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Aims/Introduction Imeglimin is a novel oral hypoglycemic agent that improves blood glucose levels through multiple mechanisms of action including the enhancement of glucose‐stimulated insulin secretion (GSIS), however, the details of this mechanism have not been clarified. In the process of GSIS, activation of the transient receptor potential melastatin 2 (TRPM2) channel, a type of non‐selective cation channel (NSCCs) in β‐cells, promotes plasma membrane depolarization. The present study aimed to examine whether imeglimin potentiates GSIS via the TRPM2 channel in β‐cells. Materials and Methods Pancreatic islets were isolated by collagenase digestion from male wild‐type and TRPM2‐knockout (KO) mice. Insulin release and nicotinamide adenine dinucleotide (NAD+) production in islets were measured under static incubation. NSCC currents in mouse single β‐cells were measured by patch‐clamp experiments. Results Batch‐incubation studies showed that imeglimin enhanced GSIS at stimulatory 16.6 mM glucose, whereas it did not affect basal insulin levels at 2.8 mM glucose. Imeglimin increased the glucose‐induced production of NAD+, a precursor of cADPR, in islets and the insulinotropic effects of imeglimin were attenuated by a cADPR inhibitor 8‐Br‐cADPR. Furthermore, imeglimin increased NSCC current in β‐cells, and abolished this current in TRPM2‐KO mice. Imeglimin did not potentiate GSIS in the TRPM2‐KO islets, suggesting that imeglimin’s increase of NSCC currents through the TRPM2 channel is causally implicated in its insulin releasing effects. Conclusions Imeglimin may activate TRPM2 channels in β‐cells via the production of NAD+/cADPR, leading to the potentiation of GSIS. Developing approaches to stimulate cADPR‐TRPM2 signaling provides a potential therapeutic tool to treat type 2 diabetes., Imeglimin may activate TRPM2 channels in β‐cells via the production of NAD+/cADPR, leading to the potentiation of GSIS.

Aims/Introduction Recent studies have identified genomic and transcript level changes along with alterations in insulin secretion in patients with diabetes and in rodent models of diabetes. It is important to establish an efficient system for testing functional consequences of these changes. We aimed to generate such a system using insulin‐secreting MIN6 cells. Materials and Methods MIN6 cells were first engineered to have a tetracycline‐regulated expression system. Then, we used the recombination‐mediated cassette exchange strategy to explore the silencing‐resistant site in the genome and generated a master cell line based on this site. Results We identified a site 10.5 kbps upstream from the Zxdb gene as a locus that allows homogenous transgene expression from a tetracycline responsible promoter. Placing the Flip/Frt‐based platform on this locus using CRISPR/Cas9 technology generated modified MIN6 cells applicable to achieving cassette exchange on the genome. Using this cell line, we generated MIN6 subclones with over‐ or underexpression of glucokinase. By analyzing a mixed population of these cells, we obtained an initial estimate of effects on insulin secretion within 6 weeks. Furthermore, we generated six MIN6 cell sublines simultaneously harboring genes of inducible overexpression with unknown functions in insulin secretion, and found that Cited4 and Arhgef3 overexpressions increased and decreased insulin secretion, respectively. Conclusions We engineered MIN6 cells, which can serve as a powerful tool for testing genetic alterations associated with diabetes, and studied the molecular mechanisms of insulin secretion., A platform for recombinase‐mediated cassette exchange is placed by a genetic editing method. This allows easy and efficient generation of insulin‐secreting cells with modified expressions of genes of interest.

Objective The role of cell death‐inducing DFF45‐like effector C (CIDEC) in insulin resistance has been established, and it is considered to be an important trigger factor for the progression of diabetic nephropathy (DN). We intend to explore whether CIDEC plays an important role in the regulation of DN and its potential mechanism. Methods High‐fat diet and low dose streptozotocin were used to establish type 2 diabetic rat model. We investigate the role of CIDEC in the pathogenesis and process of DN through histopathological analysis, western blot and gene silencing. Meanwhile, the effect of CIDEC on renal tubular epithelial cells stimulated by high glucose was also verified. Results DM group exhibited glucose and lipid metabolic disturbance, with hypertrophy of kidneys, damaged renal function, increased apoptosis, decreased autophagy, glomerulosclerosis and interstitial fibrosis. CIDEC gene silencing improved metabolic disorder and insulin resistance, alleviated renal hypertrophy and renal function damage, decreased glomerular and tubular apoptosis, increased autophagy and inhibited renal fibrosis. At the cellular level, high glucose stimulation increased CIDEC expression in renal tubular epithelial cells, accompanied by increased apoptosis and decreased autophagy. CIDEC gene silencing can improve autophagy and reduce apoptosis. At the molecular level, CIDEC gene silencing also decreased the expression of early growth response factor (EGR)1 and increased the expression of adipose triglyceride lipase (ATGL). Conclusion CIDEC gene silencing may delay the progression of DN by restoring autophagy activity and inhibiting apoptosis with the participation of EGR1and ATGL., In diabetic rats, CIDEC gene silencing may prevent apoptosis by inhibiting the expression of EGR1, while restore autophagy activity and inhibit apoptosis by increasing the expression of ATGL, thereby improving renal glomerulosclerosis and tubulointerstitial fibrosis and delaying the progression of DN. CIDEC may be a valuable new target for DN treatment.

Aims/Introduction It was reported previously that N4bp2l1 expression increases in 3T3‐L1 cells in a differentiation‐dependent manner and N4bp2l1 knockdown suppresses adipocyte differentiation. However, the physiological function of N4BP2L1 in adipocytes remains unknown. This study aimed to elucidate the physiological mechanism of N4bp2l1 expression and the role of N4BP2L1 in the physiological function of adipocytes. Materials and Methods Analysis of gene expression levels of N4bp2l1 in adipose tissue during feeding in mice was conducted. Identification of transcription factors that regulate N4bp2l1 expression was conducted using a reporter assay. Investigation of N4BP2L1‐interacting proteins was carried out using immunoprecipitation. A GLUT4 translocation assay and a glucose uptake assay in 3T3‐L1 adipocytes were performed using N4bp2l1 overexpression and knockdown adenovirus. Results The results indicated that N4bp2l1 is a novel FoxO1 target gene and its expression is controlled by the insulin‐mediated regulation of FoxO1. N4BP2L1 interacts with dynactin, which binds to the microtubule motor dynein, indicating that N4BP2L1 is involved in GLUT4 trafficking and glucose uptake in 3T3‐L1 adipocytes. Conclusions Our results suggest that N4BP2L1 is involved in adipocyte homeostasis by interacting with dynein–dynactin and affecting GLUT4‐mediated glucose uptake and the insulin signaling pathway., N4bp2l1 is a novel FoxO1 target gene that is controlled by insulin‐mediated regulation of FoxO1. N4BP2L1 interacts with dynactin, which binds to the microtubule motor dynein, suggesting that N4BP2L1 is involved in GLUT4 trafficking and glucose uptake.

Aims/Introduction Brown adipose tissue (BAT) utilizes large amounts of fuel for thermogenesis, but the mechanism by which fuel substrates are switched in response to changes in energy status is poorly understood. We have now investigated the role of Kruppel‐like factor 15 (KLF15), a transcription factor expressed at a high level in adipose tissue, in the regulation of fuel utilization in BAT. Materials and Methods Depletion or overexpression of KLF15 in HB2 differentiated brown adipocytes was achieved by adenoviral infection. Glucose and fatty acid oxidation were measured with radioactive substrates, pyruvate dehydrogenase complex activity was determined with a colorimetric assay, and gene expression was examined by reverse transcription and real‐time polymerase chain reaction analysis. Results Knockdown of KLF15 in HB2 cells attenuated fatty acid oxidation in association with downregulation of the expression of genes related to this process including Acox1 and Fatp1, whereas it increased glucose oxidation. Expression of the gene for pyruvate dehydrogenase kinase 4 (PDK4), a negative regulator of pyruvate dehydrogenase complex, was increased or decreased by KLF15 overexpression or knockdown, respectively, in HB2 cells, with these changes being accompanied by a respective decrease or increase in pyruvate dehydrogenase complex activity. Chromatin immunoprecipitation showed that Pdk4 is a direct target of KLF15 in HB2 cells. Finally, fasting increased expression of KLf15, Pdk4 and genes involved in fatty acid utilization in BAT of mice, whereas refeeding suppressed Klf15 and Pdk4 expression. Conclusions Our results implicate KLF15 in the regulation of fuel switching between glucose and fatty acids in response to changes in energy status in BAT., Brown adipose tissue (BAT) utilizes large amounts of fuel for thermogenesis with fatty acids and glucose being the major substrates for this process, but the mechanism by which fuel substrates are switched in response to changes in energy status is poorly understood. We here show that KLF15 increases fatty acid oxidation through the regulation of genes related to fatty acid utilization, whereas this transcription factor inhibits glucose oxidation via direct up‐regulation of PDK4 expression and attenuation of PDC activity, in HB2 differentiated brown adipocytes. Given that KLF15 expression in BAT was up‐regulated in response to fasting and down‐regulated after subsequent refeeding in mice and that these changes were accompanied by alterations in the expression of genes related to glucose and lipid utilization, KLF15 might play an important role in the regulation of fuel switching between glucose and fatty acids in response to changes in energy status in BAT.

Aims/Introduction Myopathy is a common complication of any diabetes type, consisting in failure to preserve mass and muscular function. Oxidative stress has been considered one of the main causes for this condition. This study aimed to search if Nicorandil, a KATP channel opener, could protect slow‐ and fast‐twitch diabetic rat muscles from oxidative stress, and to unveil its possible mechanisms. Materials and Methods Diabetes was induced in male Wistar rats by applying intraperitoneally streptozotocin (STZ) at 100 mg/kg doses. Nicorandil (3 mg/kg/day) was administered along 4 weeks. An insulin tolerance test and assessment of fasting blood glucose (FBG), TBARS, reduced (GSH), and disulfide (GSSG) glutathione levels, GSH/GSSG ratio, and mRNA expression of glutathione metabolism‐related genes were performed at end of treatment in soleus and gastrocnemius muscles. Results Nicorandil significantly reduced FBG levels and enhanced insulin tolerance in diabetic rats. In gastrocnemius and soleus muscles, Nicorandil attenuated the oxidative stress by decreasing lipid peroxidation (TBARS), increasing total glutathione and modulating GPX1‐mRNA expression in both muscle’s types. Nicorandil also increased GSH and GSH/GSSG ratio and downregulated the GCLC‐ and GSR‐mRNA in gastrocnemius, without significative effect on those enzymes’ mRNA expression in diabetic soleus muscle. Conclusions In diabetic rats, Nicorandil attenuates oxidative stress in slow‐ and fast‐twitch skeletal muscles by improving the glutathione system functioning. The underlying mechanisms for the modulation of glutathione redox state and the transcriptional expression of glutathione metabolism‐related genes seem to be fiber type‐dependent.

Aims/Introduction Type 2 diabetes mellitus is a group of metabolism abnormalities in carbohydrates and energy. Our aim was to investigate resting energy expenditure (REE) and blood glucose changes after biliary diversion in mice with diabetes. Materials and Methods Male mice with diabetes were randomly divided into biliary diversion and sham groups. REE was detected by indirect calorimetry, the levels of fasting blood glucose, total bile acids and triiodothyronine were analyzed. After mice were killed, the weight amount of brown adipose tissue (BAT) and gastrocnemius was measured, and the expression level of G protein‐coupled bile acid receptor and type 2 iodothyronine deiodinase in BAT and gastrocnemius were examined. Results The two groups of mice were pair‐fed, the bodyweights (P, Biliary diversion increased resting energy expenditure in mice with diabetes. Total bile acids increased after biliary diversion. G protein‐coupled bile acid receptor highly expressed in adipose tissue and gastrocnemius after surgery.

Aims/Introduction Glutamine is the most abundant amino acid in the circulation. In this study, we investigated cell signaling in the amplification of insulin secretion by glutamine. Materials and Methods Clonal pancreatic β‐cells MIN6‐K8, wild‐type B6 mouse islets, glutamate dehydrogenase (GDH) knockout clonal β‐cells (Glud1KOβCL), and glutamate‐oxaloacetate transaminase 1 (GOT1) knockout clonal β‐cells (Got1KOβCL) were studied. Insulin secretion from these cells and islets was examined under various conditions, and intracellular glutamine metabolism was assessed by metabolic flux analysis. Intracellular Ca2+ concentration ([Ca2+]i) was also measured. Results Glutamine dose‐dependently amplified insulin secretion in the presence of high glucose in both MIN6‐K8 cells and Glud1KOβCL. Inhibition of glutaminases, the enzymes that convert glutamine to glutamate, dramatically reduced the glutamine‐amplifying effect on insulin secretion. A substantial amount of glutamate was produced from glutamine through direct conversion by glutaminases. Glutamine also increased [Ca2+]i at high glucose, which was abolished by inhibition of glutaminases. Glutamic acid dimethylester (dm‐Glu), a membrane permeable glutamate precursor that is converted to glutamate in cells, increased [Ca2+]i as well as induced insulin secretion at high glucose. These effects of glutamine and dm‐Glu were dependent on calcium influx. Glutamine also induced insulin secretion in clonal β‐cells MIN6‐m14, which otherwise exhibit no insulin secretory response to glucose. Conclusions Glutamate converted from glutamine is an essential mediator that enhances calcium signaling in the glutamine‐amplifying effect on insulin secretion. Our data also suggest that glutamine exerts a permissive effect on glucose‐induced insulin secretion., Glutamine amplifies glucose‐induced insulin secretion through its conversion to glutamate. Glutamate converted from glutamine functions as an essential mediator that enhances calcium signaling in glutamine‐amplified insulin secretion.

Aims/Introduction Obesity is characterized by disturbed adipocytokine expression and insulin resistance in adipocytes. Growth arrest‐specific 6 (GAS6) is a gene encoding the Gas6 protein, which is expressed in fibroblasts, and its related signaling might be associated with adipose tissue inflammation, glucose intolerance and insulin resistance. The aim of this study was to investigate the associations among Gas6, adipocytokines and insulin resistance in adipocytes. Materials and Methods Mature Simpson Golabi Behmel Syndrome adipocytes were treated with high levels of insulin to mimic insulin resistance, and were examined for the expressions of Gas6, cytokines and adipocytokines from preadipocytes in differentiation. In an animal study, high‐fat diet‐induced obese mice were used to verify the Gas6 expression in vitro. Results During the differentiation of adipocytes, the expression of Gas6 gradually decreased, and was obviously downregulated with adipocyte inflammation and insulin resistance. Gas6 levels were found to be in proportion to the expression of adiponectin, which has been regarded as closely relevant to improved insulin sensitivity after metformin treatment. Similar results were also confirmed in the animal study. Conclusions Our results suggest that Gas6 might modulate the expression of adiponectin, and might therefore be associated with insulin resistance in adipose tissues., Gas6 might modulate the expression of adiponectin and could therefore be associated with insulin resistance in adipose tissues.

Aims/Introduction We aimed to determine whether glucokinase is required for β‐cell mass expansion induced by high‐starch diet (HSTD)‐feeding, as has been shown in its high‐fat diet‐induced expansion. Materials and Methods Eight‐week‐old male wild‐type (Gck+/+ ) or glucokinase haploinsufficient (Gck+/− ) mice were fed either a normal chow (NC) or an HSTD for 15 weeks. The bodyweight, glucose tolerance, insulin sensitivity, insulin secretion and β‐cell mass were assessed. Results Both HSTD‐fed Gck+/+ and Gck+/− mice had significantly higher bodyweight than NC‐fed mice. Insulin and oral glucose tolerance tests revealed that HSTD feeding did not affect insulin sensitivity nor glucose tolerance in either the Gck+/+ or Gck+/− mice. However, during the oral glucose tolerance test, the 15‐min plasma insulin concentration after glucose loading was significantly higher in the HSTD group than that in the NC group for Gck+/+ , but not for Gck+/− mice. β‐Cell mass was significantly larger in HSTD‐fed Gck+/+ mice than that in NC‐fed Gck+/+ mice. In contrast, the β‐cell mass of the HSTD‐fed Gck+/− mice was not different from that of the NC‐fed Gck+/− mice. Conclusions The results showed that HSTD feeding would increase pancreatic β‐cell mass and insulin secretion in Gck+/+ , but not Gck+/− mice. This observation implies that glucokinase in β‐cells would be required for the increase in β‐cell mass induced by HSTD feeding., Our results indicated that high‐starch diet feeding augmented insulin secretion and increased pancreatic beta‐cell mass in wild‐type mice, but not in the beta cell‐specific glucokinase haploinsufficient mice. These findings demonstrated that glucokinase in beta cells is required for increasing beta cell mass induced by high‐starch diet feeding.

Aims/Introduction Diabetic cardiomyopathy is a type of myocardial disease. It causes left ventricular hypertrophy, followed by diastolic and systolic dysfunction, eventually leading to congestive heart failure. However, the underlying mechanism still requires further elucidation. Materials and Methods A high‐glucose zebrafish model was constructed by administering streptozocin intraperitoneally to enhance the development of cardiomyopathy and then treated with adenosine monophosphate‐activated protein kinase (AMPK) activator. Cardiac structure and function, and protein and gene expression were then analyzed. Cardiomyocytes (CMs) culture in vitro using lentivirus were used for detection of AMPK, p53 and Krüppel‐like factor 2a (klf2a) gene expression. Results In the hyperglycemia group, electrocardiogram findings showed arrhythmia, echocardiography results showed heart enlargement and dysfunction, and many differences, such as increased apoptosis and myocardial fiber loss, were observed. The phospho‐AMPK and klf2a expression were downregulated, and p53 expression was upregulated. Activation of phospho‐AMPK reduced p53 and increased klf2a expression, alleviated apoptosis in CMs and improved cardiac function in the hyperglycemic zebrafish. In vitro knockdown system of AMPK, p53 and klf2a using lentivirus illustrated an increased p53 expression and decreased klf2a expression in CMs by inhibiting AMPK. Repression of p53 and upregulation of klf2a expression were observed, but no changes in the expression of AMPK and its phosphorylated type. Conclusions In the model of streptozocin‐induced hyperglycemia zebrafish, the reduction of phosphorylated AMPK increased p53, which led to KLF2a decrease to facilitate apoptosis of CMs, inducing the cardiac remodeling and cardiac dysfunction. These results can be reversed by AMPK activator, which means the AMPK–p53–klf2a pathway might be a potential target for diabetic cardiomyopathy intervention., The adenosine monophosphate‐activated protein kinase–p53–Krüppel‐like factor 2a signaling pathway was involved in hyperglycemia, induced cardiomyopathy in adult zebrafish and it might be a potential target for diabetic cardiomyopathy intervention.

Aims/Introduction β‐Cell dysfunction is a hallmark of type 2 diabetes. In a previous pilot study, we identified an association between genetic variants within the human DACH1 gene and young‐onset type 2 diabetes. Here, we characterized the function of dachb, the only dach homologue to be expressed in the pancreas, in developing zebrafish embryos. Materials and Methods We injected one‐cell stage embryos with a dachb‐morpholino (MO) or with the dachb‐MO and dachb messenger ribonucleic acid, and determined the effect on the development of the pancreatic islet. We also carried out quantitative polymerase chain reaction and ribonucleic acid sequencing on the dachb‐MO group to determine the effect of dachb knockdown on gene expression. Results MO‐mediated dachb knockdown resulted in impaired islet cell development, with a significant decrease in both the β‐cell and islet cell numbers. This islet developmental defect was rescued when embryos were co‐injected with dachb‐MO and dachb messenger ribonucleic acid. Knockdown of dachb was associated with a significant downregulation of the β‐cell specific marker gene, insa, and the somatostatin cell marker, sst2, as well as regulators of pancreas development, ptf1a, neuroD, pax6a and nkx6.1, and the cell cycle gene, insm1a. Furthermore, ribonucleic sequencing analysis showed an upregulation of genes enriched in the forkhead box O and mitogen‐activated protein kinase signaling pathways in the dachb‐MO group, when compared with the control groups. Conclusions Together, our results suggest the possible role of dachb in islet development in zebrafish., Morpholino (MO)‐mediated knock‐down of dachb resulted in impaired islet cell development, with a significant decrease in both the β‐cell and islet cell numbers in zebrafish. Embryos co‐injected with dachb mRNA and dachb‐MO expressed a similar phenotype as the uninjected control groups, indicating rescue of the islet developmental defect. The studies highlight the role of dachb in islet development in zebrafish.

Aims/Introduction Glucagon‐like peptide‐1 receptor agonists (GLP‐1RA) are used for treatment of type 2 diabetes mellitus worldwide. However, some patients do not respond well to the therapy, and caution must be taken for certain patients, including those with reduced insulin secretory capacity. Thus, it is clinically important to predict the efficacy of GLP‐1RA therapy. GLP‐1R‐targeted imaging has recently emerged to visualize and quantify β‐cells. We investigated whether GLP‐1R‐targeted imaging can predict the efficacy of GLP‐1RA treatment. Materials and Methods We developed 111Indium‐labeled exendin‐4 derivative (111In‐Ex4) as a GLP‐1R‐targeting probe. Diabetic mice were selected from NONcNZO10/LtJ male mice that were fed for different durations with 11% fat chow. After 3‐week administration of dulaglutide as GLP‐1RA therapy, mice with non‐fasting blood glucose levels 300 mg/dL were defined as responders and non‐responders, respectively. In addition, ex vivo 111In‐Ex4 pancreatic accumulations (111In‐Ex4 pancreatic values) were examined. Results The non‐fasting blood glucose levels after treatment were 172.5 ± 42.4 mg/dL in responders (n = 4) and 330.8 ± 20.7 mg/dL in non‐responders (n = 5), respectively. Ex vivo 111In‐Ex4 pancreatic values showed significant correlations with post‐treatment glycohemoglobin and glucose area under curve during an oral glucose tolerance test (R 2 = 0.76 and 0.80; P, 111Indium‐labeled exendin‐4 derivative pancreatic uptake, which is significantly correlated with glucagon‐like peptide‐1 receptor messenger ribonucleic acid expression and β‐cell mass, reflected in vivo glucose‐lowering effects of dulaglutide in diabetic mice. Glucagon‐like peptide‐1 receptor‐targeted imaging can be utilized for better selection of antidiabetic medications in patients with type 2 diabetes.

Aims/Introduction Abnormalities in Ca2+ signaling have a key role in hemodynamic dysfunction in diabetic heart. The purpose of this study was to explore the effects of streptozotocin (STZ)‐induced diabetes on Ca2+ signaling in epicardial (EPI) and endocardial (ENDO) cells of the left ventricle after 5–6 months of STZ injection. Materials and Methods Whole‐cell patch clamp was used to measure the L‐type Ca2+ channel (LTCC) and Na+/Ca2+ exchanger currents. Fluorescence photometry techniques were used to measure intracellular free Ca2+ concentration. Results Although the LTCC current was not significantly altered, the amplitude of Ca2+ transients increased significantly in EPI‐STZ and ENDO‐STZ compared with controls. Time to peak LTCC current, time to peak Ca2+ transient, time to half decay of LTCC current and time to half decay of Ca2+ transients were not significantly changed in EPI‐STZ and ENDO‐STZ myocytes compared with controls. The Na+/Ca2+ exchanger current was significantly smaller in EPI‐STZ and in ENDO‐STZ compared with controls. Conclusions STZ‐induced diabetes resulted in an increase in amplitude of Ca2+ transients in EPI and ENDO myocytes that was independent of the LTCC current. Such an effect can be attributed, at least in part, to the dysfunction of the Na+/Ca2+ exchanger. Additional studies are warranted to improve our understanding of the regional impact of diabetes on Ca2+ signaling, which will facilitate the discovery of new targeted treatments for diabetic cardiomyopathy., This is the first report to investigate the effects of streptozotocin‐induced diabetes on L‐type Ca2+ current simultaneously measured with Ca2+ transients in epicardial and endocardial left ventricular myocytes isolated from rats 5–6 months after the induction of diabetes. Streptozotocin‐induced diabetes causes a progressive increase in the amplitude of endocardial and epicardial myocyte Ca2+ transients that is independent of the L‐type Ca2+ current. The effect of streptozotocin‐induced diabetes on Ca2+ handling might be attributed, at least in part, to the dysfunction of the Na+/Ca2+ exchanger.

Aims/Objective The development of type 2 diabetes is a result of insulin resistance in various tissues, including skeletal muscle and liver. Apolipoprotein M (ApoM) plays an important role in the function of high‐density lipoprotein, and also affects hepatic lipid and glucose metabolism. In this study, we aimed to investigate whether ApoM overexpression modulates glucose metabolism and improves insulin sensitivity. Materials and Methods The Goto‐Kakizaki (GK) rats were transfected with adeno‐associated virus (AAV) encoding rat ApoM gene or control blank. The oral glucose tolerance test (OGTT) and hyperinsulinemic‐euglycemic clamp (HEC) experiment were used to assess the insulin sensitivity of GK rats. Results The results show that ApoM messenger ribonucleic acid and protein were significantly overexpressed in the pancreatic tissues. Overexpression of ApoM decreased fasting blood glucose and random blood glucose, improved glucose tolerance, and increased bodyweight and insulin levels in GK rats. The glucose infusion rate of rats in the AAV encoding rat ApoM gene group during HEC test was 1.04‐, 1.23‐ and 1.95‐fold higher than that in the AAV control blank group at 1–3 weeks after injection of AAV, respectively. A Wes‐ProteinSimple assay and quantification was carried out to assess phosphorylated protein kinase B/protein kinase B protein levels in the muscle tissues of ApoM‐overexpressing GK rats, and they were found to be higher than those of the control group at the seventh week after AAV injection. Conclusions ApoM overexpression through adeno‐associated virus gene transfer might improve insulin secretion and insulin sensitivity in GK rats., Apolipoprotein M overexpression decreased fasting blood glucose and random blood glucose, and increased insulin in Goto‐Kakizaki rats. Apolipoprotein M overexpression increased the phosphorylated protein kinase B/protein kinase B protein levels in the muscle tissues of Goto‐Kakizaki rats. Apolipoprotein M overexpression might improve insulin sensitivity in Goto‐Kakizaki rats.

Aims/Introduction Incretin therapy is a common treatment for type 2 diabetes mellitus. We have previously reported an anti‐prostate cancer effect of glucagon‐like peptide‐1 receptor (GLP‐1R) agonist exendin‐4. The attenuation of cell proliferation in the prostate cancer cell line was dependent on GLP‐1R expression. Here, we examined the relationship between human prostate cancer severity and GLP‐1R expression, as well as the effect of forced expression of GLP‐1R using a lentiviral vector. Materials and Methods Prostate cancer tissues were extracted by prostatectomy and biopsy. GLP‐1R was overexpressed in ALVA‐41 cells using a lentiviral vector (ALVA‐41‐GLP‐1R cells). GLP‐1R expression was detected by immunohistochemistry and quantitative polymerase chain reaction. Cell proliferation was examined by growth curves and bromodeoxyuridine incorporation assays. Cell cycle distribution and regulators were examined by flow cytometry and western blotting. In vivo experiments were carried out using a xenografted model. Results GLP‐1R expression levels were significantly inversely associated with the Gleason score of human prostate cancer tissues. Abundant GLP‐1R expression and functions were confirmed in ALVA‐41‐GLP‐1R cells. Exendin‐4 significantly decreased ALVA‐41‐GLP‐1R cell proliferation in a dose‐dependent manner. DNA synthesis and G1‐to‐S phase transition were inhibited in ALVA‐41‐GLP‐1R cells. SKP2 expression was decreased and p27Kip1 protein was subsequently increased in ALVA‐41‐GLP‐1R cells treated with exendin‐4. In vivo experiments carried out by implanting ALVA‐41‐GLP‐1R cells showed that exendin‐4 decreased prostate cancer growth by activation of GLP‐1R overexpressed in ALVA41‐GLP‐1R cells. Conclusions Forced expression of GLP‐1R attenuates prostate cancer cell proliferation by inhibiting cell cycle progression in vitro and in vivo. Therefore, GLP‐1R activation might be a potential therapy for prostate cancer., Forced expression of glucagon‐like peptide‐1 receptor attenuates prostate cancer cell proliferation by inhibiting cell cycle progression both in vitro and in vivo. Glucagon‐like peptide‐1 receptor activation might be a potential therapy for not only type 2 diabetes, but also prostate cancer.

Background Although the measuring free cortisol is ideal for assessment of hypothalamicpituitary-adrenal function, it is not routinely measured. Salivary cortisol correlates well with the biologically active free cortisol. Therefore, this study measured the morning basal as well as adrenocorticotropic hormone-stimulated salivary cortisol levels in mechanically ventilated patients and compared the results with non-critically ill patients. Methods We prospectively enrolled 49 mechanically ventilated patients and 120 patients from the outpatient clinic. Serum and saliva samples were collected between 8 AM and 10 AM. Salivary cortisol levels were measured using an enzyme immunoassay kit. The salivary samples were insufficient in 15 mechanically ventilated patients (30.6%), and these patients were excluded from the final analysis. Results Mechanically ventilated patients (n=34) were significantly older and had lower body mass index and serum albumin levels and higher serum creatinine levels than non-critically ill patients (n=120). After adjustment for these parameters, both basal and stimulated salivary and serum cortisol levels were higher in mechanically ventilated patients. The increase in cortisol was not significantly different between the two groups. Serum cortisol levels showed a positive correlation with salivary cortisol levels. Among mechanically ventilated patients, both basal serum and salivary cortisol levels were lower in survivors than in non-survivors. Conclusions Both basal total serum and salivary cortisol levels were elevated in mechanically ventilated patients and in non-survivors.

Aims/Introduction Sodium–glucose cotransporter 2 inhibitor (SGLT2i) lowers blood glucose and causes a whole‐body energy deficit by boosting renal glucose excretion, thus affecting glucose and energy metabolism. This energy deficit not only decreases bodyweight, but also increases food intake. This food intake increase offsets the SGLT2i‐induced bodyweight decrease, but the effect of the food intake increase on the SGLT2i regulation of glucose metabolism remains unclear. Materials and Methods We administered SGLT2i (luseogliflozin) for 4 weeks to hepatic gluconeogenic enzyme gene G6pc reporter mice with/without obesity, which were either fed freely or under a 3‐hourly dietary regimen. The effect of feeding condition on the gluconeogenic response to SGLT2i was evaluated by plasma Gaussia luciferase activity, an index of the hepatic gluconeogenic response, in G6pc reporter mice. Energy expenditure was measured by indirect calorimetry. Results In the lean mice under controlled feeding, SGLT2i decreased bodyweight and plasma glucose, and increased the hepatic gluconeogenic response while decreasing blood insulin. SGLT2i also increased oxygen consumption under controlled feeding. However, free feeding negated all of these effects of SGLT2i. In the obese mice, SGLT2i decreased bodyweight, blood glucose and plasma insulin, ameliorated the upregulated hepatic gluconeogenic response, and increased oxygen consumption under controlled feeding. Under free feeding, although blood glucose was decreased and plasma insulin tended to decrease, the effects of SGLT2i – decreased bodyweight, alleviation of the hepatic gluconeogenic response and increased oxygen consumption – were absent. Conclusions Food intake management is crucial for SGLT2i to affect glucose and energy metabolism during type 2 diabetes treatment., This study showed that increased food intake negates the regulatory effects of sodium–glucose cotransporter 2 inhibitor on glucose metabolism and hepatic gluconeogenesis. Food intake management is crucial for sodium–glucose cotransporter 2 inhibitor to affect glucose and energy metabolism in the treatment of type 2 diabetes.

Aims/Introduction Under irremediable endoplasmic reticulum (ER) stress, hyperactivated inositol‐requiring enzyme 1α (IRE1α) triggers the terminal unfolded protein response (T‐UPR), causing crucial cell dysfunction and apoptosis. We hypothesized that nicotinic acetylcholine receptor (nAChR) signaling regulates IRE1α activation to protect β‐cells from the T‐UPR under ER stress. Materials and Methods The effects of nicotine on IRE1α activation and key T‐UPR markers, thioredoxin‐interacting protein and insulin/proinsulin, were analyzed by real‐time polymerase chain reaction and western blotting in rat INS‐1 and human EndoC‐βH1 β‐cell lines. Doxycycline‐inducible IRE1α overexpression or ER stress agents were used to induce IRE1α activation. An α7 subunit‐specific nAChR agonist (PNU‐282987) and small interfering ribonucleic acid for α7 subunit‐specific nAChR were used to modulate nAChR signaling. Results Nicotine inhibits the increase in thioredoxin‐interacting protein and the decrease in insulin 1/proinsulin expression levels induced by either forced IRE1α hyperactivation or ER stress agents. Nicotine attenuated X‐box‐binding protein‐1 messenger ribonucleic acid site‐specific splicing and IRE1α autophosphorylation induced by ER stress. Furthermore, PNU‐282987 attenuated T‐UPR induction by either forced IRE1α activation or ER stress agents. The effects of nicotine on attenuating thioredoxin‐interacting protein and preserving insulin 1 expression levels were attenuated by pharmacological and genetic inhibition of α7 nAChR. Finally, nicotine suppressed apoptosis induced by either forced IRE1α activation or ER stress agents. Conclusions Our findings suggest that nAChR signaling regulates IRE1α activation to protect β‐cells from the T‐UPR and apoptosis under ER stress partly through α7 nAChR. Targeting nAChR signaling to inhibit the T‐UPR cascade may therefore hold therapeutic promise by thwarting β‐cell death in diabetes., Nicotinic acetylcholine receptor signaling attenuated inositol‐requiring enzyme 1α signaling and Terminal Unfolded Protein Response (T‐UPR) under irremediable endoplasmic reticulum stress in rat INS‐1 and human EndoC‐βH1 β‐cell lines. The effects of nicotine on terminal unfolded protein response were reduced by pharmacological and genetic inhibition of nicotinic acetylcholine receptor α7 subunit. Finally, nicotine suppressed apoptosis induced by either forced inositol requiring enzyme 1α activation or endoplasmic reticulum stress.

Aims/Introduction Pancreatic β‐cells are sensitive to endoplasmic reticulum (ER) stress, which has a major role in the context of β‐cell death. Adrenomedullin (ADM) has been shown to exert a cytoprotective effect under various pathophysiological conditions. Several studies have suggested that thiazolidinediones have protective effects on β‐cells. During the course to elucidate the molecular mechanisms by which pioglitazone prevents β‐cell death, ADM emerged as a candidate. Here, we studied the regulation of ADM expression, including the effects of pioglitazone, and its role in pancreatic islets. Materials and Methods We analyzed ADM expression in islet cell lines treated with pioglitazone. The effects of ER stress on ADM and ADM receptor expressions were investigated by analyzing thapsigargin‐treated MIN6 cells and islets isolated from Wfs1−/− and db/db mice. To study the anti‐apoptotic effect of ADM, ER stress‐exposed MIN6 cells were treated with ADM peptides or transfected with ADM expression plasmid. Results Pioglitazone increased the production and secretion of ADM in islets through peroxisome‐proliferator activated receptor‐γ‐dependent mechanisms. Thapsigargin treatment increased expressions of both ADM and ADM receptor, composed of Ramp2, Ramp3 and Crlr, in MIN6 cells. ADM and ADM receptor expressions were also increased in isolated islets from Wfs1−/− and db/db mice. ADM peptides and ADM overexpression protected MIN6 cells from thapsigargin‐induced apoptosis. Conclusions ER stress stimulates ADM production and secretion in islets. ADM signaling might protect β‐cells from ER stress‐induced apoptosis, and might be one of the self‐protective mechanisms. β‐Cell protection by pioglitazone is partly through induction of ADM. ADM‐based therapy could be a novel strategy for treating diabetes., Pioglitazone increased the production and secretion of adrenomedullin (ADM) in pancreatic islets through peroxisome‐proliferator activated receptor‐γ‐dependent mechanisms in vivo and in vitro. The expression levels of Adm and the ADM receptor, which is composed of Ramp2, Ramp3 and Crlr, were significantly increased in islets isolated from Wfs1−/− and db/db mice, and were also remarkably induced by thapsigargin‐evoked endoplasmic reticulum stress in a β‐cell line, MIN6 cells. ADM peptides and ADM overexpression protected MIN6 cells from thapsigargin‐induced apoptosis, partly through intracellular cyclic adenosine monophosphate elevation.

Aims/Introduction Pancreatic islets are heterogenous. To clarify the relationship between islet heterogeneity and incretin action in the islets, we studied gene expression and metabolic profiles of non‐large and enlarged islets of the Zucker fatty diabetes mellitus rat, an obese diabetes model, as well as incretin‐induced insulin secretion (IIIS) in these islets. Materials and Methods Pancreatic islets of control (fa/+) and fatty (fa/fa) rats at 8 and 12 weeks‐of‐age were isolated. The islets of fa/fa rats at 12 weeks‐of‐age were separated into non‐large islets (≤200 μm in diameter) and enlarged islets (>300 μm in diameter). Morphological analyses, insulin secretion experiments, transcriptome analysis, metabolome analysis and oxygen consumption analysis were carried out on these islets. Results The number of enlarged islets was increased with age in fatty rats, and IIIS was significantly reduced in the enlarged islets. Markers for β‐cell differentiation were markedly decreased in the enlarged islets, but those for cell proliferation were increased. Glycolysis was enhanced in the enlarged islets, whereas the tricarboxylic acid cycle was suppressed. The oxygen consumption rate under glucose stimulation was reduced in the enlarged islets. Production of glutamate, a key signal for IIIS, was decreased in the enlarged islets. Conclusions The enlarged islets of Zucker fatty diabetes mellitus rats, which are defective for IIIS, show tumor cell‐like metabolic features, including a dedifferentiated state, accelerated aerobic glycolysis and impaired mitochondrial function. The age‐dependent increase in such islets could contribute to the pathophysiology of obese diabetes., The enlarged islets of Zucker fatty diabetes mellitus rats, which are defective for incretin‐induced insulin secretion, show tumor cell‐like metabolic features, including a dedifferentiated state, accelerated aerobic glycolysis and impaired mitochondrial function. The age‐dependent increase in such islets could contribute to the pathophysiology of obese diabetes.

Aims/Introduction The association between diabetes and periodontal disease is considered to be bidirectional. However, there is still controversy surrounding the relationship between periodontal disease and type 1 diabetes. We investigated whether insulin improves periodontitis without any local treatments for periodontitis under type 1 diabetes conditions using the ligature‐induced experimental periodontitis model. Materials and Methods Type 1 diabetic rats were induced by streptozotocin injection. Experimental periodontitis was induced by ligature in normal and diabetic rats. Half of the diabetic rats were treated with insulin. Two weeks after the ligature, periodontitis was evaluated. Results Insulin treatment significantly improved inflammatory cell infiltration and inflammatory cytokine gene expression, leading to suppression of alveolar bone loss, in the periodontitis of diabetic rats. Insulin also suppressed the periodontitis‐increased nitric oxide synthase‐positive cells in periodontal tissue of the diabetic rats. Even without induction of periodontitis, diabetic rats showed decreased gingival blood flow and an increased number of nitric oxide synthase‐positive cells in the gingiva and alveolar bone loss compared with normal rats, all of which were ameliorated by insulin treatment. We further confirmed that insulin directly suppressed lipopolysaccharide‐induced inflammatory cytokine expressions in THP‐1 cells. Conclusions There were abnormalities of periodontal tissue even without the induction of periodontitis in streptozotocin‐induced diabetic rats. Insulin treatment significantly ameliorated periodontitis without local periodontitis treatment in diabetic rats. These data suggest the therapeutic impacts of insulin on periodontitis in type 1 diabetes., There was a slight increase in the numbers of inflammatory cells in the gingiva on the periodontitis side of normal rats and on the control side of diabetic rats. The inflammatory cells were markedly increased on the periodontitis side of diabetic rats. Insulin treatment decreased the inflammatory cells in the periodontitis gingiva of diabetic rats.

Aims/Introduction Electronegative low‐density lipoprotein (L5) is the most atherogenic fraction of low‐density lipoprotein and is elevated in people with metabolic syndrome (MetS), whereas the retinol‐binding protein 4 receptor (stimulated by retinoic acid 6 [STRA6]) cascade is disrupted in various organs of patients with obesity‐related diseases. Our objective was to investigate whether L5 from MetS patients capably induces pathogenesis of aorta through disrupting the STRA6 cascade. Material and Methods We examined the in vivo and in vitro effects of L5 on the STRA6 cascade and aortic atherogenic markers. To investigate the role of this cascade on atherosclerotic formation, crbp1 transfection was carried out in vitro. Results This study shows that L5 activates atherogenic markers (p38 mitogen‐activated protein kinases, pSmad2 and matrix metallopeptidase 9) and simultaneously suppresses STRA6 signals (STRA6, cellular retinol‐binding protein 1, lecithin‐retinol acyltransferase, retinoic acid receptor‐α and retinoid X receptor‐α) in aortas of L5‐injected mice and L5‐treated human aortic endothelial cell lines and human aortic smooth muscle cell lines. These L5‐induced changes of the STRA6 cascade and atherogenic markers were reversed in aortas of LOX1−/− mice and in LOX1 ribonucleic acid‐silenced human aortic endothelial cell lines and human aortic smooth muscle cell lines. Furthermore, crbp1 gene transfection reversed the disruption of the STRA6 cascade, the phosphorylation of p38 mitogen‐activated protein kinases and Smad2, and the elevation of matrix metallopeptidase 9 in L5‐treated human aortic endothelial cell lines. Conclusions This study shows that L5 from MetS patients induces atherogenic markers by disrupting STRA6 signaling. Suppression of STRA6 might be one novel pathogenesis of aorta in patients with MetS., Dyslipoproteinemia can cause aortic injuries by utilizing electronegative low‐density lipoprotein caused disruption of stimulated by retinoic acid 6 signaling. The suppression of stimulated by retinoic acid 6 might be one novel pathogenesis of aorta in people with metabolic syndrome.

Aims/Introduction Diabetic hearts are more vulnerable to ischemia‐reperfusion injury (I/RI). The activation of nucleotide‐binding oligomerization domain‐like receptor protein 3 (NLRP3) inflammasome can mediate the inflammatory process, and hence might contribute to myocardial I/RI. Activation of autophagy can eliminate excess reactive oxygen species and alleviate myocardial I/RI in diabetes. The present study aimed to investigate whether the activation of autophagy can alleviate diabetic myocardial I/RI through inhibition of NLRP3 inflammasome activation. Materials and Methods A dose of 65 mg/kg streptozotocin was given by tail vein injection to establish a type 1 diabetes model in the rats. The left anterior descending coronary artery was ligated for 30 min followed by reperfusion for 2 h to establish a myocardial I/RI model. H9C2 cardiomyocytes were exposed to high glucose (33 mmol/L) and subjected to hypoxia–reoxygenation (6 h hypoxia followed by 4 h reoxygenation). Results The diabetic rats showed significant inhibition of cardiac autophagy (decreased LC3‐II/I and increased p62) that was concomitant with increased activation of NLRP3 inflammasome (increased NLRP3, apoptosis‐related spots protein cleaved caspase‐1, interleukin‐18, interleukin‐1β) and more severe myocardial I/RI (elevated creatine kinase myocardial band, lactate dehydrogenase and larger infarct size). However, administration of rapamycin, an inhibitor of the autophagy, to activate autophagy resulted in the inhibition of NLRP3 inflammasome, and finally alleviated myocardial I/RI. In vitro, high glucose inhibited autophagy, while activating NLRP3 inflammasome in H9C2 cardiomyocytes and aggravating hypoxia–reoxygenation injury, but rapamycin reversed these adverse effects of high glucose. Conclusion Activation of autophagy can suppress the formation of NLRP3 inflammasome, which in turn attenuates myocardial ischemia‐reperfusion injury in diabetic rats., Autophagy was inhibited in the myocardium of 8‐week diabetic rats, with activated NLRP3 inflammasome and aggravated myocardial ischemia‐reperfusion injury. Activation of autophagy can reduce the activation of NLRP3 inflammasome and therefore increase the tolerance to myocardial ischemia in diabetes.

Aims/Introduction Taste receptors, T1rs and T2rs, and the taste‐selective G‐protein, α‐gustducin, are expressed outside the taste‐sensing system, such as enteroendocrine L cells. Here, we examined whether α‐gustducin also affects nutrition sensing and insulin secretion by pancreatic β‐cells. Materials and Methods The expression of α‐gustducin and taste receptors was evaluated in β‐cell lines, and in rat and mouse islets either by quantitative polymerase chain reaction or fluorescence immunostaining. The effects of α‐gustducin knockdown on insulin secretion and on cyclic adenosine monophosphate and intracellular Ca2+ levels in rat INS‐1 cells were estimated. Sucralose (taste receptor agonist)‐induced insulin secretion was investigated in INS‐1 cells with α‐gustducin suppression and in islets from mouse disease models. Results The expression of Tas1r3 and α‐gustducin was confirmed in β‐cell lines and pancreatic islets. Basal levels of cyclic adenosine monophosphate, intracellular calcium and insulin secretion were significantly enhanced with α‐gustducin knockdown in INS‐1 cells. The expression of α‐gustducin was decreased in high‐fat diet‐fed mice and in diabetic db/db mice. Sucralose‐induced insulin secretion was not attenuated in INS‐1 cells with α‐gustducin knockdown or in mouse islets with decreased expression of α‐gustducin. Conclusions α‐Gustducin is involved in the regulation of cyclic adenosine monophosphate, intracellular calcium levels and insulin secretion in pancreatic β‐cells in a manner independent of taste receptor signaling. α‐Gustducin might play a novel role in β‐cell physiology and the development of type 2 diabetes., The expression of α‐gustducin in pancreatic islets decreases with high‐fat diet feeding and obesity.

Aims/Introduction Clinical studies have shown that treatment with inhibitors of sodium–glucose cotransporter 2 (SGLT2) significantly increases the hematocrit in patients with type 2 diabetes. To investigate whether SGLT2 inhibitors directly promote erythropoietin production independently on blood glucose reduction, the hematopoietic effect of the specific SGLT2 inhibitor, luseogliflozin, was examined in non‐diabetic rats with renal anemia. Materials and Methods Renal anemia was induced by treatment with adenine (200 or 600 mg/kg/day, orally for 10 days) in non‐diabetic Wistar–Kyoto or Wistar rats, respectively. Luseogliflozin (10 mg/kg bodyweight) or vehicle (0.5% carboxymethyl cellulose) was then administered for 6 weeks. The hematocrit and the hemoglobin (Hb), blood urea nitrogen, plasma creatinine, and plasma erythropoietin levels were monitored. Results Treatment with adenine decreased the hematocrit and the Hb level, which were associated with increases in the blood urea nitrogen and plasma creatinine levels. In Wistar–Kyoto rats treated with 200 mg/kg/day adenine, administration of luseogliflozin induced glycosuria, but did not change the blood urea nitrogen, plasma creatinine levels, hematocrit, Hb or plasma erythropoietin levels. Similarly, luseogliflozin treatment failed to change the hematocrit or the Hb levels in Wistar rats with renal anemia induced by 600 mg/kg/day of adenine. Plasma erythropoietin concentrations were also not different between luseogliflozin‐ and vehicle‐treated rats. Similarly, in human erythropoietin‐producing cells derived from pluripotent stem cells, luseogliflozin treatment did not change the erythropoietin level in the medium. Conclusions These data suggest that SGLT2 inhibitor fails to exert hematopoietic effects in non‐diabetic conditions., Treatment with a sodium–glucose cotransporter 2 inhibitor fails to increase erythropoietin in non‐diabetic rats with renal anemia.

Aims/Introduction Transplantation of stem cells promotes axonal regeneration and angiogenesis in a paracrine manner. In the present study, we examined whether the secreted factors in conditioned medium of stem cells from human exfoliated deciduous teeth (SHED‐CM) had beneficial effects on diabetic polyneuropathy in mice. Materials and Methods Conditioned medium of stem cells from human exfoliated deciduous teeth was collected 48 h after culturing in serum‐free Dulbecco's modified Eagle's medium (DMEM), and separated into four fractions according to molecular weight. Dorsal root ganglion neurons from C57BL/6J mice were cultured with SHED‐CM or DMEM to evaluate the effect on neurite outgrowth. Streptozotocin‐induced diabetic mice were injected with 100 μL of SHED‐CM or DMEM into the unilateral hindlimb muscles twice a week over a period of 4 weeks. Peripheral nerve functions were evaluated by the plantar test, and motor and sensory nerve conduction velocities. Intraepidermal nerve fiber densities, capillary number‐to‐muscle fiber ratio, capillary blood flow and morphometry of sural nerves were also evaluated. Results Conditioned medium of stem cells from human exfoliated deciduous teeth significantly promoted neurite outgrowth of dorsal root ganglion neurons compared with DMEM. Among four fractions of SHED‐CM, the only fraction of, In this study, we examined whether the secreted factors could be collected from conditioned medium of stem cells from human exfoliated deciduous teeth had beneficial effects on diabetic polyneuropathy. Conditioned medium of stem cells from human exfoliated deciduous teeth might have a therapeutic effect on diabetic polyneuropathy through promoting neurite outgrowth, and the increase in capillaries might contribute to the improvement of neural function.

Aims/Introduction Although increased reactive oxygen species (ROS) generation is a major mechanism leading to cardiac remodeling in diabetes mellitus, research into the effects of anti‐oxidation on diabetic cardiac remodeling remains scarce and controversial. Glucagon‐like peptide‐1 (GLP‐1) shows potential anti‐oxidative effects besides lowering blood glucose. The objective of this research was to investigate the effects of GLP‐1 on cardiac remodeling and the molecular mechanism involved in diabetes mellitus. Materials and Methods Streptozotocin‐induced diabetic rats received exenatide treatment for 3 months. Cardiac function, cardiac weight index and myocardial interstitial fibrosis were measured. Cardiomyocytes were cultured in high‐glucose medium with GLP‐1 treatment. The ROS production, apoptosis and the levels of mammalian target of rapamycin complex 1/p70 ribosomal protein S6 kinase protein expression in cardiomyocytes were analyzed. Results Experimental diabetes mellitus showed impaired cardiac diastolic function, increased brain natriuretic peptide expression and increased interstitial collagen deposition in the myocardium, which were ameliorated by exenatide treatment. Exenatide reduced myocardial ROS production and apoptosis in diabetes mellitus. Also, high glucose‐induced ROS generation and apoptosis in cardiomyocytes were inhibited by GLP‐1, as well as the levels of mammalian target of rapamycin complex 1/p70 ribosomal protein S6 kinase phosphorylation. Furthermore, GLP‐1 treatment upregulated adenosine monophosphate‐activated protein kinase activity in high‐glucose‐induced cardiomyocyte. Conclusions Glucagon‐like peptide‐1 protects the cardiomyocytes from oxidative stress and apoptosis in diabetes mellitus, which might contribute to the improvement of cardiac remodeling. The cardiac protection of GLP‐1 might be dependent on inhibition of mammalian target of rapamycin complex 1/p70 ribosomal protein S6 kinase, through an adenosine monophosphate‐activated protein kinase‐mediated pathway., Glucagon‐like peptide‐1 protects cardiomyocytes against oxidative stress, apoptosis and the resultant myocardial fibrosis in diabetes mellitus, which might contribute to the improvement of cardiac remodeling. The protective effects of glucagon‐like peptide‐1 are dependent on downstream inhibition of mammalian target of rapamycin complex 1/p70 ribosomal protein S6 kinase, through an adenosine monophosphate‐activated protein kinase‐mediated pathway.

Introduction As a lytic inflammatory cell death, pyroptosis has been recently described but has not been unequivocally elucidated in diabetic nephropathy (DN). VX‐765 is a safe and effective inhibitor of caspase‐1, that was well tolerated in a phase II clinical trial in patients with epilepsy, but its application in DN is still undefined. Materials and Methods Immunoblot, co‐immunoprecipitation, confocal microscope and flow cytometry were used to analyze the effects of glucose on pyroptosis in renal tubular epithelia (HK‐2). In vitro, selective caspase‐1 inhibitors VX‐765 and Z‐YVAD‐FMK were administered. Pyroptosis and fibrogenesis were determined by immunoblot, ELISA, cytotoxicity assay and flow cytometry. In vivo, diabetic mice were administered with 100 mg/kg VX‐765. Renal function, pathological changes, and the expressions of NLRC4, GSDMD, IL‐1β, collagen I, fibronectin and CD45 in renal cortex were evaluated. Results We identified NLRC4 as a sensor for caspase‐1 activation. Moreover, we provided morphological and molecular evidence for pyroptosis in glucose‐stressed tubular cells, including ballooned cell membrane, caspase‐1 immunoreactivity, GSDMD cleavage, and the release of inflammatory cytokine and cellular contents. All these effects were prevented by treatment with VX‐765 or Z‐YVAD‐FMK, confirming that caspase‐1 effectively regulates the occurrence of pyroptosis in HK‐2 cells. In vivo, treatment of diabetic animals with VX‐765 ameliorated renal function, suppressed inflammatory cell infiltration and pyroptosis‐associated protein expression, and mitigated tubulointerstitial fibrosis. Conclusions This work revealed that caspase‐1‐mediated pyroptosis drives renal inflammation and fibrosis in diabetes. Our results are the first demonstration of VX‐765 representing a promising therapeutic opportunity for alleviating the progression of DN., VX‐765 represents a promising therapeutic opportunity for alleviating the progression of diabetic nephropathy via inhibiting caspase‐1‐mediated pyroptosis, renal inflammation and fibrosis.

Aims/Introduction Chronic inflammation of the liver is often observed with obesity or type 2 diabetes. In these pathological conditions, the immunological cells, such as macrophages, play important roles in the development or growth of liver cancer. Recently, it was reported that hypoxia‐inducible factor‐1α (HIF‐1α) is a key molecule for the acquisition of inflammatory M1 polarity of macrophages. In the present study, we examined the effects of altered macrophage polarity on obesity‐ and diabetes‐associated liver cancer using macrophage‐specific HIF‐1α knockout (KO) mice. Materials and Methods To induce liver cancer in the mice, diethylnitrosamine, a chemical carcinogen, was used. Both KO mice and wild‐type littermates were fed either a high‐fat diet (HFD) or normal chow. They were mainly analyzed 6 months after HFD feeding. Results Development of liver cancer after HFD feeding was 45% less in KO mice than in wild‐type littermates mice. Phosphorylation of extracellular signal‐regulated kinase 2 was also lower in the liver of KO mice. Those effects of HIF‐1α deletion in macrophages were not observed in normal chow‐fed mice. Furthermore, the size of liver tumors did not differ between KO and wild‐type littermates mice, even those on a HFD. These results suggest that the activation of macrophage HIF‐1α by HFD is involved not in the growth, but in the development of liver cancer with the enhanced oncogenic extracellular signal‐regulated kinase 2 signaling in hepatocytes. Conclusions The activation of macrophage HIF‐1α might play important roles in the development of liver cancer associated with diet‐induced obesity and diabetes.

Aims/Introduction Fat‐specific protein 27 (FSP27) α is the major isoform of FSP27 in white adipose tissue (WAT), and is essential for large unilocular lipid droplet (LD) formation in white adipocytes. In contrast, FSP27β is abundantly expressed in brown adipose tissue (BAT), and plays an important role in small multilocular LD formation. In FSP27 KO mice in which FSP27α and β are both depleted, WAT is characterized by multilocular LD formation, and by increased mitochondrial abundance and energy expenditure, whereas BAT conversely manifests large oligolocular LDs and reduced energy expenditure. Materials and Methods We investigated the effects of autophagy in WAT and BAT of wild type (WT) and FSP27 knockout (KO) mice. In addition, we examined the effects of FSP27α and FSP27β to the induction of autophagy in COS cells. Results Food deprivation induced autophagy in BAT of WT mice, as well as in WAT of FSP27 KO mice, suggesting that enhanced autophagy is characteristic of adipocytes with small multilocular LDs. Pharmacological inhibition of autophagy attenuated the fasting‐induced loss of LD area in adipocytes with small multilocular LDs (BAT of WT mice and WAT of FSP27 KO mice), without affecting that in adipocytes with large unilocular or oligolocular LDs (WAT of WT mice or in BAT of FSP27 KO mice). Overexpression of FSP27α inhibited autophagy induction by serum deprivation in COS cells, whereas that of FSP27β had no such effect. Conclusions The present results thus showed that FSP27α inhibits autophagy and might thereby contribute to the energy‐storage function of WAT.

Aims/Introduction Dental pulp stem cells (DPSCs) can be easily obtained from teeth for general orthodontic reasons. We have previously reported the therapeutic effects of DPSC transplantation for diabetic polyneuropathy. As abundant secretomes from DPSCs are considered to play a central role in the improvement of diabetic polyneuropathy, we investigated whether direct injection of DPSC‐conditioned media (DPSC‐CM) into hindlimb skeletal muscles ameliorates diabetic polyneuropathy in diabetic rats. Materials and Methods DPSCs were isolated from the dental pulp of Sprague–Dawley rats. Eight weeks after the induction of diabetes, DPSC‐CM was injected into the unilateral hindlimb skeletal muscles in both normal and diabetic rats. The effects of DPSC‐CM on diabetic polyneuropathy were assessed 4 weeks after DPSC‐CM injection. To confirm the angiogenic effect of DPSC‐CM, the effect of DPSC‐CM on cultured human umbilical vascular endothelial cell proliferation was investigated. Results The administration of DPSC‐CM into the hindlimb skeletal muscles significantly ameliorated sciatic motor/sensory nerve conduction velocity, sciatic nerve blood flow and intraepidermal nerve fiber density in the footpads of diabetic rats. We also showed that DPSC‐CM injection significantly increased the capillary density of the skeletal muscles, and suppressed pro‐inflammatory reactions in the sciatic nerves of diabetic rats. Furthermore, an in vitro study showed that DPSC‐CM significantly increased the proliferation of umbilical vascular endothelial cells. Conclusions We showed that DPSC‐CM injection into hindlimb skeletal muscles has a therapeutic effect on diabetic polyneuropathy through neuroprotective, angiogenic and anti‐inflammatory actions. DPSC‐CM could be a novel cell‐free regenerative medicine treatment for diabetic polyneuropathy.

Aims/Introduction We have previously reported that glycine suppresses the advanced glycation end‐products signaling pathway and mitigates subsequent oxidative stress in the kidneys of diabetic rats. In the present study, we investigated whether this beneficial effect was associated with upregulation of glyoxalase‐1 (Glo1) and activation of the nuclear factor erythroid 2‐related factor 2 (Nrf2). Materials and Methods Both healthy rats and streptozotocin‐induced diabetic rats were administrated with glycine (1% added to the drinking water) for 12 weeks. The function of Glo1, messenger ribonucleic acid (mRNA) and protein expressions of Nrf2, and markers of oxidative status were measured in the kidneys. The mRNA expressions of other downstream signaling molecules of the Nrf2 pathway were also determined. Results The mRNA and protein expressions, as well as the activity of Glo1, were decreased in the kidneys of diabetic rats, accompanied by diminished glutathione levels. After glycine treatment, these parameters of Glo1 function were markedly increased. Compared with the control group, the levels of Nrf2 mRNA and protein in the total kidney lysis were both markedly elevated in the diabetic group and glycine‐treated group. However, the nuclear translocation of Nrf2 was significantly increased in the glycine‐treated group than in the diabetic group. In addition, the anti‐oxidant capacity and the expressions of other downstream molecules of the Nrf2 signaling pathway were significantly increased after glycine treatment. Conclusions The present study shows that glycine might enhance the function of Glo1 and restore anti‐oxidant defense by promoting the nuclear translocation of Nrf2, thus inhibiting advanced glycation end‐products formation and protecting against renal oxidative stress.

Aims/Introduction Amelioration of renal impairment is the key to diabetic nephropathy (DN) therapy. The progression of DN is closely related to podocyte dysfunction, but the detailed mechanism has not yet been clarified. The present study aimed to explore the renal impairment amelioration effect of berberine and related mechanisms targeting podocyte dysfunction under the diabetic state. Materials and Methods Streptozotocin (35 mg/kg) was used to develop a DN rat model together with a high‐glucose/high‐lipid diet. Renal functional parameters and glomerular ultrastructure changes were recorded. The alterations of phosphatidylinositol 3‐kinase (PI3K), protein kinase B (Akt) and phosphorylated Akt in the kidney cortex were determined by western blot. Meanwhile, podocyte dysfunction was induced and treated with berberine and LY294002. After that, podocyte adhesion functional parameters, protein biomarker and the alterations of the PI3K–Akt pathway were detected. Results Berberine reduces the increased levels of biochemical indicators, and significantly improves the abnormal expression of PI3K, Akt and phosphorylated Akt in a rat kidney model. In vitro, a costimulating factor could obviously reduce the podocyte adhesion activity, including decreased expression of nephrin, podocin and adhesion molecule α3β1 levels, to induce podocyte dysfunction, and the trends were markedly reversed by berberine and LY294002 therapy. Furthermore, reduction of PI3K and phosphorylated Akt levels were observed in the berberine (30 and 60 μmol/L) and LY294002 (40 μmol/L) treatment group, but the Akt protein expression showed little change. Conclusions Berberine could be a promising antidiabetic nephropathy drug through ameliorating renal impairment and inhibiting podocyte dysfunction in diabetic rats, and the underlying molecular mechanisms might be involved in the regulation of the PI3K–Akt signaling pathway., The phosphatidylinositol 3‐kinase–protein kinase B signaling pathway in podocytes might be an important therapeutic target in diabetic nephropathy treatment. Berberine will be a promising antidiabetic nephropathy drug.

Aims/Introduction The putative tumor suppressor gene, KBTBD11, might play a role in tumorigenesis, and is associated with cellular apoptosis and proliferation in colorectal cancer cells. However, the function of Kbtbd11 during adipogenesis is unknown. The aim of the present study was to investigate the role of Kbtbd11 in the differentiation of 3T3‐L1 preadipocytes. Materials and Methods For the fasting–refeeding protocol, mice were subjected to fasting for 24 h, followed by a chow diet for 12 h. Adenovirus infection methods were used to examine the effect of Kbtbd11, and 3T3‐L1 cells were analyzed with Oil Red O staining and real‐time polymerase chain reaction. Results The white adipose tissue expression of Kbtbd11 messenger ribonucleic acid (mRNA) was significantly higher in the re‐fed state than in the fasted state. Kbtbd11 mRNA levels were markedly increased in epididymal white adipose tissue of diet‐induced obesity mice compared with those in the mice fed a chow diet. In addition, Kbtbd11 mRNA expression was increased in a differentiation‐dependent manner in 3T3‐L1 cells. Knockdown of Kbtbd11 mRNA through the infection with adenoviral vectors remarkably inhibited triglyceride accumulation and adipocyte differentiation in 3T3‐L1 cells. In contrast, the overexpression of Kbtbd11 promoted the differentiation of 3T3‐L1 adipocytes. Conclusions The present findings show that Kbtbd11 expression might be involved in nutritional regulation and is increased in obese adipose tissue. In addition, Kbtbd11 appears to be required for the differentiation of adipocytes in 3T3‐L1 cells. Collectively, these results show a novel link between the expression of Kbtbd11 and fat accumulation, and suggest that Kbtbd11 is a new therapeutic target for obesity.

Two-photon intravital imaging is a powerful method by which researchers are able to directly observe biological phenomena in live organisms. Researchers in various biomedical research fields have applied two-photon imaging to a variety of target organs by utilizing this technology’s ability to penetrate to significant depths with minimal phototoxicity. The mouse respiratory system in inflammation models is a good example, as two-photon intravital imaging can provide insights as to how the immune system is activated in response to inflammation within the respiratory system. Inflammation models can be generated via influenza viral, bacterial, or lipopolysaccharide injection. To exteriorize the lungs or trachea, thoracotomy or tracheotomy is performed, respectively; the appropriate combination of inflammation induction and organ exposure is selected depending on the study purpose. On the other hand, visualizing the movement of leukocytes is also an important component; to this end, immune cell populations of interest are either labeled via the genetic attachment of fluorescent proteins or stained with antibodies or dyes. With the proper selection of methods at each step, twophoton intravital imaging can yield visual evidence regarding immune responses to inflammation.

Background Overactivation of inflammatory cells, including macrophages and neutrophils, is associated with acute lung injury. BMS-470539 is a selective agonist of melanocortin 1 receptor, which triggers the inhibition of proinflammatory responses, suppressing neutrophil infiltration and protecting tissue. This study evaluated the effects of BMS-470539 on lipopolysaccharide-induced acute lung injury in a mouse model. Methods Mice received a subcutaneous injection of saline or BMS-470539 (18.47 mg/kg) 1 hour before an intratracheal injection of saline or lipopolysaccharide (20 μg). Mice were sacrificed to analyze the severity of pulmonary edema (lung wet-to-dry weight [W/D] ratio) and inflammatory responses (level of leukocytes, polymorphonuclear neutrophils [PMNs] and tumor necrosis factor alpha [TNF-α] in bronchoalveolar lavage fluid [BALF]), and neutrophil infiltration (myeloperoxidase activity). TNF-α activation was also measured in neutrophils from bone marrow. Survival was investigated in a second-hit sepsis mouse model. Results BMS-470539 improved sepsis-induced pulmonary edema, as demonstrated by a decreased W/D ratio (5.76%±0.83% to 3.81%±0.86%, P

Aims/Introduction The preservation of pancreatic β‐cell mass is an essential factor in the onset and development of type 2 diabetes mellitus. Recently, sodium–glucose cotransporter 2 inhibitors have been launched as antihyperglycemic agents, and their organ‐protective effects are attracting attention. They are also reported to have favorable effects on the preservation of pancreatic β‐cell mass, but the appropriate timing for the administration of sodium–glucose cotransporter 2 inhibitors is obscure. Materials and Methods In the present study, we administered a sodium–glucose cotransporter 2 inhibitor, dapagliflozin, to an animal model of type 2 diabetes mellitus, db/db mice, and investigated the adequate timing and duration for its administration. We also carried out microarray analysis using pancreatic islets from db/db mice. Results We found that dapagliflozin preserved pancreatic β‐cell mass depending on the duration of administration and markedly improved blood glucose levels. If the duration was the same, the earlier administration of dapagliflozin was more effective in preserving pancreatic β‐cell mass, increasing serum insulin levels and improving blood glucose levels. From microarray analysis, we discovered that the expression of Agr2, Tff2 and Gkn3 was significantly upregulated after the early administration of dapagliflozin. This upregulated gene expression might provide a legacy effect for the preservation of pancreatic β‐cell mass. Conclusions We expect that the early administration of dapagliflozin would provide a long‐lasting effect in preserving pancreatic β‐cell mass.

Aims/Introduction Diabetes is an important risk factor for atherosclerotic disease. The initiating factor of atherosclerosis is local endothelial cell injury. The arachidonic acid metabolite, 12(S)‐hydroxyeicosatetraenoic acid (12[S]‐HETE), might be involved in this process. In recent years, some studies have discussed the effect of 12(S)‐HETE on vascular endothelial cell function. In the present study, we investigated the effect of 12(S)‐HETE on vascular endothelial cell function in high‐glucose conditions and the mechanisms involved. Materials and Methods Human umbilical vein endothelial cells were cultured in conventional M199 medium and high‐glucose M199 medium. Human umbilical vein endothelial cells were stimulated with 12(S)‐HETE and cinnamyl‐3,4‐dihydroxy‐α‐cyanocinnamate (a 12/15‐lipoxygenases inhibitor). A type 1 diabetes mellitus model was established in C57BL/6 or 12/15‐lipoxygenases knockout mice with streptozotocin. Aortic tissue was harvested for subsequent testing. The transmembrane transport of dextran and human acute monocytic leukaemia cell line (THP‐1) cells was measured. The adherens junction protein, IkBα, nuclear factor kappa Bp65 (P65), intercellular adhesion molecule 1 and vascular cell adhesion protein 1 expression and phosphorylation, and the binding/dissociation of endothelial cell components were observed. Results Transendothelial migration of dextran and THP‐1 cells was significantly increased by stimulation of human umbilical vein endothelial cell monolayers with high glucose and 12(S)‐HETE (P

Aims/Introduction A high‐carbohydrate diet is known to increase insulin secretion and induce obesity. However, whether or not a high‐carbohydrate diet affects β‐cell mass (BCM) has been little investigated. Materials and Methods Both wild‐type (WT) mice and adenosine triphosphate‐sensitive potassium channel‐deficient (Kir6.2KO) mice were fed normal chow or high‐starch (ST) diets for 22 weeks. BCM and the numbers of islets were analyzed by immunohistochemistry, and gene expression levels in islets were investigated by quantitative real‐time reverse transcription polymerase chain reaction. MIN6‐K8 β‐cells were stimulated in solution containing various concentrations of glucose combined with nifedipine and glimepiride, and gene expression was analyzed. Results Both WT and Kir6.2KO mice fed ST showed hyperinsulinemia and body weight gain. BCM, the number of islets and the expression levels of cyclinD2 messenger ribonucleic acid were increased in WT mice fed ST compared with those in WT mice fed normal chow. In contrast, no significant difference in BCM, the number of islets or the expression levels of cyclinD2 messenger ribonucleic acid were observed between Kir6.2KO mice fed normal chow and those fed ST. Incubation of MIN6‐K8 β‐cells in high‐glucose media or with glimepiride increased cyclinD2 expression, whereas nifedipine attenuated a high‐glucose‐induced increase in cyclinD2 expression. Conclusions These results show that a high‐starch diet increases BCM in an adenosine triphosphate‐sensitive potassium channel‐dependent manner, which is mediated through upregulation of cyclinD2 expression.

Aims/Introduction Type 2 diabetes mellitus is a risk factor of acute kidney injury after myocardial infarction (MI), a form of cardiorenal syndrome. Recent clinical trials have shown that a sodium–glucose cotransporter 2 (SGLT2) inhibitor improved both cardiac and renal outcomes in patients with type 2 diabetes mellitus, but effects of an SGLT2 inhibitor on cardiorenal syndrome remain unclear. Materials and Methods Type 2 diabetes mellitus (Otsuka Long‐Evans Tokushima Fatty rats [OLETF]) and control (Long‐Evans Tokushima Otsuka rats [LETO]) were treated with canagliflozin, an SGLT2 inhibitor, for 2 weeks. Renal tissues were analyzed at 12 h after MI with or without preoperative fasting. Results Canagliflozin reduced blood glucose levels in OLETF, and blood β‐hydroxybutyrate levels were increased by canagliflozin only with fasting. MI increased neutrophil gelatinase‐associated lipocalin and kidney injury molecule‐1 protein levels in the kidney by 3.2‐ and 1.6‐fold, respectively, in OLETF, but not in LETO. The renal messenger ribonucleic acid level of Toll‐like receptor 4 was higher in OLETF than in LETO after MI, whereas messenger ribonucleic acid levels of cytokines/chemokines were not significantly different. Levels of lipid peroxides, nicotinamide adenine dinucleotide phosphate oxidase (NOX)2 and NOX4 proteins after MI were significantly higher in OLETF than in LETO. Canagliflozin with pre‐MI fasting suppressed MI‐induced renal expression of neutrophil gelatinase‐associated lipocalin and kidney injury molecule‐1 in OLETF, together with reductions in lipid peroxides and NOX proteins in the kidney. Blood β‐hydroxybutyrate levels before MI were inversely correlated with neutrophil gelatinase‐associated lipocalin protein levels in OLETF. Pre‐incubation with β‐hydroxybutyrate attenuated angiotensin II‐induced upregulation of NOX4 in NRK‐52E cells. Conclusions The findings suggest that SGLT2 inhibitor treatment with a fasting period protects kidneys from MI‐induced cardiorenal syndrome, possibly by β‐hydroxybutyrate‐mediated reduction of NOXs and oxidative stress, in type 2 diabetic rats.

Background Critical illness-related corticosteroid insufficiency (CIRCI) and adrenocorticotropic hormone (ACTH)-cortisol dissociation are hormonal conditions frequently observed in patients in the intensive care unit (ICU). The aim of this study was to evaluate the association between ACTH-cortisol dissociation and clinical outcomes of critically ill patients. Methods We retrospectively reviewed the medical records of 94 ICU patients who underwent two rapid cosyntropin tests during hospital admission and compared the clinical aspects of patients with and without ACTH-cortisol dissociation. ACTH-cortisol dissociation was defined as plasma ACTH and serum cortisol concentrations of 600 nmol/L, respectively. Results Dissociation was present in 30 of the 94 patients (31.9%). Patients with ACTH-cortisol dissociation in the initial test had significantly higher hospital mortality rate than those in the control group (55% vs. 25.7%, P=0.013) There was no difference in hospital mortality between patients classified as having CIRCI and those who were not. In multivariate adjusted Cox regression analysis, the mortality risk was higher in the group with ACTH-cortisol dissociation (hazard ratio, 2.98; 95% confidence interval, 1.34 to 6.63; P=0.007). Patients with dissociation in two consecutive stimulation tests showed the highest hospital mortality rate among groups classified according to stimulation test results (100% vs. 31.3%). Conclusions The hospital mortality was higher in ICU patients diagnosed with ACTH-cortisol dissociation. It is clinically feasible to evaluate the presence of ACTH-cortisol dissociation by analyzing rapid ACTH stimulation test results in critically ill patients.

AIMS/INTRODUCTION: The purpose of the present study was to investigate the possible effect of oral magnesium sulfate (MgSO4 ) in the reduction of atherosclerosis plaques through inhibition of lectin-like low-density lipoprotein receptor-1 (LOX-1) gene expression in diabetic vessels. MATERIALS AND METHODS: A total of 50 rats were divided into five groups, including non-diabetic control, Mg-treated non-diabetic control, chronic diabetic, Mg-treated chronic diabetic and insulin-treated chronic diabetic. The induction of diabetes was carried out by streptozotocin. The Mg-treated chronic diabetic and Mg-treated non-diabetic control groups were treated with 10 g/L of MgSO4 added to their drinking water. The insulin-treated chronic diabetic group received 2.5 U/kg of insulin twice per day. The fasting blood glucose level and bodyweight were determined weekly. Blood pressure measurement and the intraperitoneal glucose tolerance test were carried out after 16 weeks, and the plasma levels of Mg, lipid profile and oxidized low-density lipoprotein cholesterol (oxLDL) were determined. The mesenteric bed was isolated and perfused according to the McGregor method. The aorta was isolated for LOX-1 genes and proteins expression, and pathological investigation. RESULTS: MgSO4 administration improved blood pressure, sensitivity to phenylephrine, intraperitoneal glucose tolerance test, lipid profile and plasma ox-LDL level, and also lowered the blood glucose level to the normal range, and decreased LOX-1 gene and protein expressions. Insulin decreased blood pressure, sensitivity to phenylephrine, blood glucose, lipid profiles and plasma oxLDL level, but it did not decrease LOX-1 gene and protein expressions. CONCLUSIONS: The present findings suggested that MgSO4 improves blood pressure and vessel structure through decreasing oxLDL, and LOX-1 gene and protein expressions; however, insulin did not repair vessel structure, and LOX-1 gene and protein expressions. © 2018 The Authors. Journal of Diabetes Investigation published by Asian Association for the Study of Diabetes (AASD) and John Wiley & Sons Australia, Ltd.

Aims/Introduction To investigate the protective effect of Polygonatum sibiricum polysaccharide (PSP) on the retina in diabetic rats. Materials and Methods A total of 120 Sprague–Dawley rats were randomly divided into blank control, control model (meaning diabetes mellitus), and diabetes mellitus with PSP intervention of high, medium and low doses groups. The difference of retinal vascularization between groups was evaluated by fluorescein isothiocyanate‐dextran perfusion. Terminal deoxynucleotidyl transferase‐mediated dUTP nick‐end labeling staining was used to assess apoptosis in the retinal ganglion cells; reverse transcriptase polymerase chain reaction and western blotting were utilized to evaluate the expression of Bcl2‐associated X protein, B‐cell lymphoma‐2 factor, epidermal growth factor, p38 mitogen‐activated protein kinases, transforming growth factor‐β and vascular endothelial growth factor at the messenger ribonucleic acid and protein level. Results Fluorescein isothiocyanate‐dextran perfusion showed retinal vascular anomaly in diabetes mellitus rats, but vascular tortuosity and leakage were relatively alleviated after PSP intervention. Terminal deoxynucleotidyl transferase‐mediated dUTP nick‐end labeling staining showed numerous terminal deoxynucleotidyl transferase‐mediated dUTP nick‐end labeling‐positive retinal cells in the diabetes mellitus group, which then were reduced by PSP treatment. Reverse transcriptase polymerase chain reaction showed that PSP intervention decreased Bcl2‐associated X protein, epidermal growth factor, p38 mitogen‐activated protein kinases, vascular endothelial growth factor and transforming growth factor‐β messenger ribonucleic acid expression, but increased B‐cell lymphoma‐2 factor messenger ribonucleic acid expression. Western blot showed that PSP intervention upregulated the expression of B‐cell lymphoma‐2 factor, and downregulated the expression of Bcl2‐associated X protein, epidermal growth factor, p38 mitogen‐activated protein kinases, vascular endothelial growth factor and transforming growth factor‐β proteins. Conclusions Polygonatum sibiricum polysaccharide shows a protective effect against diabetes‐induced retinal injury in a dose‐dependent manner. The mechanism of action deserves further study and exploration.

Aims/introduction Double C2 domain protein b (DOC2b), one of the synaptotagmins, has been shown to translocate to the plasma membrane, and to initiate membrane‐fusion processes of vesicles containing glucose transporter 4 proteins on insulin stimulation. However, the mechanism by which DOC2b is regulated remains unclear. Herein, we identified the upstream regulatory factors of DOC2b in insulin signal transduction. We also examined the role of DOC2b on systemic homeostasis using DOC2b knockout (KO) mice. Materials and Methods We first identified DOC2b binding proteins by immunoprecipitation and mutagenesis experiments. Then, DOC2b KO mice were generated by disrupting the first exon of the DOC2b gene. In addition to the histological examination, glucose metabolism was assessed by measuring parameters on glucose/insulin tolerance tests. Insulin‐stimulated glucose uptake was also measured using isolated soleus muscle and epididymal adipose tissue. Results We identified an isoform of atypical protein kinase C (protein kinase C iota) that can bind to DOC2b and phosphorylates one of the serine residues of DOC2b (S34). This phosphorylation is essential for DOC2b translocation. DOC2b KO mice showed insulin resistance and impaired oral glucose tolerance on insulin and glucose tolerance tests, respectively. Insulin‐stimulated glucose uptake was impaired in isolated soleus muscle and epididymal adipose tissues from DOC2b KO mice. Conclusions We propose a novel insulin signaling mechanism by which protein kinase C iota phosphorylates DOC2b, leading to glucose transporter 4 vesicle translocation, fusion and facilitation of glucose uptake in response to insulin. The present results also showed DOC2b to play important roles in systemic glucose homeostasis.

Aims/Introduction Non‐alcoholic steatohepatitis (NASH), which occurs in association with insulin resistance and hepatic fat accumulation, is characterized by chronic liver injury and fibrosis. NASH onset and progression is closely related to hepatic inflammation, which is partly regulated by the vagus nerve through the α7 nicotinic acetylcholine receptor (α7nAchR). Hepatic α7nAchR action is impeded in obesity and insulin resistance. In the present study, using α7nAchR knockout (α7KO) mice, we elucidated the effect of α7nAchR deficiency on NASH‐related inflammation and fibrosis. Materials and Methods α7KO mice were fed an atherogenic high‐fat diet (AD) for 32 weeks or methionine/choline‐deficient diet (MCD) for 6 weeks, both of which induce NASH. Mice were then examined for the degree of NASH‐related inflammation and fibrosis by hepatic gene expression analysis and Sirius red histological staining. Results Hepatic triglyceride accumulation and elevated plasma transaminase levels were observed in both AD and MCD mice, but the plasma transaminase level increase was higher in α7KO mice than in control mice. α7KO mice fed an AD showed significant upregulation of the Col1a1 gene encoding alpha‐1 type I collagen, which is involved in liver fibrosis, and the Ccl2 gene encoding C‐C motif chemokine ligand 2, a pro‐inflammatory chemokine; α7KO mice fed an MCD had significant upregulation of the Col1a1 gene and the Tnf gene, an inflammatory cytokine. Histological analysis showed that AD and MCD exacerbated liver fibrosis in α7KO mice. Conclusions The results of this study suggest that α7nAchR deficiency exacerbates hepatic inflammation and fibrosis in a diet‐induced mouse model of NASH.

Aims/Introduction Tubulointerstitial fibrosis is a hallmark of diabetic nephropathy and is associated with an epithelial‐to‐mesenchymal transition (EMT) program and aberrant glycolysis. Dimeric pyruvate kinase (PK) M2 (PKM2) acts as a key protein kinase in aberrant glycolysis by promoting the accumulation of hypoxia‐inducible factor (HIF)‐1α, while tetrameric PKM2 functions as a pyruvate kinase in oxidative phosphorylation. The aim of the research is to study the effect of PKM2 tetramer activation on preventing kidney fibrosis via suppression of aberrant glycolysis and the EMT program. Materials and methods In vivo: Streptozotocin (STZ) was utilized to induce diabetes in 8‐week‐old CD‐1 mice; 4 weeks after diabetes induction, proteinuria‐induced kidney fibrosis was developed by intraperitoneal injection of bovine serum albumin (BSA: 0.3 g/30 g BW) for 14 days; The PKM2 activator TEPP‐46 was also administered orally simultaneously. In vitro: HK2 cells were co‐treated with high‐glucose media or/and TGF‐β1 and TEPP46 for 48 h, cellular protein was extracted for evaluation. Results Diabetic mice developed kidney fibrosis associated with aberrant glycolysis and EMT; BSA injection accelerated kidney fibrosis in both the control and diabetic mice; TEPP‐46 rescued the kidney fibrosis. In HK2 cells, TEPP‐46 suppressed the EMT program induced by TGF‐β1 and/or high‐glucose incubation. TEPP‐46‐induced PKM2 tetramer formation and PK activity resulted in suppression of HIF‐1α and lactate accumulation. Specific siRNA‐mediated knockdown of HIF‐1α expression diminished high glucose‐induced mesenchymal protein levels. Conclusion PKM2 activation could restore the tubular phenotype via suppression of the EMT program and aberrant glycolysis, providing an alternative target to mitigate fibrosis in diabetic kidneys., Diabetic kidney disease displayed kidney fibrosis associated with aberrant glycolysis and EMT program, these alterations were related to the increased level of dimeric‐PKM2 formation, PKM2 activation by TEPP46 rescued the kidney fibrosis by promoting PKM2 tetramer formation, and inhibiting dimeric‐PKM2 which directly interacted with HIF‐1α and promoted its transcription.

Background Few studies have evaluated the effects of hypothermia on cardiac arrest (CA)-induced liver damage. This study aimed to investigate the effects of hypothermic therapy on the liver in a rat model of asphyxial cardiac arrest (ACA). Methods Rats were subjected to 5-minute ACA followed by return of spontaneous circulation (RoSC). Body temperature was controlled at 33°C±0.5°C or 37°C±0.5°C for 4 hours after RoSC in the hypothermia group and normothermia group, respectively. Liver tissues in each group were collected at 6 hours, 12 hours, 1 day, and 2 days after RoSC. To examine hepatic inflammation, mast cells were stained with toluidine blue. Superoxide anion radical production was evaluated using dihydroethidium fluorescence straining and expression of endogenous antioxidants (superoxide dismutase 1 [SOD1] and SOD2) was examined using immunohistochemistry. Results There were significantly more mast cells in the livers of the normothermia group with ACA than in the hypothermia group with ACA. Gradual increase in superoxide anion radical production was found with time in the normothermia group with ACA, but production was significantly suppressed in the hypothermia group with ACA relative to the normothermia group with ACA. SOD1 and SOD2 levels were higher in the hypothermia group with ACA than in the normothermia group with ACA. Conclusions Experimental hypothermic treatment after ACA significantly inhibited inflammation and superoxide anion radical production in the rat liver, indicating that this treatment enhanced or maintained expression of antioxidants. Our findings suggest that hypothermic therapy after CA can reduce mast cell-mediated inflammation through regulation of oxidative stress and the expression of antioxidants in the liver.

Aims/Introduction Micro‐ribonucleic acids (miRNAs) possess crucial functions in governing metabolisms associated with type 2 diabetes mellitus. This study aimed to investigate the role of miR‐23a‐3p in pyroptosis caused by nucleotide‐binding oligomerization‐like receptor family pyrin domain containing 3 (NLRP3) inflammatory body activation, thereby reducing the occurrence of type 2 diabetes mellitus. Materials and Methods miR‐23a‐3p and NIMA‐related kinase 7 (NEK7) expression in type 2 diabetes mellitus patients and rat models was examined. Dual‐luciferase reporter gene experiments were used to verify the targeting relationship between miR‐23a‐3p and NEK7. Bone marrow‐derived macrophages were transfected with miR‐23a‐3p mimic, miR‐23a‐3p inhibitor or short hairpin NEK7 and were treated with a specific activator of NLRP3 inflammatory body (lipopolysaccharide + adenosine‐5′‐triphosphate) to evaluate expression of NEK7, miR‐23a‐3p, gasdermin D p30, pro‐caspase‐1 and caspase‐1 in cells, and interleukin‐1β and tumor necrosis factor‐α in supernatant. Type 2 diabetes mellitus rat models were used to observe the influences of miR‐23a‐3p, NEK7 and NLRP3 inflammatory body on pyroptosis and type 2 diabetes mellitus in vivo. Results NEK7 was overexpressed, whereas miR‐23a‐3p was underexpressed in patients and rat models with type 2 diabetes mellitus. NEK7 was a target gene of miR‐23a‐3p. After the addition of lipopolysaccharide + adenosine‐5′‐triphosphate in bone marrow‐derived macrophages, the expression of miR‐23a‐3p subsequently declined. Furthermore, the addition of lipopolysaccharide + adenosine‐5′‐triphosphate elevated NEK7, NLRP3, pro‐caspase‐1, cle‐caspase‐1 and gasdermin D p30 expressions in bone marrow‐derived macrophages, and enhanced levels of interleukin‐1β and tumor necrosis factor‐α in the supernatant, accompanied with conspicuous cell pyroptosis, which was reversed after miR‐23a‐3p overexpression and NEK7 silencing. miR‐23a‐3p overexpression alleviated liver and kidney damage in type 2 diabetes mellitus rats, and reduced NLRP3‐induced pyroptosis. Conclusions Targeting NEK7 by miR‐23a‐3p could reduce NLRP3‐induced pyroptosis, and assuage liver and kidney injuries in type 2 diabetes mellitus rats., Targeting NIMA‐related kinase 7 by micro‐ribonucleic acid could reduce NOD‐, LRR‐ and pyrin domain‐containing protein 3‐induced pyroptosis, and assuage liver and kidney injuries in type 2 diabetes mellitus rats.

Aims/Introduction Duodenal‐jejunal bypass (DJB) surgery has been reported to effectively relieve diabetic cardiomyopathy (DCM). However, the specific mechanisms remain largely unknown. The present study was designed to determine the alterations of myocardial glucose uptake (MGU) after DJB and their effects on DCM. Materials and Methods Duodenal‐jejunal bypass and sham surgeries were carried out in diabetic rats induced by a high‐fat diet and a low dose of streptozotocin, with chow‐diet fed rats as controls. Bodyweight, food intake, glucose homeostasis and lipid profiles were measured at indicated time‐points. Cardiac function was evaluated by transthoracic echocardiography and hemodynamic measurement. Cardiac remodeling was assessed by a series of morphometric analyses along with transmission electron microscopy. Positron‐emission tomography with fluorine‐18 labeled fluorodeoxyglucose was carried out to evaluate the MGU in vivo. Furthermore, myocardial glucose transporters (GLUT; GLUT1 and GLUT4), myocardial insulin signaling and GLUT‐4 translocation‐related proteins were investigated to elucidate the underlying mechanisms. Results The DJB group showed restored systolic and diastolic cardiac function, along with significant remission in cardiac hypertrophy, cardiac fibrosis, lipid deposit and ultrastructural disorder independent of weight loss compared with the sham group. Furthermore, the DJB group showed upregulated myocardial insulin signaling, hyperphosphorylation of AKT substrate of 160 kDa (AS160) and TBC1D1, along with preserved soluble N‐ethylmaleimide‐sensitive factor attachment protein receptor proteins, facilitating the GLUT‐4 translocation to the myocardial cell surface and restoration of MGU. Conclusions The present findings provide evidence that restoration of MGU is implicated in the alleviation of DCM after DJB through facilitating GLUT‐4 translocation, suggesting a potential choice for treatment of human DCM if properly implemented.