USP13 ameliorates nonalcoholic fatty liver disease through inhibiting the activation of TAK1.

BACKGROUND: The molecular mechanisms underlying nonalcoholic fatty liver disease (NAFLD) remain to be fully elucidated. Ubiquitin specific protease 13 (USP13) is a critical participant in inflammation-related signaling pathways, which are linked to NAFLD. Herein, the roles of USP13 in NAFLD and the underlying mechanisms were investigated. METHODS: L02 cells and mouse primary hepatocytes were subjected to free fatty acid (FFA) to establish an in vitro model reflective of NAFLD. To prepare in vivo model of NAFLD, mice fed a high-fat diet (HFD) for 16 weeks and leptin-deficient (ob/ob) mice were used. USP13 overexpression and knockout (KO) strategies were employed to study the function of USP13 in NAFLD in mice. RESULTS: The expression of USP13 was markedly decreased in both in vitro and in vivo models of NAFLD. USP13 overexpression evidently inhibited lipid accumulation and inflammation in FFA-treated L02 cells in vitro. Consistently, the in vivo experiments showed that USP13 overexpression ameliorated hepatic steatosis and metabolic disorders in HFD-fed mice, while its deficiency led to contrary outcomes. Additionally, inflammation was similarly attenuated by USP13 overexpression and aggravated by its deficiency in HFD-fed mice. Notably, overexpressing of USP13 also markedly alleviated hepatic steatosis and inflammation in ob/ob mice. Mechanistically, USP13 bound to transforming growth factor ß-activated kinase 1 (TAK1) and inhibited K63 ubiquitination and phosphorylation of TAK1, thereby dampening downstream inflammatory pathways and promoting insulin signaling pathways. Inhibition of TAK1 activation reversed the exacerbation of NAFLD caused by USP13 deficiency in mice. CONCLUSIONS: Our findings indicate the protective role of USP13 in NAFLD progression through its interaction with TAK1 and inhibition the ubiquitination and phosphorylation of TAK1. Targeting the USP13-TAK1 axis emerges as a promising therapeutic strategy for NAFLD treatment.

Fat mass and obesity-associated protein promotes liver steatosis by targeting PPARα.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease worldwide. The fat mass and obesity-associated protein (FTO) has been shown to be involved in obesity; however, its role in NAFLD and the underlying molecular mechanisms remain largely unknown. METHODS: FTO expression was first examined in the livers of patients with NAFLD and animal and cellular models of NAFLD by real-time PCR and Western blotting. Next, its role in lipid accumulation in hepatocytes was assessed both in vitro and in vivo via gene overexpression and knockdown studies. RESULTS: FTO expression was obviously elevated in the livers of mice and humans with hepatic steatosis, probably due to its decreased ubiquitination. FTO overexpression in HepG2 cells induced triglyceride accumulation, whereas FTO knockdown exerted an opposing effect. Consistent with the findings of in vitro studies, adeno-associated viruses 8 (AAV8)-mediated FTO overexpression in the liver promoted hepatic steatosis in C57BL/6J mice. Mechanistically, FTO inhibited the mRNA of peroxisome proliferator-activated receptor α (PPARα) in hepatocytes. Activation of PPARα by its agonist GW7647 reversed lipid accumulation in hepatocytes induced by FTO overexpression. CONCLUSIONS: Overall, FTO expression is increased in NAFLD, and it promotes hepatic steatosis by targeting PPARα.

Triglycerides to high-density lipoprotein cholesterol ratio as a surrogate for nonalcoholic fatty liver disease: a cross-sectional study.

BACKGROUND: Triglycerides (TG) to high-density lipoprotein cholesterol (HDL-C) ratio (TG/HDL-C) has been recommended as a surrogate marker for insulin resistance. In the present study, we aimed to investigate the relationship between TG/HDL-C and NAFLD in an apparently healthy population. METHODS: A total of 18,061 subjects who participated in a health checkup program were included. NAFLD was diagnosed by ultrasonography. RESULTS: The prevalence rate of NAFLD was 24.8% in the whole population, and progressively increased across the quartiles of TG/HDL-C (4.9, 14.1, 26.8 and 53.5%, respectively, P <  0.001). After adjustment for confounding factors, TG/HDL-C was independently associated with the risk of NAFLD. Compared with the first quartile of TG/HDL-C (Q1), the odds ratios (95% confidence intervals) for NAFLD in the increasing quartiles (Q2-Q4) were 2.1(1.8-2.6), 3.6 (3.0-4.3) and 9.2(7.6-11.1), respectively. In addition, the area under receiver operator characteristic curve (95% confidence interval) of TG/HDL-C for NAFLD was 0.85 (0.84-0.86) in women and 0.79 (0.78-0.80) in men, significantly higher than that of TG, TC, LDL-C, HDL-C, ALT and AST (P <  0.05). The optimal cutoff point of TG/HDL-C for detection of NAFLD was 0.9 in women (sensitivity = 78.8%, specificity = 77.3%) and 1.4 in men (sensitivity = 70.7%, specificity = 73.5%). CONCLUSIONS: TG/HDL-C is independently associated with NAFLD in apparently healthy individuals and may be used as a surrogate for NAFLD.

Heat shock protein 70 promotes lipogenesis in HepG2 cells.

BACKGROUND: The increasing prevalence of non-alcoholic fatty liver disease (NAFLD) has followed the international rise in obesity rates. Multiple mechanisms are involved in NAFLD, including endoplasmic reticulum stress and oxidative stress. Heat shock protein 70 (HSP70), which is abundant in most organisms, is sensitive to stress. However, the role of HSP70 in NAFLD has not been investigated. Here, we investigated the possible role of HSP70 in lipid synthesis. METHODS: C57BL/6 mice were fed a high-fat diet, and HepG2 cells were treated with 0.5 mM palmitic acid (PA). HSP70 expression was detected by qPCR, Western blot and immunohistochemistry. Total cholesterol (TC) and triglyceride (TG) levels were detected by enzyme-linked immunosorbent assay (ELISA). After Hsp70 overexpression and knockdown, TC and TG levels and FAS, SCD, and ACC expression were detected. RESULTS: HSP70 expression was significantly increased in the livers of obese mice. In vitro, HSP70 expression was markedly induced by PA in HepG2 cells. Notably, HSP70 overexpression in HepG2 cells enhanced TC and TG synthesis, in parallel with the upregulation of lipogenic genes, including FAS, SCD and ACC. By contrast, HSP70 knockdown decreased the levels of cellular lipids and the expression of FAS, SCD, and ACC in HepG2 cells. Together, our results suggest that HSP70 may promote lipogenesis in HepG2 cells. CONCLUSIONS: Heat shock protein 70 promotes lipogenesis in HepG2 cells.

Rapamycin improves palmitate-induced ER stress/NF κ B pathways associated with stimulating autophagy in adipocytes.

Obesity-induced endoplasmic reticulum (ER) stress and inflammation lead to adipocytes dysfunction. Autophagy helps to adapt to cellular stress and involves in regulating innate inflammatory response. In present study, we examined the activity of rapamycin, a mTOR kinase inhibitor, against endoplasmic reticulum stress and inflammation in adipocytes. An in vitro model was used in which 3T3-L1 adipocytes were preloaded with palmitate (PA) to generate artificial hypertrophy mature adipocytes. Elevated autophagy flux and increased number of autophagosomes were observed in response to PA and rapamycin treatment. Rapamycin attenuated PA-induced PERK and IRE1-associated UPR pathways, evidenced by decreased protein levels of eIF2α phosphorylation, ATF4, CHOP, and JNK phosphorylation. Inhibiting autophagy with chloroquine (CQ) exacerbated these ER stress markers, indicating the role of autophagy in ameliorating ER stress. In addition, cotreatment of CQ abolished the anti-ER stress effects of rapamycin, which confirms the effect of rapamycin on ERs is autophagy-dependent. Furthermore, rapamycin decreased PA-induced nuclear translocation of NFκB P65 subunit, thereby NFκB-dependent inflammatory cytokines MCP-1 and IL-6 expression and secretion. In conclusion, rapamycin attenuated PA-induced ER stress/NFκB pathways to counterbalance adipocytes stress and inflammation. The beneficial of rapamycin in this context partly depends on autophagy. Stimulating autophagy may become a way to attenuate adipocytes dysfunction.

The risk factors of diabetic ketosis and diabetic ketoacidosis among patients with type 2 diabetes mellitus treated with SGLT2 inhibitors: a retrospective study.

BACKGROUND: There is limited evidence on the safety of sodium-glucose co-transporter-2 inhibitor (SGLT2i) use in the real world of China. We conducted this two-center, retrospective study to assess the incidence rate and risk factors of Dapagliflozin-associated DK/DKA among patients with type 2 diabetes mellitus (T2DM) in China. RESEARCH DESIGN AND METHODS: Patients with T2DM treated with Dapagliflozin in Shanghai General Hospital were included in this retrospective analysis. Univariate and multivariate logistic regression was performed, and odds ratio (OR) and 95% confidence interval (CI) were calculated to identify the influencing factors associated with the occurrence of DK/DKA. RESULTS: A total of 1985 T2DM patients received Dapagliflozin for the first time were included. The prevalence of DK and DKA was 2.47% and 0.35%, respectively. Multivariate logistic regression identified age <45 years [OR = 2.99, 95% CI (1.45-6.17)], concomitant use of Acarbose [OR = 2.18, 95% CI (1.06-3.38)], Metformin [OR = 1.84, 95% CI (1.01-3.38)], and Insulin [OR = 1.93, 95% CI (1.02-3.66)] as participating factors for DK/DKA. The 1:4 matched subset sensitivity analysis further confirmed the risk factors of Dapagliflozin-associated DK/DKA. CONCLUSIONS: Age less than 45 years, concomitant use of Acarbose and insulin were risk factors for Dapagliflozin-associated DK/DKA. Clinicians should watch out for high-risk features among patients with SGLT2i prescription.

G protein-coupled receptor 48 upregulates estrogen receptor alpha expression via cAMP/PKA signaling in the male reproductive tract.

The epididymis and efferent ducts play major roles in sperm maturation, transport, concentration and storage by reabsorbing water, ions and proteins produced from seminiferous tubules. Gpr48-null male mice demonstrate reproductive tract defects and infertility. In the present study, we found that estrogen receptor alpha (ERalpha) was dramatically reduced in the epididymis and efferent ducts in Gpr48-null male mice. We further revealed that ERalpha could be upregulated by Gpr48 activation via the cAMP/PKA signaling pathway. Moreover, we identified a cAMP responsive element (Cre) motif located at -1307 to -1300 bp in the ERalpha promoter that is able to interact with Cre binding protein (Creb). In conclusion, Gpr48 participates in the development of the male epididymis and efferent ducts through regulation of ERalpha expression via the cAMP/PKA signaling pathway.

Follistatin-like 1: a potential mediator of inflammation in obesity.

Obesity is associated with a state of chronic low-grade inflammation, which contributes to insulin resistance and type 2 diabetes. However, the molecular mechanisms that link obesity to inflammation are not fully understood. Follistatin-like 1 (FSTL1) is a novel proinflammatory cytokine that is expressed in adipose tissue and secreted by preadipocytes/adipocytes. We aimed to test whether FSTL1 could have a role in obesity-induced inflammation and insulin resistance. It was found that FSTL1 expression was markedly decreased during differentiation of 3T3-L1 preadipocytes but reinduced by TNF-α. Furthermore, a significant increase in FSTL1 levels was observed in adipose tissue of obese ob/ob mice, as well as in serum of overweight/obese subjects. Mechanistic studies revealed that FSTL1 induced inflammatory responses in both 3T3-L1 adipocytes and RAW264.7 macrophages. The expression of proinflammatory mediators including IL-6, TNF-α, and MCP-1 was upregulated by recombinant FSTL1 in a dose-dependent manner, paralleled with activation of the IKKß-NFκB and JNK signaling pathways in the two cell lines. Moreover, FSTL1 impaired insulin signaling in 3T3-L1 adipocytes, as revealed by attenuated phosphorylation of both Akt and IRS-1 in response to insulin stimulation. Together, our results suggest that FSTL1 is a potential mediator of inflammation and insulin resistance in obesity.

GPR48 increases mineralocorticoid receptor gene expression.

Aldosterone and the mineralocorticoid receptor (MR) are critical to the maintenance of electrolyte and BP homeostasis. Mutations in the MR cause aldosterone resistance known as pseudohypoaldosteronism type 1 (PHA1); however, some cases consistent with PHA1 do not exhibit known gene mutations, suggesting the possibility of alternative genetic variants. We observed that G protein-coupled receptor 48 (Gpr48/Lgr4) hypomorphic mutant (Gpr48(m/m)) mice had hyperkalemia and increased water loss and salt excretion despite elevated plasma aldosterone levels, suggesting aldosterone resistance. When we challenged the mice with a low-sodium diet, these features became more obvious; the mice also developed hyponatremia and increased renin expression and activity, resembling a mild state of PHA1. There was marked renal downregulation of MR and its downstream targets (e.g., the α-subunit of the amiloride-sensitive epithelial sodium channel), which could provide a mechanism for the aldosterone resistance. We identified a noncanonical cAMP-responsive element located in the MR promoter and demonstrated that GPR48 upregulates MR expression via the cAMP/protein kinase A pathway in vitro. Taken together, our data demonstrate that GPR48 enhances aldosterone responsiveness by activating MR expression, suggesting that GPR48 contributes to homeostasis of electrolytes and BP and may be a candidate gene for PHA1.

Interrelation between the lipid accumulation product index and diabetic kidney disease in patients with type 2 diabetes mellitus.

Objective: The purpose of this study was to determine the relation between the lipid accumulation product index (LAPI) and diabetic kidney disease (DKD) in patients with type 2 diabetes mellitus (T2DM). Methods: Herein, 931 patients were enrolled and their data were collected. Then the interrelation between LAPI and DKD was assessed using multivariate logistic regression analyses (LRAs) and by a restricted cubic spline (RCS). Results: In total, 931 participants (352 females and 579 males) aged 55 years on average were included in the study. After adjusting for several confounders, the odds ratio for DKD was increased evidently in the third LAPI tertile compared with that in the first LAPI tertile. In addition, the RCS revealed a positive interrelation between LAPI and DKD. In the subgroup analyses, age, sex, hyperlipidemia, hypertension, and HbA1c did not significantly interact with LAPI. Conclusions: LAPI was higher in the DKD group than in the no-DKD group, and LAPI is positively linked with DKD, which may have potential value to diagnose DKD in clinical practice.

Association between Chinese visceral adiposity index and metabolic-associated fatty liver disease in Chinese adults with type 2 diabetes mellitus.

Objective: The purpose of the study was to determine the correlation of the Chinese visceral adiposity index (CVAI) with metabolic-associated fatty liver disease (MAFLD) in Chinese adults with type 2 diabetes mellitus (T2DM). Materials/methods: In this cross-sectional study, data on sociodemographic characteristics, laboratory test results, coexisting diseases, and medical therapy were collected and analyzed. Multivariate logistic regression analyses were used to examine the correlation between CVAI and MAFLD. In order to investigate the correlation between CVAI on a continuous scale and MAFLD, a restricted cubic spline (RCS) was used. Results: A total of 679 participants were included in this study. There were 251 female participants and 428 male participants, with a median age of 55 years. In the multivariate logistic regression model, diastolic blood pressure, duration of diabetes, glycated hemoglobin, hemoglobin, alanine transaminase, aspartate aminotransferase, gamma -glutamyl transferase, albumin, blood urea nitrogen, total cholesterol, low-density lipoprotein cholesterol, statin use and metformin use were adjusted, and an evident increase in the odds ratios of MAFLD from the lowest to the highest CVAI quartile was found (P value for trend < 0.001). Moreover, the RCS curves revealed a positive correlation between CVAI and MAFLD. Conclusions: The CVAI is positively correlated with MAFLD and may be an indicator with diagnostic value for MAFLD in clinical practice in type 2 diabetic patients.

Association between use of liraglutide and liver fibrosis in patients with type 2 diabetes.

Objective: Patients with type 2 diabetes have a high risk of non-alcoholic fatty liver disease (NAFLD) and related liver fibrosis. Glucagon-like peptide-1 receptor agonists (GLP-1RAs) have demonstrated efficacy in improving NAFLD, while their effectiveness on liver fibrosis is limited in type 2 diabetic patients. Materials/Methods: A prospective cohort study was performed in type 2 diabetic patients. The study subjects were divided into two groups based on the use of liraglutide or not, and propensity score matching (PSM) was also conducted. After 12 months follow-up, liver fibrosis was assessed by NAFLD fibrosis score (NFS) fibrosis-4 (FIB-4), and liver stiffness measurement (LSM). The association between liraglutide use and liver fibrosis was analyzed by multivariable linear regression. Results: In the current study, a total of 1,765 type 2 diabetic patients were enrolled. 262 patients were liraglutide user and 1,503 were nouser. After 12 months follow-up, liraglutide use tended to be associated with reduced prevalence of advanced fibrosis (3.1% vs. 6.1%, P = 0.218). After adjustment for confounding factors, multivariable linear regression revealed that liraglutide use was negatively associated with decreased NFS (ß= -0.34, P = 0.043), FIB4 (ß= -0.26, P = 0.044) and LSM (ß= -4.95, P = 0.007) in type 2 diabetics. The results after PSM were similar to those before PSM. Conclusions: Liraglutide treatment is associated with decreased liver fibrosis in type 2 diabetic subjects.

Association Between High-Sensitivity C-Reactive Protein and Diabetic Kidney Disease in Patients With Type 2 Diabetes Mellitus.

Objective: High-sensitivity C-reactive protein (hs-CRP) is an inflammatory marker. This study aimed to identify the correlation between hs-CRP levels and diabetic kidney disease (DKD) in patients with type 2 diabetes mellitus (T2DM). Materials/Methods: This cross-sectional and observational study included 927 patients with T2DM. We collected the data of patients based on their medical data, including sociodemographic characteristics, concomitant diseases, laboratory results, and medical therapy. Multivariate logistic regression analysis was conducted to assess the relationship between hs-CRP levels and DKD. A restricted cubic spline (RCS) was used to assess the correlation of hs-CRP levels on a continuous scale with the DKD. Results: In total, 927 patients were recruited in our study. The median age of the recruited patients was 55 years, and there were 346 female patients and 581 male patients. The hs-CRP levels were evidently higher in patients with DKD than those without DKD. After adjusting for age, sex, diastolic blood pressure, systolic blood pressure, body mass index, neck circumference, waist circumference, hypertension, duration of diabetes, common carotid artery plaque, fasting plasma glucose, glycated hemoglobin, hemoglobin, erythrocyte, leukocyte, γ-glutamyl transferase, albumin, urea nitrogen, uric acid and triglyceride, a significant increase in the odds ratios (ORs) for DKD in the fourth hs-CRP quartile compared with the first quartile was observed (P value for trend= 0.003), and the ORs (95% confidence intervals) in the fourth quartile of hs-CRP were 1.968 (1.244-3.114) for DKD compared to the first quartile.. Moreover, the RCS curves presented a positive association between hs-CRP and DKD in total subjects, male subjects and female subjects, respectively. Conclusions: The results of our study indicated that hs-CRP levels were significantly and positively correlated with the presence of DKD, which may provide predictive and diagnostic values in clinical practice.

Association of the Non-Alcoholic Fatty Liver Disease Fibrosis Score with subclinical myocardial remodeling in patients with type 2 diabetes: A cross-sectional study in China.

AIMS/INTRODUCTION: Non-alcoholic fatty liver disease, especially with liver fibrosis, is associated with cardiovascular diseases. The Non-Alcoholic Fatty Liver Disease Fibrosis Score (NFS), a non-invasive marker of advanced fibrosis, was found to be associated with cardiovascular diseases in different populations. The aim of the present study was to determine whether the NFS is associated with subclinical myocardial remodeling in type 2 diabetes patients. MATERIALS AND METHODS: A cross-sectional study was carried out in type 2 diabetes patients. The NFS derived from available parameters was calculated, and the participants were divided according to the quartiles of the NFS and grades of the NFS (low, intermediate and high). Fibrosis-4 and Aspartate Aminotransferase to Platelet Ratio Index, another two liver fibrosis scores, were also calculated. Subclinical myocardial remodeling was examined by echocardiography, and its associations with NFS, Fibrosis-4 and Aspartate Aminotransferase to Platelet Ratio Index were analyzed. RESULTS: A total of 1,878 type 2 diabetes patients were enrolled in the present study. The NFS was independently associated with sex, age, body mass index, low-density lipoprotein cholesterol and glycated hemoglobin in type 2 diabetes patients. Parameters of subclinical myocardial remodeling including left atrial dimension, interventricular septum thickness, left ventricular end-diastolic diameter, left ventricular end-systolic diameter, left ventricular posterior wall thickness (LVPWT) and left ventricular mass index were all gradually increased with the increment of the NFS. Linear regression analysis further showed that the NFS was positively associated with left atrial dimension, interventricular septum thickness, left ventricular end-diastolic diameter, left ventricular end-systolic diameter, LVPWT and left ventricular mass index after adjustment for the confounding factors. Similarly, Fibrosis-4 was associated with left atrial dimension, interventricular septum thickness, LVPWT and left ventricular mass index. In contrast, the Aspartate Aminotransferase to Platelet Ratio Index was only associated with LVPWT. CONCLUSIONS: Non-invasive liver fibrosis scores, especially the NFS, are independently associated with subclinical myocardial remodeling in type 2 diabetes patients.

Helicobacter pylori Infection Is Not Associated with Non-alcoholic Fatty Liver Disease: A Cross-Sectional Study in China.

Background and Aim:Helicobacter pylori infection has been reported to promote the development of a variety of extra-digestive manifestations, including type 2 diabetes, cardiovascular and liver diseases. Recently, the association between H. pylori infection and non-alcoholic fatty liver disease (NAFLD) was also proposed. However, evidence from different studies was controversial. We therefore performed this study to investigate the relationship between them in a large population of apparently healthy subjects in China. Methods: A total of 21,456 subjects underwent a healthy checkup program were included. H. pylori infection was detected by 14C urea breath test (14C-UBT) and NAFLD was diagnosed by ultrasonography. Results: Subjects infected with H. pylori had a more unfavorable metabolic profile, including higher levels of body mass index (BMI), blood pressure, triglycerides (TG) and lower levels of high-density lipoprotein cholesterol (HDL-C), as compared with those without H. pylori infection (all P < 0.05). Moreover, the prevalence rate of NAFLD was significantly increased in subjects with H. pylori infection when compared with those without H. pylori in women (23.6% vs. 21.5%, P < 0.05), but not in men (46.5% vs. 45.5%, P > 0.05). After adjusting for confounding factors including age, sex, BMI, blood pressure and lipid profiles, multivariate logistic analysis revealed that H. pylori infection was not independently associated with the risk of NAFLD in the total population (OR = 0.9, 95% CI = 0.9-1.0, P = 0.097). Also, subgroup analysis (stratified by age, sex, BMI, and diabetes status) showed no independent association between H. pylori infection and NAFLD. Conclusion: Our data suggests that H. pylori infection is not independently associated with the risk of NAFLD in apparently healthy subjects.

Sex-Specific Association between Serum Uric Acid and Nonalcoholic Fatty Liver Disease in Type 2 Diabetic Patients.

Across-sectional study was performed in 541 type 2 diabetic patients to determine the relationship between serum uric acid (SUA) and NAFLD in type 2 diabetic patients. Clinical parameters including SUA were determined and NAFLD was diagnosed by ultrasonography. SUA was significantly higher in type 2 diabetic subjects with NAFLD than in those without NAFLD in men, but not in women. Furthermore, the prevalence rate of NAFLD increased progressively across the sex-specific SUA tertiles only in men (37.9%, 58.6%, and 72.6%, resp., P for trend < 0.001). After adjusting for confounding factors, the odd ratios (95% CI) for NAFLD were 1 (reference), 2.93 (95%CI 1.25-6.88), and 3.93 (95% CI 1.55-9.98), respectively, across the tertiles of SUA in men. Contrastingly, SUA levels in women were not independently associated with the risk of NAFLD. Our data suggests that SUA is specifically associated with NAFLD in male type 2 diabetic subjects, independent of insulin resistance and other metabolic factors.

Palmitate induces endoplasmic reticulum stress and autophagy in mature adipocytes: implications for apoptosis and inflammation.

Endoplasmic reticulum (ER) stress and inflammation induced by obesity lead to adipocyte dysfunction, with the impairment of the insulin pathway. Recent studies have indicated that understanding the physiological role of autophagy is of great significance. In the present study, an in vitro model was used in which 3T3-L1 adipocytes were pre-loaded with palmitate (PA) to generate artificially hypertrophied mature adipocytes. PA induced an autophagic flux, determined by an increased microtubule-associated protein 1 light chain 3 (LC3)-II formation, as shown by western blot analysis and fluorescence microscopy, and was confirmed using transmission electron microscopy (TEM). Using TEM and western blot analysis, we observed increased ER stress in response to PA, as indicated by the increased levels of the ER stress markers, BiP, activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP), and the phosphoralytion of eukaryotic translation initiation factor 2α and c-Jun N-terminal kinase (JNK). Of note, we observed that the PA-induced ER stress occurred prior to the activation of autophagy. We confirmed that autophagy was induced in response to JNK-dependent ER stress, as autophagy was suppressed by treatment with the ER stress inhibitor, 4-phenyl butyrate (4-PBA), and the JNK inhibitor, SP600125. Upon the inhibition of autophagy using chloroquine (CQ), we observed exacerbated ER stress and an increased level of cell death. Importantly, to determine whether autophagy is linked to inflammation, the autophagy inhibitor, 3-methyladenine (3-MA) was used. The inhibition of autophagy led to a further increase in the PA-induced expression of monocyte chemoattractant protein-1 (MCP-1) and interleukin-6 (IL-6). Consistently, such an increase was also observed following treatment with SP600125. In conclusion, our data indicate that PA elicits a ER stress-JNK-autophagy axis, and that this confers a pro-survival effect against PA-induced cell death and stress in hypertrophied adipocytes. The JNK-dependent activation of autophagy diminishes PA-induced inflammation. Therefore, the stimulation of autophagy may become a method with which to attenuate adipocyte dysfunction and inflammation.

Spexin peptide is expressed in human endocrine and epithelial tissues and reduced after glucose load in type 2 diabetes.

Spexin mRNA and protein are widely expressed in rat tissues and associate with weight loss in rodents of diet-induced obesity. Its location in endocrine and epithelial cells has also been suggested. Spexin is a novel peptide that involves weight loss in rodents of diet-induced obesity. Therefore, we aimed to examine its expression in human tissues and test whether spexin could have a role in glucose and lipid metabolism in type 2 diabetes mellitus (T2DM). The expression of the spexin gene and immunoreactivity in the adrenal gland, skin, stomach, small intestine, liver, thyroid, pancreatic islets, visceral fat, lung, colon, and kidney was higher than that in the muscle and connective tissue. Immunoreactive serum spexin levels were reduced in T2DM patients and correlated with fasting blood glucose (FBG, r=-0.686, P<0.001), hemoglobin A1c (HbA1c, r=-0.632, P<0.001), triglyceride (TG, r=-0.236, P<0.001) and low density lipoprotein-cholesterol (LDL-C, r=-0.382, P<0.001). A negative correlation of blood glucose with spexin was observed during oral glucose tolerance test (OGTT). Spexin is intensely expressed in normal human endocrine and epithelial tissues, indicating that spexin may be involved in physiological functions of endocrine and in several other tissues. Circulating spexin levels are low in T2DM patients and negatively related to blood glucose and lipids suggesting that the peptide may play a role in glucose and lipid metabolism in T2DM.

LGR4 acts as a link between the peripheral circadian clock and lipid metabolism in liver.

The circadian clock plays an important role in the liver by regulating the major aspects of energy metabolism. Currently, it is assumed that the circadian clock regulates metabolism mostly by regulating the expression of liver enzymes at the transcriptional level, but the underlying mechanism is not well understood. In this study, we showed that Lgr4 homozygous mutant (Lgr4(m/m)) mice showed alteration in the rhythms of the respiratory exchange ratio. We further detected impaired plasma triglyceride rhythms in Lgr4(m/m) mice. Although no significant changes in plasma cholesterol rhythms were observed in the Lgr4(m/m) mice, their cholesterol levels were obviously lower. This phenotype was further confirmed in the context of ob/ob mice, in which lack of LGR4 dampened circadian rhythms of triglyceride. We next demonstrated that Lgr4 expression exhibited circadian rhythms in the liver tissue and primary hepatocytes in mice, but we did not detect changes in the expression levels or circadian rhythms of classic clock genes, such as Clock, Bmal1 (Arntl), Pers, Rev-erbs, and Crys, in Lgr4(m/m) mice compared with their littermates. Among the genes related to the lipid metabolism, we found that the diurnal expression pattern of the Mttp gene, which plays an important role in the regulation of plasma lipid levels, was impaired in Lgr4(m/m) mice and primary Lgr4(m/m) hepatocytes. Taken together, our results demonstrate that LGR4 plays an important role in the regulation of plasma lipid rhythms, partially through regulating the expression of microsomal triglyceride transfer protein. These data provide a possible link between the peripheral circadian clock and lipid metabolism.

Midkine, a potential link between obesity and insulin resistance.

Obesity is associated with increased production of inflammatory mediators in adipose tissue, which contributes to chronic inflammation and insulin resistance. Midkine (MK) is a heparin-binding growth factor with potent proinflammatory activities. We aimed to test whether MK is associated with obesity and has a role in insulin resistance. It was found that MK was expressed in adipocytes and regulated by inflammatory modulators (TNF-α and rosiglitazone). In addition, a significant increase in MK levels was observed in adipose tissue of obese ob/ob mice as well as in serum of overweight/obese subjects when compared with their respective controls. In vitro studies further revealed that MK impaired insulin signaling in 3T3-L1 adipocytes, as indicated by reduced phosphorylation of Akt and IRS-1 and decreased translocation of glucose transporter 4 (GLUT4) to the plasma membrane in response to insulin stimulation. Moreover, MK activated the STAT3-suppressor of cytokine signaling 3 (SOCS3) pathway in adipocytes. Thus, MK is a novel adipocyte-secreted factor associated with obesity and inhibition of insulin signaling in adipocytes. It may provide a potential link between obesity and insulin resistance.