Xavier Escoté

Hog1 mediates cell-cycle arrest in G1 phase by the dual targeting of Sic1

Activation of stress-activated protein kinases (SAPKs) is essential for proper cell adaptation to extracellular stimuli. The exposure of yeast cells to high osmolarity, or mutations that lead to activation of the Hog1 SAPK, result in cell-cycle arrest. The mechanisms by which Hog1 and SAPKs in general regulate cell-cycle progression are not completely understood. Here we show that Hog1 regulates cell cycle progression at the G1 phase by a dual mechanism that involves downregulation of cyclin expression and direct targeting of the CDK-inhibitor protein Sic1. Hog1 interacts physically with Sic1 in vivo and in vitro, and phosphorylates a single residue at the carboxyl terminus of Sic1, which, in combination with the downregulation of cyclin expression, results in Sic1 stabilization and inhibition of cell-cycle progression. Cells lacking Sic1 or containing a Sic1 allele mutated in the Hog1 phosphorylation site are unable to arrest at G1 phase after Hog1 activation, and become sensitive to osmostress. Together, our data indicate that the Sic1 CDK-inhibitor is the molecular target for the SAPK Hog1 that is required to modulate cell-cycle progression in response to stress.

Phosphorylation of Hsl1 by Hog1 leads to a G2 arrest essential for cell survival at high osmolarity

Control of cell cycle progression by stress‐activated protein kinases (SAPKs) is essential for cell adaptation to extracellular stimuli. Exposure of yeast to osmostress leads to activation of the Hog1 SAPK, which controls cell cycle at G1 by the targeting of Sic1. Here, we show that survival to osmostress also requires regulation of G2 progression. Activated Hog1 interacts and directly phosphorylates a residue within the Hsl7‐docking site of the Hsl1 checkpoint kinase, which results in delocalization of Hsl7 from the septin ring and leads to Swe1 accumulation. Upon Hog1 activation, cells containing a nonphosphorylatable Hsl1 by Hog1 are unable to promote Hsl7 delocalization, fail to arrest at G2 and become sensitive to osmostress. Together, we present a novel mechanism that regulates the Hsl1–Hsl7 complex to integrate stress signals to mediate cell cycle arrest and, demonstrate that a single MAPK coordinately modulates different cell cycle checkpoints to improve cell survival upon stress.

Study of the Potential Association of Adipose Tissue GLP-1 Receptor with Obesity and Insulin Resistance

The increase in glucagon-like peptide-1 (GLP-1) activity has emerged as a useful therapeutic tool for the treatment of type 2 diabetes mellitus. The actions of GLP-1 on β-cells and the nervous and digestive systems are well known. The action of this peptide in adipose tissue (AT), however, is still poorly defined. Furthermore, no relationship has been established between GLP-1 receptor (GLP-1R) in AT and obesity and insulin resistance (IR). We provide evidence for the presence of this receptor in AT and show that its mRNA and protein expressions are increased in visceral adipose depots from morbidly obese patients with a high degree of IR. Experiments with the 3T3-L1 cell line showed the lipolytic and lipogenic dose-dependent effect of GLP-1. Moreover, GLP-1 stimulated lipolysis in 3T3-L1 adipocytes in a receptor-dependent manner involving downstream adenylate cyclase/cAMP signaling. Our data also demonstrate that the expression of the GLP-1R in AT correlated positively with the homeostasis model assessment index in obese IR subjects. Furthermore, prospective studies carried out with patients that underwent biliopancreatic diversion surgery showed that subjects with high levels of GLP-1R expression in AT, which indicates a deficit of GLP-1 in this tissue, were those whose insulin sensitivity improved after surgery, suggesting the potential relationship between AT GLP-1R and insulin sensitivity amelioration in obese subjects. Altogether these results indicate that the GLP-1/GLP-1R system in AT represents another potential candidate for improving insulin sensitivity in obese patients.

A role for adipocyte-derived lipopolysaccharide-binding protein in inflammation- and obesity-associated adipose tissue dysfunction

Aims/hypothesisCirculating lipopolysaccharide-binding protein (LBP) is an acute-phase reactant known to be increased in obesity. We hypothesised that LBP is produced by adipose tissue (AT) in association with obesity.MethodsLBP mRNA and LBP protein levels were analysed in AT from three cross-sectional (n = 210, n = 144 and n = 28) and three longitudinal (n = 8, n = 25, n = 20) human cohorts; in AT from genetically manipulated mice; in isolated adipocytes; and in human and murine cell lines. The effects of a high-fat diet and exposure to lipopolysaccharide (LPS) and peroxisome proliferator-activated receptor (PPAR)γ agonist were explored. Functional in vitro and ex vivo experiments were also performed.ResultsLBP synthesis and release was demonstrated to increase with adipocyte differentiation in human and mouse AT, isolated adipocytes and human and mouse cell lines (Simpson–Golabi–Behmel syndrome [SGBS], human multipotent adipose-derived stem [hMAD] and 3T3-L1 cells). AT LBP expression was robustly associated with inflammatory markers and increased with metabolic deterioration and insulin resistance in two independent cross-sectional human cohorts. AT LBP also increased longitudinally with weight gain and excessive fat accretion in both humans and mice, and decreased with weight loss (in two other independent cohorts), in humans with acquired lipodystrophy, and after ex vivo exposure to PPARγ agonist. Inflammatory agents such as LPS and TNF-α led to increased AT LBP expression in vivo in mice and in vitro, while this effect was prevented in Cd14-knockout mice. Functionally, LBP knockdown using short hairpin (sh)RNA or anti-LBP antibody led to increases in markers of adipogenesis and decreased adipocyte inflammation in human adipocytes.Conclusions/interpretationCollectively, these findings suggest that LBP might have an essential role in inflammation- and obesity-associated AT dysfunction.

Enhanced fatty acid oxidation in adipocytes and macrophages reduces lipid-induced triglyceride accumulation and inflammation.

Lipid overload in obesity and type 2 diabetes is associated with adipocyte dysfunction, inflammation, macrophage infiltration, and decreased fatty acid oxidation (FAO). Here, we report that the expression of carnitine palmitoyltransferase 1A (CPT1A), the rate-limiting enzyme in mitochondrial FAO, is higher in human adipose tissue macrophages than in adipocytes and that it is differentially expressed in visceral vs. subcutaneous adipose tissue in both an obese and a type 2 diabetes cohort. These observations led us to further investigate the potential role of CPT1A in adipocytes and macrophages. We expressed CPT1AM, a permanently active mutant form of CPT1A, in 3T3-L1 CARΔ1 adipocytes and RAW 264.7 macrophages through adenoviral infection. Enhanced FAO in palmitate-incubated adipocytes and macrophages reduced triglyceride content and inflammation, improved insulin sensitivity in adipocytes, and reduced endoplasmic reticulum stress and ROS damage in macrophages. We conclude that increasing FAO in adipocytes and macrophages improves palmitate-induced derangements. This indicates that enhancing FAO in metabolically relevant cells such as adipocytes and macrophages may be a promising strategy for the treatment of chronic inflammatory pathologies such as obesity and type 2 diabetes.

Cyclin G2 Regulates Adipogenesis through PPARγ Coactivation

Cell cycle regulators such as cyclins, cyclin-dependent kinases, or retinoblastoma protein play important roles in the differentiation of adipocytes. In the present paper, we investigated the role of cyclin G2 as a positive regulator of adipogenesis. Cyclin G2 is an unconventional cyclin which expression is up-regulated during growth inhibition or apoptosis. Using the 3T3-F442A cell line, we observed an up-regulation of cyclin G2 expression at protein and mRNA levels throughout the process of cell differentiation, with a further induction of adipogenesis when the protein is transiently overexpressed. We show here that the positive regulatory effects of cyclin G2 in adipocyte differentiation are mediated by direct binding of cyclin G2 to peroxisome proliferator-activated receptor γ (PPARγ), the key regulator of adipocyte differentiation. The role of cyclin G2 as a novel PPARγ coactivator was further demonstrated by chromatin immunoprecipitation assays, which showed that the protein is present in the PPARγ-responsive element of the promoter of aP2, which is a PPARγ target gene. Luciferase reporter gene assays, showed that cyclin G2 positively regulates the transcriptional activity of PPARγ. The role of cyclin G2 in adipogenesis is further underscored by its increased expression in mice fed a high-fat diet. Taken together, our results demonstrate a novel role for cyclin G2 in the regulation of adipogenesis.

Zinc-alpha 2-glycoprotein gene expression in adipose tissue is related with insulin resistance and lipolytic genes in morbidly obese patients.

Objective Zinc-α2 glycoprotein (ZAG) stimulates lipid loss by adipocytes and may be involved in the regulation of adipose tissue metabolism. However, to date no studies have been made in the most extreme of obesity. The aims of this study are to analyze ZAG expression levels in adipose tissue from morbidly obese patients, and their relationship with lipogenic and lipolytic genes and with insulin resistance (IR). Methods mRNA expression levels of PPARγ, IRS-1, IRS-2, lipogenic and lipolytic genes and ZAG were quantified in visceral (VAT) and subcutaneous adipose tissue (SAT) of 25 nondiabetic morbidly obese patients, 11 with low IR and 14 with high IR. Plasma ZAG was also analyzed. Results The morbidly obese patients with low IR had a higher VAT ZAG expression as compared with the patients with high IR (p = 0.023). In the patients with low IR, the VAT ZAG expression was greater than that in SAT (p = 0.009). ZAG expression correlated between SAT and VAT (r = 0.709, p<0.001). VAT ZAG expression was mainly predicted by insulin, HOMA-IR, plasma adiponectin and expression of adiponectin and ACSS2. SAT ZAG expression was only predicted by expression of ATGL. Conclusions ZAG could be involved in modulating lipid metabolism in adipose tissue and is associated with insulin resistance. These findings suggest that ZAG may be a useful target in obesity and related disorders, such as diabetes.

FABP4 Dynamics in Obesity: Discrepancies in Adipose Tissue and Liver Expression Regarding Circulating Plasma Levels

Background FABP4 is predominantly expressed in adipose tissue, and its circulating levels are linked with obesity and a poor atherogenic profile. Objective In patients with a wide BMI range, we analyze FABP4 expression in adipose and hepatic tissues in the settings of obesity and insulin resistance. Associations between FABP4 expression in adipose tissue and the FABP4 plasma level as well as the main adipogenic and lipolytic genes expressed in adipose tissue were also analyzed. Methods The expression of several lipogenic, lipolytic, PPAR family and FABP family genes was analyzed by real time PCR. FABP4 protein expression in total adipose tissues and its fractions were determined by western blot. Results In obesity FABP4 expression was down-regulated (at both mRNA and protein levels), with its levels mainly predicted by ATGL and inversely by the HOMA-IR index. The BMI appeared as the only determinant of the FABP4 variation in both adipose tissue depots. FABP4 plasma levels showed a significant progressive increase according to BMI but no association was detected between FABP4 circulating levels and SAT or VAT FABP4 gene expression. The gene expression of FABP1, FABP4 and FABP5 in hepatic tissue was significantly higher in tissue from the obese IR patients compared to the non-IR group. Conclusion The inverse pattern in FABP4 expression between adipose and hepatic tissue observed in morbid obese patients, regarding the IR context, suggests that both tissues may act in a balanced manner. These differences may help us to understand the discrepancies between circulating plasma levels and adipose tissue expression in obesity.

Adipocyte fatty acid-binding protein as a determinant of insulin sensitivity in morbid-obese women.

The aim of the study was to evaluate human plasma circulating levels of adipocyte fatty acid‐binding protein (A‐FABP) and its relationship with proinflammatory adipocytokines and insulin resistance in a severely obese cohort, before and 1 year after a surgical gastric bypass. Plasmatic levels of A‐FABP were measured in 77 morbid‐obese women before and 1 year after bariatric surgery. Anthropometrical parameters and body composition by bioelectrical impedance analysis were determined. Circulating levels of soluble tumor necrosis factor receptor 2 (sTNFR2), Interleukin 18 (IL‐18), adiponectin, and high‐sensitive C‐reactive protein (hsCRP) were also analyzed. Insulin resistance by homeostasis model assessment of insulin resistance (HOMA‐IR) index was calculated. After massive weight loss, A‐FABP plasmatic levels decreased significantly [7.6 (8.9) vs. 4.3 (5.1); P < 0,001] but no association with circulating adipokines or proinflammatory cytokines, both at the beginning and at the end of follow‐up, was observed. A decrease in sTNFR2, IL‐18, hsCRP, and an increase in adiponectin levels (P < 0.001 in all cases) were observed after the gastric bypass. HOMA‐IR index improved 1 year after surgery and after multiple regression analysis remained associated with A‐FABP after controlling for confounding variables (β = 0.322, P = 0.014; R2 for the model 0.281). In morbid‐obese women, plasma A‐FABP concentrations were dramatically reduced after gastric bypass surgery. After weight loss this protein contributed to HOMA‐IR index independently of proinflammatory/antinflammatory cytokine profile. Further studies are warranted to elucidate the role of A‐FABP in the pathogenesis of insulin resistance in morbid obesity.

CDK4 is an essential insulin effector in adipocytes

Insulin resistance is a fundamental pathogenic factor that characterizes various metabolic disorders, including obesity and type 2 diabetes. Adipose tissue contributes to the development of obesity-related insulin resistance through increased release of fatty acids, altered adipokine secretion, and/or macrophage infiltration and cytokine release. Here, we aimed to analyze the participation of the cyclin-dependent kinase 4 (CDK4) in adipose tissue biology. We determined that white adipose tissue (WAT) from CDK4-deficient mice exhibits impaired lipogenesis and increased lipolysis. Conversely, lipolysis was decreased and lipogenesis was increased in mice expressing a mutant hyperactive form of CDK4 (CDK4(R24C)). A global kinome analysis of CDK4-deficient mice following insulin stimulation revealed that insulin signaling is impaired in these animals. We determined that insulin activates the CCND3-CDK4 complex, which in turn phosphorylates insulin receptor substrate 2 (IRS2) at serine 388, thereby creating a positive feedback loop that maintains adipocyte insulin signaling. Furthermore, we found that CCND3 expression and IRS2 serine 388 phosphorylation are increased in human obese subjects. Together, our results demonstrate that CDK4 is a major regulator of insulin signaling in WAT.