134 individuals with T2DM without NAFLD, and 152 individuals with T2DM and NAFLD. External validation encompassed 172 participants. NAFLD patients were divided based on liver fibrosis scores. The analytical approach employed univariate testing, orthogonal partial least squares-discriminant analysis, logistic regression, receiver operating characteristic curve analysis, and decision curve analysis to pinpoint and assess the diagnostic value of serum biomarkers. RESULTS: Compared to HCs, both T2DM and NAFLD groups exhibited diminished levels of specific BAs. In UFAs, particular acids exhibited a positive correlation with NAFLD risk in T2DM, while the ω-6:ω-3 UFA ratio demonstrated a negative correlation. Levels of α-linolenic acid and γ-linolenic acid were linked to significant liver fibrosis in NAFLD. The validation cohort substantiated the predictive efficacy of these biomarkers for assessing NAFLD risk in T2DM patients. CONCLUSION: This study underscores the connection between altered BA and UFA profiles and the presence of NAFLD in individuals with T2DM, proposing their potential as biomarkers in the pathogenesis of NAFLD.

Ubiquitin fold modifier 1 activates NF-κB pathway by down-regulating LZAP expression in the macrophage of diabetic mouse model.

Inflammatory response is closely related with the development of many serious health problems worldwide including diabetes mellitus (DM). Ubiquitin-fold modifer 1 (Ufm1) is a newly discovered ubiquitin-like protein, while its function remains poorly investigated, especially in inflammatory response and DM. In the present study, we analyzed the role of Ufm1 on inflammatory response in DM, and found that the proinflammatory cytokine levels (tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and IL-1ß) and Ufm1 expression were highly increased both in the peritoneal macrophages of db/db mice and Raw264.7 cells induced by lipopolysaccharide (LPS). Western blot and luciferase reporter assay showed that NF-κB pathway was obviously activated in macrophages and the expression of LZAP, an inhibitor of NF-κB pathway, was down-regulated. With the LZAP knockdown plasmid and activation plasmid, we demonstrated that NF-κB/p65 activation was inhibited by LZAP in macrophages. The interaction of Ufm1 and LZAP was further proved with co-immunoprecipitation assay in HEK293 and Raw264.7 cells. The LZAP expression was also related with the presence of Ufm1 demonstrated by Ufm1 knockdown plasmid and activation plasmid. Besides that, we finally proved that the expression and activation of Ufm1 induced by LPS were regulated by JNK/ATF2 and JNK/c-Jun pathway with the use of SP600125. In conclusion, the present study demonstrated that Ufm 1 could activate NF-κB pathway by down-regulating LZAP in macrophage of diabetes, and its expression and activation were regulated by JNK/ATF2 and c-Jun pathway.

Resveratrol Modulates the Gut Microbiota and Inflammation to Protect Against Diabetic Nephropathy in Mice.

Oral administration of resveratrol is able to ameliorate the progression of diabetic nephropathy (DN); however, its mechanisms of action remain unclear. Recent evidence suggested that the gut microbiota is involved in the metabolism therapeutics. In the current study, we sought to determine whether the anti-DN effects of resveratrol are mediated through modulation of the gut microbiota using the genetic db/db mouse model of DN. We demonstrate that resveratrol treatment of db/db mice relieves a series of clinical indicators of DN. We then show that resveratrol improves intestinal barrier function and ameliorates intestinal permeability and inflammation. The composition of the gut microbiome was significantly altered in db/db mice compared to control db/m mice. Dysbiosis in db/db mice characterized by low abundance levels of Bacteroides, Alistipes, Rikenella, Odoribacter, Parabacteroides, and Alloprevotella genera were reversed by resveratrol treatment, suggesting a potential role for the microbiome in DN progression. Furthermore, fecal microbiota transplantation, derived from healthy resveratrol-treated db/m mice, was sufficient to antagonize the renal dysfunction, rebalance the gut microbiome and improve intestinal permeability and inflammation in recipient db/db mice. These results indicate that resveratrol-mediated changes in the gut microbiome may play an important role in the mechanism of action of resveratrol, which provides supporting evidence for the gut-kidney axis in DN.

High glucose induces apoptosis of HUVECs in a mitochondria-dependent manner by suppressing hexokinase 2 expression.

Hyperglycemia in patients with diabetes induces vascular endothelial cell apoptosis and subsequent vasculopathy. The aim of the current study was to investigate the pathological mechanism of hyperglycemia-induced endothelial cell apoptosis and vasculopathy using human umbilical vein endothelial cells. As high glucose-induced apoptosis is caused by elevated mitochondrial permeability-mediated release of mitochondrial cytochrome c, the current study examined voltage-dependent anion channel (VDAC1), the controller of mitochondrial permeability, and its regulators, hexokinase2 (HK2), Bcl-2 and Bax. The current study demonstrated that HK2 may be involved in high glucose-induced cell apoptosis, as HK2 overexpression partially reversed high glucose-induced downregulation of mitochondrial/cellular HK2 and Bcl-2 as well as upregulation of mitochondrial Bax. These results suggest that HK2 overexpression partially reversed the reduced binding of HK2 and Bcl-2 and the enhanced binding of Bax to VDAC1, which reduced the high mitochondrial permeability observed under high-glucose conditions. Furthermore, high glucose reduced HK2 transcription via down-regulation of the HK2 transcriptional factor, peroxisome proliferator activated receptor γ (PPARγ). Taken together, these results suggest that PPARγ/HK2 may be novel targets for the prevention of diabetic vasculopathy.

MicroRNA-134-5p promotes high glucose-induced podocyte apoptosis by targeting bcl-2.

Podocyte apoptosis is a typical early feature of diabetic nephropathy (DN), with loss of nephrin integrity contributing to increased proteinuria in patients with DN. Emerging evidence shows that microRNAs (miRNAs) play vital roles in the pathogenesis of DN. Thus, we aimed to further elucidate the role of miRNAs in podocyte apoptosis in DN. We used db/db and db/m mice maintained under a continuous feeding regime for 12 weeks. Using microarray analysis, we found several miRNAs potentially related to podocyte apoptosis. In addition, we cultured a conditionally immortalized human podocyte cell line in 30 mM D-glucose and found that miR-134-5p was upregulated in both db/db mice and high-glucose (HG)-treated podocytes. Upregulation of miR-134-5p was accompanied by podocyte apoptosis and downregulation of nephrin. Inhibition of miR-134-5p produced the opposite effect. Dual-luciferase reporter assays showed that miR-134-5p directly targeted the 3'-untranslated region of the B-cell lymphoma-2 gene (BCL2), and further study confirmed an increase in bcl-2 protein level in HG-treated podocytes transfected with anti-miR-134-5p. Knockdown of BCL2 impeded the antiapoptotic effect of anti-miR-134-5p. Finally, we found that miR-134-5p might regulate apoptosis in db/db mice and podocytes by targeting BCL2. Taken together, our findings suggest that miR-134-5p promotes podocyte apoptosis under HG conditions by targeting BCL2. Our study provides a meaningful approach to interpret the mechanisms of action of miRNAs involved in DN.

Hypoxia-induced pulmonary hypertension in type 2 diabetic mice.

Hypoxia-induced pulmonary hypertension (HPH) is a progressive disease that is mainly caused by chronic exposure to high altitude, chronic obstructive lung disease, and obstructive sleep apnea. The increased pulmonary vascular resistance and increased pulmonary arterial pressure result in increased right ventricular afterload, leading to right heart failure and increased morbidity. There are several clinical reports suggesting a link between PH and diabetes, insulin resistance, or obesity; however, it is unclear whether HPH is associated with diabetes as a progressive complication in diabetes. The major goal of this study is to examine the effect of diabetic "preconditioning" or priming effect on the progression of HPH and define the molecular mechanisms that explain the link between diabetes and HPH. Our data show that HPH is significantly enhanced in diabetic mice, while endothelium-dependent relaxation in pulmonary arteries is significantly attenuated in chronically hypoxic diabetic mice (DH). In addition, we demonstrate that mouse pulmonary endothelial cells (MPECs) isolated from DH mice exhibit a significant increase in mitochondrial reactive oxygen species (ROS) concentration and decreased SOD2 protein expression. Finally, scavenging mitochondrial ROS by mitoTempol restores endothelium-dependent relaxation in pulmonary arteries that is attenuated in DH mice. These data suggest that excessive mitochondrial ROS production in diabetic MPECs leads to the development of severe HPH in diabetic mice exposed to hypoxia.

Akt signals upstream of L-type calcium channels to optimize insulin secretion.

OBJECTIVES: Both the serine/threonine protein kinase Akt and the voltage-gated L-type calcium channel act as important players in glucose-stimulated insulin secretion. Akt recruits the L-type calcium channel to and maintains them in the plasma membrane. This study aimed to characterize the role of L-type calcium channels in mediation of Akt signaling in glucose-stimulated insulin secretion. METHODS: Insulin secretion was evaluated in rat pancreatic islets and INS-1 pancreatic ß cells by a standard insulin radioimmunoassay. RESULTS: Akt inhibition effectively abrogates not only glucose-stimulated but also potassium depolarization-stimulated insulin secretion from rat islets, the latter critically relying on the voltage-gated calcium channel-mediated Ca(2+) influx without involvement of glucose metabolism. Likewise, Akt inhibition also reduces both glucose-stimulated and potassium depolarization-stimulated insulin secretion from INS-1 cells. Importantly, pharmacological ablation of L-type calcium channels partially blocks Akt inhibition-induced reduction in glucose-stimulated insulin secretion but completely prevents that in potassium depolarization-evoked insulin release from INS-1 cells. Furthermore, Akt inhibition does not influence calcium ionophore A23187-induced insulin secretion from INS-1 cells, which occurred without involvement of L-type calcium channels. CONCLUSIONS: Akt signals upstream of L-type calcium channels to optimize glucose-stimulated insulin secretion.

Subthreshold α2-adrenergic activation counteracts glucagon-like peptide-1 potentiation of glucose-stimulated insulin secretion.

The pancreatic ß cell harbors α2-adrenergic and glucagon-like peptide-1 (GLP-1) receptors on its plasma membrane to sense the corresponding ligands adrenaline/noradrenaline and GLP-1 to govern glucose-stimulated insulin secretion. However, it is not known whether these two signaling systems interact to gain the adequate and timely control of insulin release in response to glucose. The present work shows that the α2-adrenergic agonist clonidine concentration-dependently depresses glucose-stimulated insulin secretion from INS-1 cells. On the contrary, GLP-1 concentration-dependently potentiates insulin secretory response to glucose. Importantly, the present work reveals that subthreshold α2-adrenergic activation with clonidine counteracts GLP-1 potentiation of glucose-induced insulin secretion. This counteractory process relies on pertussis toxin- (PTX-) sensitive Gi proteins since it no longer occurs following PTX-mediated inactivation of Gi proteins. The counteraction of GLP-1 potentiation of glucose-stimulated insulin secretion by subthreshold α2-adrenergic activation is likely to serve as a molecular mechanism for the delicate regulation of insulin release.

Alteration of CXCR7 expression mediated by TLR4 promotes tumor cell proliferation and migration in human colorectal carcinoma.

The link between inflammation and colorectal carcinoma has been acknowledged. However, the impact of bacterial lipopolysaccharide (LPS) binding to Toll-like receptor 4 (TLR4) on chemokine receptors in human colorectal carcinoma cells still remains to be elucidated. The present study shows that exposure to LPS elevated CXC chemokine receptor 7 (CXCR7) expression in colorectal carcinoma SW480 and Colo 205 cell lines expressing TLR4/myeloid differential protein (MD-2). CXCR7 is associated with SW480 cell proliferation and migration. However, exposure of SW480 and Colo 205 cells to LPS had no effect on CXCR4 expression. To further support the above results, the expression of TLR4, MD-2, and CXCR7 was analyzed in human colorectal carcinoma tissues. Higher rates of TLR4 (53%), MD-2 (70%), and CXCR7 (29%) expression were found in colorectal carcinoma tissues than in normal tissues. We demonstrated that the recombination of TLR4, MD-2 and CXCR7 strongly correlated with tumor size, lymph node metastasis and distant metastasis in colorectal carcinoma tissue samples (p = 0.037, p = 0.002, p = 0.042, resp.). Accordingly, simultaneous examination of the expression of TLR4, MD-2 and CXCR7 in cancer tissues of colorectal carcinoma may provide valuable prognostic diagnosis of carcinoma growth and metastasis. Interplay of TLR4, MD-2 and CXCR7 may be of interest in the context of novel immunomodulatory therapies for colorectal carcinoma.