Qichen Fang

APPL1 binds to adiponectin receptors and mediates adiponectin signalling and function.

Adiponectin, also known as Acrp30, is an adipose tissue-derived hormone with anti-atherogenic, anti-diabetic and insulin sensitizing properties. Two seven-transmembrane domain-containing proteins, AdipoR1 and AdipoR2, have recently been identified as adiponectin receptors, yet signalling events downstream of these receptors remain poorly defined. By using the cytoplasmic domain of AdipoR1 as bait, we screened a yeast two-hybrid cDNA library derived from human fetal brain. This screening led to the identification of a phosphotyrosine binding domain and a pleckstrin homology domain-containing adaptor protein, APPL1 (adaptor protein containing pleckstrin homology domain, phosphotyrosine binding (PTB) domain and leucine zipper motif). APPL1 interacts with adiponectin receptors in mammalian cells and the interaction is stimulated by adiponectin. Overexpression of APPL1 increases, and suppression of APPL1 level reduces, adiponectin signalling and adiponectin-mediated downstream events (such as lipid oxidation, glucose uptake and the membrane translocation of glucose transport 4 (GLUT4)). Adiponectin stimulates the interaction between APPL1 and Rab5 (a small GTPase) interaction, leading to increased GLUT4 membrane translocation. APPL1 also acts as a critical regulator of the crosstalk between adiponectin signalling and insulin signalling pathways. These results demonstrate a key function for APPL1 in adiponectin signalling and provide a molecular mechanism for the insulin sensitizing function of adiponectin.

Adiponectin activates AMP-activated protein kinase in muscle cells via APPL1/LKB1-dependent and phospholipase C/Ca2+/Ca2+/calmodulin-dependent protein kinase kinase-dependent pathways

The binding of the adaptor protein APPL1 to adiponectin receptors is necessary for adiponectin-induced AMP-activated protein kinase (AMPK) activation in muscle, yet the underlying molecular mechanism remains unknown. Here we show that in muscle cells adiponectin and metformin induce AMPK activation by promoting APPL1-dependent LKB1 cytosolic translocation. APPL1 mediates adiponectin signaling by directly interacting with adiponectin receptors and enhances LKB1 cytosolic localization by anchoring this kinase in the cytosol. Adiponectin also activates another AMPK upstream kinase Ca2+/calmodulin-dependent protein kinase kinase by activating phospholipase C and subsequently inducing Ca2+ release from the endoplasmic reticulum, which plays a minor role in AMPK activation. Our results show that in muscle cells adiponectin is able to activate AMPK via two distinct mechanisms as follows: a major pathway (the APPL1/LKB1-dependent pathway) that promotes the cytosolic localization of LKB1 and a minor pathway (the phospholipase C/Ca2+/Ca2+/calmodulin-dependent protein kinase kinase-dependent pathway) that stimulates Ca2+ release from intracellular stores.

Fibroblast growth factor 21 is regulated by the IRE1α-XBP1 branch of the unfolded protein response and counteracts endoplasmic reticulum stress-induced hepatic steatosis.

Background: Although both are involved in metabolic homeostasis, the interconnection between ER stress and FGF21 remains incompletely understood. Results: Directly up-regulated by the IRE1α-XBP1 pathway, FGF21 could alleviate ER stress-induced liver steatosis. Conclusion: FGF21 acts as a metabolic effector of the UPR program, exerting feedback effects upon lipid metabolism. Significance: These findings reveal a regulatory mechanism linking FGF21 actions to metabolic ER stress. Endoplasmic reticulum (ER) stress activates the adaptive unfolded protein response (UPR) and represents a critical mechanism that underlies metabolic dysfunctions. Fibroblast growth factor 21 (FGF21), a hormone that is predominantly secreted by the liver, exerts a broad range of effects upon the metabolism of carbohydrates and lipids. Although increased circulating levels of FGF21 have been documented in animal models and human subjects with obesity and nonalcoholic fatty liver disease, the functional interconnections between metabolic ER stress and FGF21 are incompletely understood. Here, we report that increased ER stress along with the simultaneous elevation of FGF21 expression were associated with the occurrence of nonalcoholic fatty liver disease both in diet-induced obese mice and human patients. Intraperitoneal administration of the ER stressor tunicamycin in mice resulted in hepatic steatosis, accompanied by activation of the three canonical UPR branches and increased the expression of FGF21. Furthermore, the IRE1α-XBP1 pathway of the UPR could directly activate the transcriptional expression of Fgf21. Administration of recombinant FGF21 in mice alleviated tunicamycin-induced liver steatosis, in parallel with reduced eIF2α-ATF4-CHOP signaling. Taken together, these results suggest that FGF21 is an integral physiological component of the cellular UPR program, which exerts beneficial feedback effects upon lipid metabolism through counteracting ER stress.

Autophagy-mediated insulin receptor down-regulation contributes to endoplasmic reticulum stress-induced insulin resistance.

Endoplasmic reticulum (ER) stress is associated with obesity-induced insulin resistance, yet the underlying mechanisms remain to be fully elucidated. Here we show that ER stress-induced insulin receptor (IR) down-regulation may play a critical role in obesity-induced insulin resistance. The expression levels of IR are negatively associated with the ER stress marker C/EBP homologous protein (CHOP) in insulin target tissues of db/db mice and mice fed a high-fat diet. Significant IR down-regulation was also observed in fat tissue of obese human subjects and in 3T3-L1 adipocytes treated with ER stress inducers. ER stress had little effect on IR tyrosine phosphorylation per se but greatly reduced IR downstream signaling. The ER stress-induced reduction in IR cellular levels was greatly alleviated by the autophagy inhibitor 3-methyladenine but not by the proteasome inhibitor N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal (MG132). Inhibition of autophagy prevented IR degradation but did not rescue IR downstream signaling, consistent with an adaptive role of autophagy in response to ER stress-induced insulin resistance. Finally, chemical chaperone treatment protects cells from ER stress-induced IR degradation in vitro and obesity-induced down-regulation of IR and insulin action in vivo. Our results uncover a new mechanism underlying obesity-induced insulin resistance and shed light on potential targets for the prevention and treatment of obesity-induced insulin resistance and type 2 diabetes.

Mechanism and clinical evidence of lipocalin‐2 and adipocyte fatty acid‐binding protein linking obesity and atherosclerosis

Obesity is considered to be a chronic inflammatory state in which the dysfunction of adipose tissue plays a central role. The adipokines, which are cytokines secreted by adipose tissue, are key links between obesity and related diseases such as metabolic syndrome and atherosclerosis. LCN2 and A‐FABP, both of which are major adipokines predominantly produced in adipose tissue, have recently been shown to be pivotal modulators of vascular function. However, different adipokines modulate the development of atherosclerosis in distinctive manners, which are partly attributable to their unique regulatory mechanisms and functions. This review highlights recent advances in the understanding of the role of two adipokines in mediating chronic inflammation and the pathogenesis of atherosclerosis. Copyright

Elevated Circulating Lipocalin-2 Levels Independently Predict Incident Cardiovascular Events in Men in a Population-Based Cohort

Objective—Adipose tissue inflammation and perturbation of adipokine secretion may contribute to the pathogenesis of cardiovascular diseases (CVD). Lipocalin-2 (LCN2), mainly released from adipocytes, has been shown to be positively associated with CVD in cross-sectional studies. We aimed to evaluate the association of LCN2 with CVD involving a population-based cohort recruited from the Shanghai Diabetes Study. Approach and Results—Serum LCN2 levels were measured using ELISA. Independent predictors of CVD development were identified using Cox proportion hazards regression. The predictive performances of the various models were assessed by Kaplan–Meier analysis. At baseline, circulating LCN2 was significantly associated with a cluster of traditional cardiovascular risk factors. Baseline LCN2 levels in male subjects who developed CVD events during follow-up were significantly higher than those who did not develop CVD events (P=0.012). However, such difference was not significant in female subjects. LCN2 was a predictor of CVD in men, which remained statistically significant after adjustment for traditional cardiovascular risk factors (hazard ratio, 1.038 [95% confidence interval, 1.017–1.060]). LCN2 remained significantly associated with incident CVD even after adjustment for renal function, adiponectin, and high-sensitivity C-reactive protein levels. Kaplan–Meier analysis suggested combination of LCN2 and high-sensitivity C-reactive protein might improve the prediction of CVD events in male subjects. Conclusions—Elevated circulating LCN2 level is an independent predictor of CVD events in men in a population-based cohort and adds to the prognostic value of high-sensitivity C-reactive protein, which is currently the most extensively studied biomarker of CVD. Measurement of serum LCN2 might be useful for early detection and intervention of CVD.

Relationship between serum osteocalcin levels and non-alcoholic fatty liver disease in Chinese men.

The present study was designed to investigate the relationship between serum osteocalcin levels and non‐alcoholic fatty liver disease (NAFLD) in Chinese men. In all, 1558 men (21–78 years old) were recruited to the study. Serum osteocalcin, glucose and lipid profiles were determined. Demographic and clinical characteristics were recorded. All participants underwent hepatic ultrasonographic examination. Serum osteocalcin levels were significantly lower in subjects with NAFLD than those without (P < 0.01). All study subjects were divided into four subgroups according to quartiles of serum osteocalcin levels. The frequency of NAFLD increased progressively with declining serum osteocalcin levels (Ptrend < 0.01). Serum osteocalcin levels were inversely correlated with NAFLD (P < 0.01). However, the significant association between serum osteocalcin levels and NAFLD disappeared in logistic regression analyses. Furthermore, multiple stepwise regression analysis showed that serum osteocalcin levels were independently associated with serum alanine aminotransferase levels in Chinese men (P < 0.01). The findings of the present study suggest that serum osteocalcin levels are not directly correlated with NAFLD.

Negative Regulation of DsbA-L Gene Expression by the Transcription Factor Sp1

Disulfide-bond A oxidoreductase-like protein (DsbA-L) possesses beneficial effects such as promoting adiponectin multimerization and stability, increasing insulin sensitivity, and enhancing energy metabolism. The expression level of DsbA-L is negatively correlated with obesity in mice and humans, but the underlying mechanisms remain unknown. To address this question, we generated reporter gene constructs containing the promoter sequence of the mouse DsbA-L gene. Deletion analysis showed that the proximal promoter of mouse DsbA-L is located between −186 and −34 bp relative to the transcription start site. In silico analysis identified a putative Sp1 transcription factor binding site in the first intron of the DsbA-L gene. Electrophoretic mobility shift assay and chromatin immunoprecipitation analysis indicated that Sp1 bound to this intron region in vitro and in intact cells. Overexpression of Sp1 or suppressing Sp1 expression by siRNA reduced or increased DsbA-L promoter activity, respectively. The binding activity of Sp1 was gradually decreased during 3T3-L1 cell differentiation and was significantly increased in adipose tissues of obese mice. Our results identify Sp1 as an inhibitor of DsbA-L gene transcription, and the Sp1-mediated inhibition of DsbA-L gene expression may provide a mechanism underlying obesity-induced adiponectin downregulation and insulin resistance.

Enhanced expression of Survivin has distinct roles in adipocyte homeostasis

Although precisely controlled lipolysis is crucial for maintaining physiological levels of circulating free fatty acids in response to energetic stress, the underlying mechanisms by which this process is governed remain poorly understood. Survivin is a gene that has been found to be highly expressed in the most common human tumors, and it is considered to be associated with tumorigenesis. Survivin expression in normal tissue is developmentally downregulated and is undetectable in most terminally differentiated adult tissues. Here, we report that Survivin expression in mature adipocytes from murine white adipose tissue can be highly induced under high-fat diet feeding conditions. During the adipocyte differentiation of 3T3-L1 preadipocytes and mesenchymal C3H10T1/2 cells, Survivin expression is gradually decreased and almost undetectable in fully differentiated adipocytes. However, it can be expressed again upon insulin exposure, through the PI3K/mTOR signaling pathway. Nevertheless, Survivin overexpression is sensitive to nutritional deprivation, and expression markedly decreases in response to starvation with Hanks buffered salt solution challenge. The ectopic expression of Survivin downregulates expression of Adrb3 and then decreases the production of cAMP, while Fsp27 protein levels are upregulated as a result of reduced protein degradation. This in turn inhibits isoproterenol-stimulated adipocyte lipolysis. Survivin also attenuates DNA damage related to PARP activation and inhibits TNFα-induced lipolysis, suggesting that Survivin may facilitate adipocyte maintenance in response to inflammatory stimuli. Further studies will be undertaken to determine whether Survivin is critical for lipid storage to maintain metabolic homeostasis in vivo.

Complementary Role of Fibroblast Growth Factor 21 and Cytokeratin 18 in Monitoring the Different Stages of Nonalcoholic Fatty Liver Disease.

Fibroblast growth factor 21 (FGF21) and cytokeratin 18 (CK18) were previously reported to be elevated in nonalcoholic fatty liver disease (NAFLD). We aim to analyze the differential roles of FGF21, cell apoptosis marker CK18 fragment M30 and total cell death marker CK18 M65ED in monitoring the different stages of NAFLD spectrum in a population-based prospective cohort comprising 808 Chinese subjects. Predictive performances for monitoring the different stages of NAFLD were assessed by logistic regression and receiver-operating characteristic (ROC) curves. We found baseline FGF21 but not CK18 level was an independent predictor for the development of simple steatosis. NAFLD patients who had remission during follow-up had significantly lower baseline M30 levels than those who sustained NAFLD (84.74U/L [53.26–135.79] vs. 118.47U/L [87.16–188.89], P = 0.012). M65ED was independently predictive of progressing to suspected non-alcoholic steatohepatitis (NASH) in NAFLD patients. These results suggest that FGF21 can be used for early identification of hepatic steatosis. On the other hand, CK18 including M30 and M65ED, are predictive of the prognosis of NAFLD patients. FGF21 and CK18 might play differential roles and have complementary value in non-invasive identification and monitoring the outcome of NAFLD patients.