Dysregulation of sterol regulatory element binding protein-1c in livers of morbidly obese women is associated with altered suppressor of cytokine signaling-3 and signal transducer and activator of transcription-1 signaling

Abstract

We compared hepatic expression of genes that regulate lipid biosynthesis and metabolic signaling in liver biopsy specimens from women who were undergoing gastric bypass surgery (GBP) for morbid obesity with that in women undergoing ventral hernia repair who had experienced massive weight loss (MWL) after prior GBP. Comprehensive metabolic profiles of morbidly obese (MO) (22 subjects) and MWL (9 subjects) were also compared. Analyses of gene expression in liver biopsies from MO and MWL were accomplished by Affymetrix microarray, real-time polymerase chain reaction, and Western blotting techniques. After GBP, MWL subjects had lost on average 102 lb as compared with MO subjects. This was accompanied by effective reversal of the dyslipidemia and insulin resistance that were present in MO. As compared with MWL, livers of MO subjects exhibited increased expression of sterol regulatory element binding protein (SREBP)-1c and its downstream lipogenic targets, fatty acid synthase and acetyl-coenzyme A-carboxylase-1. Livers of MO subjects also exhibited enhanced expression of suppressor of cytokine signaling-3 protein and attenuated Janus kinase signal transducer and activator of transcription (JAK/STAT) signaling. Consistent with these findings, we found that the human SREBP-1c promoter was positively regulated by insulin and negatively regulated by STAT3. These data support the hypothesis that suppressor of cytokine signaling-3-mediated attenuation of the STAT signaling pathway and resulting enhanced expression of SREBP-1c, a key regulator of de novo lipid biosynthesis, are mechanistically related to the development of hepatic insulin resistance and dyslipidemia in MO women.

Figure 1. Weight loss following GBP effectively reverses abnormal glucose and insulin tolerance following oral glucose challenge in morbidly obese women

Data are mean ± S.E.M. of (A) plasma glucose, (B) insulin and (C) C-peptide following oral glucose challenge (OGTT) in morbidly obese (Obese) women undergoing gastric bypass surgery (GBP) and in those undergoing ventral hernia repair after experiencing significant weight loss (Post-obese) following prior GBP. Significance of differences between responses to oral glucose challenge was assessed by 2-way Analysis of Variance (Group versus Time).

Figure 2. Expression of hepatic proteins related to lipid metabolism and metabolic signaling in livers of Morbidly Obese (MO) and Massive Weight Loss (MWL) patients

Panel A. Data are Western blots of total cell lysate from liver of MO (N=11) and MWL (N=4) subjects. Representative Western blots from six subjects are shown. Blots show expression of Sterol-Regulatory-Protein-1c and 2 (SREBP-1c / 2), Fatty acid synthase (FAS), Acetyl-CoA Carboxylase-1 (ACC-1), Insulin-induced gene-2 (INSIG-2), Apolipoprotein A1 (ApoA), Apolipoprotein E (ApoE), Suppressor of Cytokine Signaling -1 and 3 (SOCS-1, 3), STAT-3 and phospho-STAT-3 (pSTAT3). Panels B–D show results of densitometry scanning for Lipogenic Genes (panel B), Apolipoprotein genes (Panel C), and STAT signaling genes (Panel D). *P< 0.05 MO vs. MWL.

Figure 3. The human SREBP-1c promoter is responsive to insulin treatment in vitro

A) A schematic representation of the major cis-acting elements of the human and rat SREBP-1c promoter. The transcription initiation site of the human promoter, determined by TRANSFAC analysis, was designated as +1. Insulin-mediated activation of the human SREBP-1c promoter may involve a number of binding sites known to regulate the rat and mouse promoter. These include: STATRE, signal transducer and activator of transcription response element; LXRE, liver X receptor response element; SRE, sterol regulatory element; SP1, specificity protein-1; NF-Y, nuclear factor-Y. The locations of these cis-acting elements are not depicted to scale on the promoter models. B) Primary rat hepatocytes were either transfected with human (hSREBP-1c) or rat (rSREBP-1c) SREBP-1c promoter-luciferase reporter constructs and incubated with and without insulin (100 nM) or LXR agonist TO901317 (10 µM) for 24 h. Luciferase activity was measured as described in Methods. Both human and rat SREBP-1c promoter constructs responded comparably to both insulin and TO901317. Data are mean ± SEM of luciferase activity relative to untreated (control) hepatocytes (N=12 determinations from 4 separate hepatocyte preparations). P<0.05 vs. control

Figure 4. STAT3 represses the human SREBP-1c promoter

Primary rat hepatocytes were transfected with hSREBP1c-luc DNA along with plasmids expressing wild type or dominant negative STAT3 proteins, or empty vector. Twenty-four hours after transfection cells were harvested and the luciferase activity was measured. Cells were co-transfected with a control Renilla expression plasmid, pRL-TK, and transfection results were normalized to Renilla luciferase activity. The results represent the mean ±S.E.M. for five independent experiments, with fold induction over the level observed with the reporter construct alone.