Decreased Vagal Activity and Deviation in Sympathetic Activity Precedes Development of Diabetes.

OBJECTIVE: The objective of this study was to examine whether altered heart rate variability (HRV) could predict the risk of diabetes in Asians. RESEARCH DESIGN AND METHODS: A cohort study was conducted in 54,075 adults without diabetes who underwent 3-min HRV measurement during health checkups between 2011 and 2014 at Kangbuk Samsung Hospital. We analyzed the time domain (SD of the normal-to-normal interval [SDNN] and root mean square differences of successive normal-to-normal intervals [RMSSD]) and the frequency domain (total power, normalized low-frequency power [LF], and normalized high-frequency power [HF] and LF/HF ratio). We compared the risk of diabetes until 2017 according to tertiles of heart rate and HRV variables, with tertile 1 serving as the reference group. RESULTS: During 243,758.2 person-years, 1,369 subjects were diagnosed with diabetes. Both time and frequency domain variables were lower in the group with diabetes, with the exception of those with normalized LF and LF/HF ratio. In Cox analysis, as SDNN, RMSSD, and normalized HF tertiles increased, the risk of diabetes decreased (hazard ratios [95% CIs] of tertile 3: 0.81 [0.70-0.95], 0.76 [0.65-0.90], and 0.78 [0.67-0.91], respectively), whereas the risk of diabetes increased in the case of heart rate, normalized LF, and LF/HF ratio (hazard ratios [95% CIs] of tertile 3: 1.41 [1.21-1.65], 1.32 [1.13-1.53], and 1.31 [1.13-1.53), respectively) after adjusting for age, sex, BMI, smoking, drinking, systolic blood pressure, lipid level, CRP, and HOMA of insulin resistance. CONCLUSIONS: Abnormal HRV, especially decreased vagal activity and deviation in sympathovagal imbalance to sympathetic activity, might precede incident diabetes.

Resveratrol, an activator of SIRT1, improves ER stress by increasing clusterin expression in HepG2 cells.

Endoplasmic reticulum stress (ER stress) is involved in lipid metabolism and lipotoxicity and can lead to apoptosis. Resveratrol, a sirtuin 1 (SIRT1) agonist, prevents ER stress and improves ER stress-induced hepatic steatosis and cell death. Clusterin is a secreted chaperone and has roles in various physiological processes. However, changes in the expression of clusterin upon ER stress and the connection between SIRT1 and clusterin in protection against ER stress are not well known. In cells treated with tunicamycin, resveratrol increased the expression of clusterin mRNA and protein and the secreted clusterin protein level in conditioned medium. Resveratrol decreased protein expression of the ER stress markers, p-PERK, p-IRE1α, and CHOP, and increased the expression of the ER-associated degradation (ERAD) factors, SEL1L and HRD1, in tunicamycin-treated cells. However, no changes in the expression of these genes were observed in clusterin siRNA-transfected cells. Moreover, increased LAMP2 and LC3 expression and decreased Rubicon expression were observed in cells treated with resveratrol or secreted clusterin. These data suggest that SIRT1 activation by resveratrol attenuates ER stress by promoting protective processes such as ERAD and autophagy pathways and that these protective effects are mediated by clusterin.

Metabolic Obesity Phenotypes and Thyroid Cancer Risk: A Cohort Study.

BACKGROUND: No cohort studies have evaluated the effect of obesity on the incidence of thyroid cancer according to metabolic health status. Therefore, this study examined the association of body mass index (BMI) and metabolic health status with thyroid cancer risk. METHODS: A cohort study was performed involving 255,051 metabolically healthy (MH) and metabolically unhealthy (MUH) adults free of thyroid cancer at baseline who were followed for a median of 5.3 years. A parametric proportional hazard model was used to estimate the adjusted hazard ratio (aHR) and confidence interval (CI). RESULTS: During 1,402,426.3 person-years of follow-up, 2927 incident thyroid cancers were identified. Among men, the multivariable aHR for thyroid cancer comparing obesity, defined as a BMI ≥25 kg/m2, with a BMI of 18.5-22.9 kg/m2 was 1.47 [CI 1.12-1.93] in MH individuals, whereas the corresponding HR in MUH individuals was 1.26 [CI 1.03-1.53]. Among women, the corresponding HR in MH individuals was 1.05 [CI 0.80-1.36], whereas the corresponding HR in MUH individuals was 1.43 [CI 1.22-1.69]. Increasing quartiles of waist circumference were positively associated with risk of thyroid cancer in MUH men and women (p for trend <0.005) but not in MH individuals. CONCLUSIONS: In both MH and MUH men, obesity was associated with an increased risk of incident thyroid cancer, indicating excessive adiposity per se as an independent risk factor for thyroid cancer. Conversely, women with MUH obesity but not MH obesity were found to have an increased risk of thyroid cancer, indicating that obesity with accompanying metabolic abnormalities may affect thyroid cancer risk in women.

Deficiency of Sphingosine-1-Phosphate Reduces the Expression of Prohibitin and Causes ß-Cell Impairment via Mitochondrial Dysregulation.

BACKGROUND: Emerging evidence suggests that sphingolipids may be involved in type 2 diabetes. However, the exact signaling defect through which disordered sphingolipid metabolism induces ß-cell dysfunction remains unknown. The current study demonstrated that sphingosine-1-phosphate (S1P), the product of sphingosine kinase (SphK), is an essential factor for maintaining ß-cell function and survival via regulation of mitochondrial action, as mediated by prohibitin (PHB). METHODS: We examined ß-cell function and viability, as measured by mitochondrial function, in mouse insulinoma 6 (MIN6) cells in response to manipulation of cellular S1P and PHB levels. RESULTS: Lack of S1P induced by sphingosine kinase inhibitor (SphKi) treatment caused ß-cell dysfunction and apoptosis, with repression of mitochondrial function shown by decreases in cellular adenosine triphosphate content, the oxygen consumption rate, the expression of oxidative phosphorylation complexes, the mitochondrial membrane potential, and the expression of key regulators of mitochondrial dynamics (mitochondrial dynamin-like GTPase [OPA1] and mitofusin 1 [MFN1]). Supplementation of S1P led to the recovery of mitochondrial function and greatly improved ß-cell function and viability. Knockdown of SphK2 using small interfering RNA induced mitochondrial dysfunction, decreased glucose-stimulated insulin secretion (GSIS), and reduced the expression of PHB, an essential regulator of mitochondrial metabolism. PHB deficiency significantly reduced GSIS and induced mitochondrial dysfunction, and co-treatment with S1P did not reverse these trends. CONCLUSION: Altogether, these data suggest that S1P is an essential factor in the maintenance of ß-cell function and survival through its regulation of mitochondrial action and PHB expression.

Exendin-4 improves ER stress-induced lipid accumulation and regulates lipin-1 signaling in HepG2 cells.

Lipin-1 performs dual function during lipid metabolism, i.e., it functions as a transcriptional coactivator and as a phosphatidate phosphatase during triglyceride biosynthesis. We investigated whether exendin-4 prevented endoplasmic reticulum (ER) stress-induced hepatic steatosis and whether the protective effects of exendin-4 were associated with lipin-1 signaling. Tunicamycin and thapsigargin, ER stress inducers, increased triglycerides (TG) content and expression of genes encoding lipid droplet surface proteins. Exendin-4 decreased the expression of ER stress markers phosphorylated PKR like ER kinase (PERK), phosphorylated inositol-requiring enzyme 1 alpha (IRE1α), and glucose-regulated protein 78 kDa (GRP78) proteins and spliced X-box binding protein 1 (XBP-1s) mRNA and increased the expression of genes encoding lipolytic enzymes hormone-sensitive lipase (HSL) and monoacylglycerol lipase (MGL) and VLDL assembly-associated proteins microsomal triglyceride transfer protein (MTP) and apolipoprotein B (APOB) in tunicamycin-pretreated cells. Moreover, exendin-4 significantly decreased lipin-1ß/α ratio by increasing SFRP10 and increased lipin-1 nuclear localization. The decrease in lipin-1ß/α ratio was also observed in SIRT1 and AMPK agonist-treated cells. These data suggest that exendin-4 improves ER stress-induced hepatic lipid accumulation by increasing lipolysis and VLDL assembly, which is partially mediated by the regulation of lipin-1 signaling.

Pioglitazone Attenuates Palmitate-Induced Inflammation and Endoplasmic Reticulum Stress in Pancreatic ß-Cells.

BACKGROUND: The nuclear receptor peroxisome proliferator-activator gamma (PPARγ) is a useful therapeutic target for obesity and diabetes, but its role in protecting ß-cell function and viability is unclear. METHODS: To identify the potential functions of PPARγ in ß-cells, we treated mouse insulinoma 6 (MIN6) cells with the PPARγ agonist pioglitazone in conditions of lipotoxicity, endoplasmic reticulum (ER) stress, and inflammation. RESULTS: Palmitate-treated cells incubated with pioglitazone exhibited significant improvements in glucose-stimulated insulin secretion and the repression of apoptosis, as shown by decreased caspase-3 cleavage and poly (adenosine diphosphate [ADP]-ribose) polymerase activity. Pioglitazone also reversed the palmitate-induced expression of inflammatory cytokines (tumor necrosis factor α, interleukin 6 [IL-6], and IL-1ß) and ER stress markers (phosphor-eukaryotic translation initiation factor 2α, glucose-regulated protein 78 [GRP78], cleaved-activating transcription factor 6 [ATF6], and C/EBP homologous protein [CHOP]), and pioglitazone significantly attenuated inflammation and ER stress in lipopolysaccharide- or tunicamycin-treated MIN6 cells. The protective effect of pioglitazone was also tested in pancreatic islets from high-fat-fed KK-Ay mice administered 0.02% (wt/wt) pioglitazone or vehicle for 6 weeks. Pioglitazone remarkably reduced the expression of ATF6α, GRP78, and monocyte chemoattractant protein-1, prevented α-cell infiltration into the pancreatic islets, and upregulated glucose transporter 2 (Glut2) expression in ß-cells. Moreover, the preservation of ß-cells by pioglitazone was accompanied by a significant reduction of blood glucose levels. CONCLUSION: Altogether, these results support the proposal that PPARγ agonists not only suppress insulin resistance, but also prevent ß-cell impairment via protection against ER stress and inflammation. The activation of PPARγ might be a new therapeutic approach for improving ß-cell survival and insulin secretion in patients with diabetes mellitus.

Association Between Coronary Artery Calcification and the Hemoglobin Glycation Index: The Kangbuk Samsung Health Study.

Context: The hemoglobin glycation index (HGI) is known to be correlated with the risk for cardiovascular disease. Objective: To analyze the association between incident coronary artery calcification (CAC) and the changes in HGI among participants without diabetes, over 4 years. Design, Setting, Participants, and Outcome Measures: A retrospective study of 2052 nondiabetic participants in whom the coronary artery calcium score was measured repeatedly over 4 years, as part of a health checkup program in Kangbuk Samsung Hospital in Korea, and who had no CAC at baseline. The HGI was defined as the difference between the measured and predicted hemoglobin A1c (HbA1c) levels. Results: A total of 201 participants developed CAC after 4 years, and the mean baseline HGI was significantly higher in those patients. The incidence of CAC gradually increased from the first to the fourth quartile groups of baseline HGI. The odds ratio (OR) for incident CAC was the highest among the four groups divided by the quartiles of the baseline HGI and was significant after adjustment for confounding variables (vs first quartile group: OR, 1.632; 95% confidence interval, 1.024 to 2.601). The incidence of and risk for CAC development were significantly higher than in other groups compared with the low-to-low group after adjustment for confounding factors; however, when baseline HbA1c level was included in the model, only participants with a low-to-high HGI over 4 years showed a significantly increased OR for CAC development compared with the low-to-low group (OR, 1.722; 95% confidence interval, 1.046 to 2.833). Conclusions: The participants with a high baseline HGI and consistently high HGI showed a higher risk for incident CAC than those with a low baseline HGI. An increased HGI over 4 years significantly increased the risk for CAC regardless of the baseline HbA1c levels.

Effects of Low-density Lipoprotein Cholesterol on Coronary Artery Calcification Progression According to High-density Lipoprotein Cholesterol Levels.

BACKGROUND AND AIM: Previous studies reported that many patients are at high risk for cardiovascular disease (CVD) despite achieving recommended low-density lipoprotein cholesterol (LDL-C) levels. Therefore, we investigated whether the association between LDL-C and the risk for incident CVD differed according to high-density lipoprotein cholesterol (HDL-C) levels using coronary artery calcium score (CACS) progression as a surrogate marker for predicting CVD. METHODS: We investigated 2132 Korean men in a health screening program, in which CACS was measured at baseline and after 4 years. Coronary artery calcification (CAC) progression was defined as a change in CACS ≥0 over 4 years. We divided the subjects into nine groups according to baseline HDL-C and LDL-C levels and compared their risks for CAC progression. RESULTS: After 4 years, 475 subjects (22.3%) exhibited CAC progression. We identified a positive relationship between baseline LDL-C levels and the risk for incident CAC. However, this association was attenuated by high baseline HDL-C levels. Multivariate logistic regression analysis adjusted for age, body mass index, systolic blood pressure, fasting glucose, smoking, and exercise status revealed that the odds ratios for incident CAC in the lowest HDL-C tertile were 3.08 for LDL-C tertile 3 and 2.02 for LDL-C tertile 2 compared to LDL-C tertile 1. However, these differences disappeared in the highest HDL-C tertile (HDL-C ≥54.0 mg/dL). CONCLUSIONS: In this longitudinal study, we found that the positive relationship between LDL-C and the relative risk for incident CAC was attenuated by higher HDL-C levels. Therefore, HDL-C levels should be considered when estimating CVD risk.

Additive effect of non-alcoholic fatty liver disease on the development of diabetes in individuals with metabolic syndrome.

INTRODUCTION: Non-alcoholic fatty liver disease (NAFLD) is considered as the hepatic manifestation of the metabolic syndrome (MetS), with insulin resistance as the common pathophysiology. In a current longitudinal cohort study, we evaluated the separate and combined effects of MetS and NAFLD on incident diabetes risk. METHODS: Participants were categorized into four groups on the basis of the presence of NAFLD and MetS at baseline (i.e., with NAFLD, with MetS, with both, or without either). We compared the development of diabetes among these four groups. RESULTS: During the mean follow up of 4years, 435 of the 7849 participants (5.5%) developed diabetes. The age, sex, and smoking-adjusted risk of incident diabetes was higher in the NAFLD only group (HR 1.51, 95% CI 1.14-1.99), MetS only group (HR 2.82, 95% CI 2.01-3.95), and both group (HR 5.45, 95% CI 4.32-6.82) compared with the group of neither. When compared with the NAFLD only group, the adjusted HR for incident diabetes was 1.87 (95% CI 1.29-2.72) in the MetS only group and 3.62 (95% CI 2.74-4.77) in both group. Among individuals with MetS, the presence of NAFLD showed a significant increase in risk of incident diabetes even after further adjustment for MetS components including fasting glucose, TG, BMI, systolic BP, and HDL-C (HR 1.53, 95% CI 1.09-2.16). CONCLUSION: The presence of NAFLD further increased the risk of incident diabetes in individuals with metabolic syndrome. Our results suggest that the coexistence of NAFLD has an additive effect on the development of diabetes in individuals with MetS.

Exendin-4 Inhibits Hepatic Lipogenesis by Increasing ß-Catenin Signaling.

The aim of this study is to investigate whether the beneficial effect of exendin-4 on hepatic steatosis is mediated by ß-catenin signaling. After the HepG2 human hepatoma cells were treated with PA for 24 hours, total triglycerides levels were increased in a dose-dependent manner, and the expression levels of perilipin family members were upregulated in cells treated with 400 µM PA. For our in vitro model of hepatic steatosis, HepG2 cells were treated with 400 µM palmitic acid (PA) in the presence or absence of 100 nM exendin-4 for 24 hours. PA increased the expression of lipogenic genes, such as sterol regulatory element-binding protein 1c (SREBP-1c), peroxisome proliferator-activated receptor gamma (PPARγ), stearoyl-CoA desaturase 1 (SCD1), fatty acid synthase (FAS), and acetyl-CoA carboxylase (ACC) and triglyceride synthesis-involved genes, such as diacylglycerol acyltransferase 1 (DGAT1) and diacylglycerol acyltransferase 2 (DGAT2) in HepG2 cells, whereas exendin-4 treatment significantly prevented the upregulation of SREBP-1c, PPARγ, SCD1, FAS, ACC, DGAT1 and DGAT2. Moreover, exendin-4 treatment increased the expression of phosphorylated glycogen synthase kinase-3 beta (GSK-3ß) in the cytosolic fraction and the expression of ß-catenin and transcription factor 4 (TCF4) in the nuclear fraction. In addition, siRNA-mediated inhibition of ß-catenin upregulated the expression of lipogenic transcription factors. The protective effects of exendin-4 on intracellular triglyceride content and total triglyceride levels were not observed in cells treated with the ß-catenin inhibitor IWR-1. These data suggest that exendin-4 treatment improves hepatic steatosis by inhibiting lipogenesis via activation of Wnt/ß-catenin signaling.

Increased risk for development of coronary artery calcification in insulin-resistant subjects who developed diabetes: 4-year longitudinal study.

OBJECTIVE: Coronary artery calcification (CAC) is considered a surrogate marker for atherosclerotic burden. The aim of this study was to analyze the risk of incident CAC associated with diabetes development in non-diabetic subjects with zero CAC score (CACS) at baseline. METHODS: 2076 non-diabetic participants (mean age 40 years) in a health screening program in whom CACS were repeatedly measured by multi-detector computed tomography in four years of intervals and with zero CACS at baseline, were retrospectively analyzed. Glycemic status was assessed in both years, with subjects divided into three groups: subjects with 'no progression', 'normal to impaired fasting glucose (IFG)' and 'progression to diabetes'. Insulin resistance was assessed by homeostasis model assessment-insulin resistance (HOMA-IR) index. RESULTS: Over 4 years, 204 subjects (9.8%) developed CAC. Subjects who developed diabetes showed the highest proportion of subjects with incident CAC among the three groups (21.0% vs. 9.3 and 10.4% in non-progressors and subjects from normal to IFG). The subjects with HOMA-IR level in higher half at baseline showed significantly increased risk for incident CAC in subjects who progressed from normal to IFG and in subjects who developed diabetes (1.740; 95% CI 1.014-2.985, 2.449; 95% CI 1.159-5.174) even after adjustment for confounding variables, whereas subjects with HOMA-IR level in lower half at baseline showed no significantly increased risk for incident CAC even in subjects who developed diabetes. CONCLUSIONS: In this non-diabetic population, we found that increased risk for incident CAC in relation to diabetes development over 4 years was pronounced only in subjects with insulin resistance at baseline.

AMP-activated protein kinase suppresses the expression of LXR/SREBP-1 signaling-induced ANGPTL8 in HepG2 cells.

ANGPTL8 is a liver-derived secretory protein that leads to elevated serum triglyceride and the level of circulating ANGPTL8 is strongly associated with obesity and diabetes. Here we investigated the mechanisms of activation and inhibition of ANGPTL8 expression in hepatocytes. The expression of ANGPTL8 was significantly increased in HepG2 cells exposed to palmitic acid, tunicamycin, or T0901317, and was reversed in cells treated with AICAR. Palmitic acid, tunicamycin, and T0901317 increased LXRα and SREBP-1c mRNA expression. The inhibitory effect of AICAR on the expression of T0901317-induced ANGPTL8 was most strongly evident in cells that were transfected with SREBP-1 siRNA. AICAR increased phosphorylation of PPARα and the effect of AICAR was not observed in cells treated with PPARα inhibitor. Metformin had a similar effect on ANGPTL8 expression to that of AICAR. These data suggest that AMPK can suppress the expression of LXR/SREBP-1 signal-induced ANGPTL8 in HepG2 cells.

Higher association of coronary artery calcification with non-alcoholic fatty liver disease than with abdominal obesity in middle-aged Korean men: the Kangbuk Samsung Health Study.

BACKGROUND: It is uncertain whether non-alcoholic fatty liver disease (NAFLD) or abdominal obesity is more associated with atherosclerosis. The aim of this study was to determine whether NAFLD or abdominal obesity is more strongly associated with subclinical atherosclerosis represented by coronary artery calcification (CAC). METHODS: A total of 21,335 male participants in a health screening program (mean age 41 years) were enrolled. Ultrasonographic measurements of fatty liver and multi-detector computed tomography were performed to determine the coronary artery calcium score (CACS). The presence of CAC was defined as CACS > 0. Subjects were divided into four groups according to the presence or absence of NAFLD and/or abdominal obesity as assessed by waist-hip ratio (WHR) > 0.9. RESULTS: The presence of CAC was detected in 2,385 subjects (11.2%). The proportion of subjects with CAC was highest in the abdominal obesity only group (23.2%). After adjustment for age, diabetes history, hypertension, cigarette smoking, and physical inactivity, the odds ratio (OR) for CAC was the highest in the group with both abnormalities [1.465 (1.324-1.623)]. The NAFLD only group showed significantly increased OR for CAC compared to that in the abdominal obesity only group [1.286 (1.151-1.436) vs. 1.076 (0.939-1.233)]. CONCLUSION: Non-alcoholic fatty liver disease is more closely associated with CAC than abdominal obesity as assessed by the WHR. NAFLD could be considered an independent determinant of subclinical atherosclerosis as assessed by CAC.

Exendin-4 Inhibits the Expression of SEPP1 and Fetuin-A via Improvement of Palmitic Acid-Induced Endoplasmic Reticulum Stress by AMPK.

BACKGROUND: Selenoprotein P (SEPP1) and fetuin-A, both circulating liver-derived glycoproteins, are novel biomarkers for insulin resistance and nonalcoholic fatty liver disease. However, the effect of exendin-4 (Ex-4), a glucagon-like peptide-1 receptor agonist, on the expression of hepatokines, SEPP1, and fetuin-A, is unknown. METHODS: The human hepatoma cell line HepG2 was treated with palmitic acid (PA; 0.4 mM) and tunicamycin (tuni; 2ug/ml) with or without exendin-4 (100 nM) for 24 hours. The change in expression of PA-induced SEPP1, fetuin-A, and endoplasmic reticulum (ER) stress markers by exendin-4 treatment were evaluated using quantitative real-time reverse transcription polymerase chain reaction and Western blotting. Transfection of cells with AMP-activated protein kinase (AMPK) small interfering RNA (siRNA) was performed to establish the effect of exendin-4-mediated AMPK in the regulation of SEPP1 and fetuin-A expression. RESULTS: Exendin-4 reduced the expression of SEPP1, fetuin-A, and ER stress markers including PKR-like ER kinase, inositol-requiring kinase 1α, activating transcription factor 6, and C/EBP homologous protein in HepG2 cells. Exendin-4 also reduced the expression of SEPP1 and fetuin-A in cells treated with tunicamycin, an ER stress inducer. In cells treated with the AMPK activator 5-aminoidazole-4-carboxamide ribonucleotide (AICAR), the expression of hepatic SEPP1 and fetuin-A were negatively related by AMPK, which is the target of exendin-4. In addition, exendin-4 treatment did not decrease SEPP1 and fetuin-A expression in cells transfected with AMPK siRNA. CONCLUSION: These data suggest that exendin-4 can attenuate the expression of hepatic SEPP1 and fetuin-A via improvement of PA-induced ER stress by AMPK.

Ezetimibe stimulates intestinal glucagon-like peptide 1 secretion via the MEK/ERK pathway rather than dipeptidyl peptidase 4 inhibition.

OBJECTIVE: Ezetimibe is known as a Niemann-Pick C1-Like 1 (NPC1L1) inhibitor and has been used as an agent for hypercholesterolemia. In our previous study, ezetimibe administration improved glycemic control and increased glucagon like peptide-1 (GLP-1), an incretin hormone with anti-diabetic properties. However, the mechanisms by which ezetimibe stimulates GLP-1 secretion are not fully understood. Thus, the specific aim of this study was to investigate the mechanism(s) by which ezetimibe stimulates GLP-1 secretion. MATERIALS/METHODS: Male KK/H1J mice were divided into following groups: AIN-93G (NC), NC with ezetimibe (10 mg/kg/day), 45% high fat (HF) diet, and HF diet with ezetimibe. To investigate the role of ezetimibe in glucose homeostasis and GLP-1 secretion, an insulin tolerance test was performed and serum and intestinal GLP-1 levels and intestinal mRNA expression involved in GLP-1 synthesis were measured after 6 weeks of ezetimibe treatment. In vivo and in vitro dipeptidyl peptidase-4 (DPP-4) inhibition assays were employed to demonstrate the association between ezetimibe-induced GLP-1 change and DPP-4. The molecular mechanism by which ezetimibe affects GLP-1 secretion was evaluated by using human enteroendocrine NCI-H716 cells. RESULTS: Ezetimibe supplementation significantly ameliorated HF-increased glucose and insulin resistance in the type 2 diabetic KK/H1J mouse model. Serum and intestinal active GLP-1 levels were significantly increased by ezetimibe in HF-fed animals. However, mRNA expression of genes involved in intestinal GLP-1 synthesis was not altered. Furthermore, ezetimibe did not inhibit the activity of either in vivo or in vitro dipeptidyl peptidase-4 (DPP-4). The direct effects of ezetimibe on GLP-1 secretion and L cell secretory mechanisms were examined in human NCI-H716 intestinal cells. Ezetimibe significantly stimulated active GLP-1 secretion, which was accompanied by the activation of mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK)/extracellular signal-regulated kinase (ERK). Ezetimibe-increased GLP-1 secretion was abrogated by inhibiting the MEK/ERK pathway with PD98059. CONCLUSION: These findings suggest a possible novel biological role of ezetimibe in glycemic control to stimulate intestinal GLP-1 secretion via the MEK/ERK signaling pathway.

Preventive effects of bitter melon (Momordica charantia) against insulin resistance and diabetes are associated with the inhibition of NF-κB and JNK pathways in high-fat-fed OLETF rats.

Bitter melon (BM; Momordica charantia) has been used as a treatment method for various diseases including cancer and diabetes. The objective of this study was to investigate whether BM has preventive effects against insulin resistance and diabetes and to identify the underlying mechanism by which BM ameliorates insulin resistance in obese and diabetic rats. The rats were separated into three groups as follows: (a) high-fat (HF) diet control, (b) HF diet and 1% BM and (c) HF diet and 3% BM. After 6 weeks of assigned treatments, body weight and food intake were not altered by BM administration. Bitter melon treatment significantly improved glucose tolerance and insulin sensitivity. The levels of proinflammatory cytokines were significantly down-regulated in liver, muscle and epididymal fats from BM-treated rats. The activation of nuclear factor-κB (NF-κB) in the liver and muscle was decreased by BM compared with HF controls. The 3% BM supplementation significantly increased the levels of phospho-insulin receptor substrate-1 (Tyr612) and phospho-Akt (Ser473). It also significantly decreased the levels of phospho-NF-κB (p65) (Ser536) and phospho-c-Jun N-terminal kinase (JNK) (Thr183/Tyr185) in liver, muscle and epididymal fats. The findings of this study indicate that BM exerted preventive effects against insulin resistance and diabetes through the modulation of NF-κB and JNK pathways. Therefore, BM may be useful in the prevention of insulin resistance and diabetes.

Comparison of Serum Adipocytokine Levels according to Metabolic Health and Obesity Status.

BACKGROUND: Metabolic health is an emerging concept that is highly correlated with various metabolic complications, and adipocytokines have been causally linked to a wide range of metabolic diseases. Thus, this study compared serum adipocytokine levels according to metabolic health and obesity status. METHODS: Four hundred and fifty-six nondiabetic subjects (mean age, 40.5 years) were categorized into four groups according to metabolic health and obesity status: metabolically healthy nonobese (MHNO), metabolically healthy obese (MHO), metabolically unhealthy nonobese (MUHNO), and metabolically unhealthy obese (MUHO). Being metabolically healthy was defined as the presence of fewer than two of the following five metabolic abnormalities: high blood pressure, high fasting blood glucose, high triglyceride, low high density lipoprotein cholesterol, and being in the highest decile of the homeostatic model assessment of insulin resistance index. Obesity status was assessed using body mass index (BMI), with obesity defined as a BMI higher than 25 kg/m². Levels of serum interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor α (TNF-α), and adipocyte fatty acid binding protein (A-FABP) were also evaluated. RESULTS: Of the 456 subjects, 247 (54.2%) were in the MHNO group, 66 (14.5%) were in the MHO group, 66 (14.5%) were in the MUHNO group, and 77 (16.9%) were in the MUHO group. There were no significant differences in IL-6 or MCP-1 levels among the groups, but levels of TNF-α and A-FABP were significantly higher in the MUHNO group compared to the MHNO group. CONCLUSION: High TNF-α and A-FABP levels are significantly associated with metabolically unhealthiness in nonobese Korean individuals.

Activation of AMP-Activated Protein Kinase Attenuates Tumor Necrosis Factor-α-Induced Lipolysis via Protection of Perilipin in 3T3-L1 Adipocytes.

BACKGROUND: Tumor necrosis factor (TNF)-α and AMP-activated protein kinase (AMPK) are known to stimulate and repress lipolysis in adipocytes, respectively; however, the mechanisms regulating these processes have not been completely elucidated. METHODS: The key factors and mechanism of action of TNF-α and AMPK in lipolysis were investigated by evaluating perilipin expression and activity of protein kinase RNA-like endoplasmic reticulum kinase (PERK)/eukaryotic initiation factor 2 α (eIF2α) by Western blot and an immunofluorescence assay in 24-hour TNF-α-treated 3T3-L1 adipocytes with artificial manipulation of AMPK activation. RESULTS: Enhancement of AMPK activity by the addition of activator minoimidazole carboxamide ribonucleotide (AICAR) suppressed TNF-α-induced lipolysis, whereas the addition of compound C, an inhibitor of AMPK phosphorylation, enhanced lipolysis. Perilipin, a lipid droplet-associated protein, was decreased by TNF-α and recovered following treatment with AICAR, showing a correlation with the antilipolytic effect of AICAR. Significant activation of PERK/eIF2α, a component of the unfolded protein response signaling pathway, was observed in TNF-α or vesicle-treated 3T3-L1 adipocytes. The antilipolytic effect and recovery of perilipin expression by AICAR in TNF-α-treated 3T3-L1 adipocytes were significantly diminished by treatment with 2-aminopurine, a specific inhibitor of eIF2α. CONCLUSION: These data indicated that AICAR-induced AMPK activation attenuates TNF-α-induced lipolysis via preservation of perilipin in 3T3-L1 adipocytes. In addition, PERK/eIF2α activity is a novel mechanism of the anti-lipolytic effect of AICAR.

Association of osteoporosis susceptibility genes with bone mineral density and bone metabolism related markers in Koreans: the Chungju Metabolic Disease Cohort (CMC) study.

In this study, we evaluated the association between bone mineral density (BMD) and 10 single-nucleotide polymorphisms (SNPs) within eight osteoporosis susceptibility genes that were previously identified in genome-wide association studies (GWASs). A total of 494 men and 493 postmenopausal women participating in the Chungju Metabolic Disease cohort study in Korea were included. The following 10 SNPs were genotyped: ZBTB40 rs6426749, MEF2C rs1366594, ESR1 rs2941740, TNFRSF11B rs3134070, TNFRSF11B rs2073617, SOX6 rs711785, LRP5 rs599083, TNFSF11 rs227438, TNFSF11 rs9594782, and FOXL1 rs10048146; and the association between these SNPs and bone metabolism-related markers was assessed. Two SNPs, TNFSF11 rs2277438 and FOXL1 rs1004816, were associated with lumbar spine BMD. TNFSF11 rs2277438 in men and SOX6 rs7117858 and FOXL1 rs10048146 in postmenopausal women were found to be associated with lumbar BMD. ZBTB40 rs6426749, MEF2C rs1366594, and LRP5 rs599083 showed significant associations with femur neck BMD. These three SNPs in men and MEF2C rs1366594 and ESR1 rs2941740 in postmenopausal women were associated with femur neck BMD. A significant association between MEF2C rs1366594 and serum calcium levels was observed in men. Serum phosphorus levels were related to SOX6 rs7117858. Serum PTH levels were significantly associated with TNFRSF11B rs3134070 in men, and SOX6 rs711858 in postmenopausal women. In conclusion, our study independently confirmed associations between several SNPs: ZBTB40, MEF2C, ESR1, SOX6, LRP5, TNFSF11, and FOXL1 and bone marrow density in the Korean population.

Restoration of adiponectin expression via the ERK pathway in TNFα-treated 3T3-L1 adipocytes.

Adiponectin and tumor necrosis factor­α (TNF­α) exert opposite effects on obesity­associated inflammation and insulin signaling. The purpose of the present study was to investigate the effects of chronic TNF­α on adiponectin levels in 3T3­L1 adipocytes, as well as the potential reversal mechanisms. Differentiated 3T3­L1 adipocytes were exposed to TNF­α for three different incubation times and then to various wash­off periods with or without mitogen­activated protein kinase (MAPK) inhibitors. TNF­α significantly reduced adiponectin gene expression in a dose­ and time­dependent manner and activated c­Jun N­terminal kinases (JNK), extracellular signal­regulated kinases (ERK) and p38 MAPK. A 16 h restoration period fully reversed the decrease in adiponectin levels following 16 h treatment with TNF­α; however, 16 h withdrawal of TNF­α following 32 or 48 h treatment did not completely reverse the TNF­α­induced decrease in adiponectin levels. In 3T3­L1 adipocytes, 32 or 48 h wash­off periods were required following 32 or 48 h TNF­α treatments, respectively. The pattern of ERK activation following TNF­α exposure and removal was similar to the pattern of adiponectin expression. Furthermore, ERK1/2 inhibition accelerated the recovery of adiponectin levels compared with the levels in the untreated control adipocytes. Therefore, the inhibitory effects of TNF­α on adiponectin levels in differentiated 3T3­L1 cells were fully reversed following a wash­out period equivalent to the TNF­α treatment time, potentially through the ERK 1/2 pathway.