Transforming growth factor-beta 1: A new factor reducing hepatic SHBG production in liver fibrosis.

Low plasma sex hormone-binding globulin (SHBG) levels are present in fatty liver disease, which represents a spectrum of diseases ranging from hepatocellular steatosis through steatohepatitis to fibrosis and irreversible cirrhosis. We have previously determined that fat accumulation reduces SHBG production in different nonalcoholic fatty liver disease mouse models. In the present work, we are interested in elucidating the molecular mechanisms reducing SHBG plasma levels in liver fibrosis. For this purpose, in vivo studies were performed using the human SHBG transgenic mice developing liver fibrosis induced by carbon tetrachloride (CCl4 ). Our results clearly showed that CCl4 induced liver fibrosis and reduced SHBG production by reducing hepatocyte nuclear factor 4 alpha (HNF-4α). The SHBG reduction could be influenced by the increase in transforming growth factor-beta 1 (TGF-ß1), which was increased in mice developing liver fibrosis. Therefore, we decided to evaluate the role of TGF-ß1 in regulating hepatic SHBG production. Results obtained in both HepG2 cells and human SHBG transgenic mice showed that TGF-ß1 reduced significantly SHBG messenger RNA and protein levels. Mechanistically TGF-ß1 downregulated P1-HNF-4α isoforms and increased P2-HNF-4α isoforms via Smad3 and Stat3 pathways through TGF-ß1 receptor I, resulting in transcriptional repression of the SHBG gene. Taken together, we found for the first time that TGF-ß1 is a new factor regulating hepatic SHBG production in liver fibrosis. Further research is needed to determine the role of this reduction in hepatic SHBG production in the progression of nonalcoholic steatohepatitis.

Relationship between adiponectin, TNFα, and SHBG in prepubertal children with obesity.

BACKGROUND: Sex hormone-binding globulin (SHBG) levels are low in adult subjects with obesity when compared to normal-weight individuals. Obesity is associated with higher tumor necrosis factor alpha (TNFα) plasma levels and lower adiponectin levels. Moreover, we have recently elucidated the molecular mechanisms by which TNFα and adiponectin regulate hepatic SHBG production. AIM: The main objective of this study was to assess if the adult associations between TNFα, adiponectin, and SHBG are present in prepubertal children. METHODS: We determined several morphometric and biochemical parameters in normal-weight (n=15) and obese prepubertal (n=51) children, as well as quantified plasma SHBG, TNFα receptor 1 (TNFα-R1), and adiponectin levels. RESULTS: Our results showed that prepubertal children with obesity had decreased plasma SHBG levels compared to normal-weight controls (67 nmol/L vs 172 nmol/L). Importantly, SHBG plasma levels correlated significantly (P < 0.05) with TNFα (negatively, ßstd= - 0.31) and adiponectin (positively, ßstd= 0.58) suggesting an important role of these two cytokines in determining plasma SHBG levels in prepubertal children. CONCLUSIONS: Our results suggest that plasma adiponectin levels may play a more important role than TNFα in influencing plasma SHBG levels in our prepubertal population with obesity.

Sex hormone-binding globulin overexpression protects against high-fat diet-induced obesity in transgenic male mice.

Obese subjects of all ages and sex have reduced plasma SHBG levels. Whether these low plasma SHBG levels play a role in obesity development is unknown. In the present work we wanted to explore if SHBG overexpression could prevent obesity development induced by high fat diet (HFD). To do so, we fed humanized SHBG transgenic male mice and their wild-type littermates with control diet (CD) or HFD over the course of 8 weeks. The results showed that SHBG overexpression protected against body weight gain and fat accumulation induced by HFD. In addition, SHBG overexpression also abrogated the increase in insulin, leptin and resistin levels, as well as the reduction in adiponectin, induced by HFD. Mechanistically, the SHBG protection against HFD-induced obesity was achieved by stimulating lipolysis in white adipose tissue. Furthermore, we have demonstrated the SHBG cell-autonomous effect using human primary visceral adipocytes. Taking together, our results demonstrate that SHBG overexpression protects against diet-induced obesity and improves the metabolic profile of male mice fed a HFD diet.

Caffeine Upregulates Hepatic Sex Hormone-Binding Globulin Production by Increasing Adiponectin Through AKT/FOXO1 Pathway in White Adipose Tissue.

SCOPE: Epidemiological studies have shown that caffeine increases serum sex hormone-binding globulin (SHBG) levels. The relationship between caffeine and SHBG production has never been studied before at molecular level. The aim of this study is to examine whether caffeine regulates SHBG production and to determine the associated molecular mechanisms. METHODS AND RESULTS: Two different studies are performed; in vitro studies using human HepG2 cells treated with caffeine (100 and 500 µm) and in vivo studies using a humanized SHBG transgenic mice drinking caffeine in the water (0.1 mg mL-1 ) for 12 days. The results show that caffeine does not change SHBG production in HepG2 cells. By contrast, caffeine treatment increases significantly hepatic SHBG production in human SHBG transgenic mice when compared with control mice. Caffeine increases adiponectin levels in epididymal adipose tissue of human SHBG transgenic mice. Moreover, caffeine increases adiponectin production by reducing protein kinase B (AKT) phosphorylation which increases forkhead box protein O1 (FOXO1) protein levels in 3T3-L1 mature adipocytes and human SHBG transgenic mice. Finally, caffeine-induced increase in adiponectin in turn upregulates hepatic hepatocyte nuclear receptor 4-alpha (HNF-4α) levels in human SHBG transgenic mice. CONCLUSIONS: The results show that caffeine upregulates hepatic SHBG expression by increasing adiponectin production through AKT/FOXO1 pathway in the adipose tissue.

Sex Hormone-Binding Globulin Expression Correlates With Acetyl-Coenzyme A Carboxylase and Triglyceride Content in Human Liver.

CONTEXT: There is emerging evidence that SHBG is substantially reduced in chronic metabolic diseases, including obesity and nonalcoholic fatty liver disease (NAFLD). We have recently reported, through use of in vitro (HepG2 cells) and in vivo (SHBG-C57BL/ksJ-db/db mice) models, that SHBG could play a role in arresting the progression of NAFLD by downregulating lipogenesis. OBJECTIVE: The main aim of this study was to investigate the mechanisms by which SHBG prevents hepatic lipogenesis by examining the relationship between SHBG and a key lipogenic enzyme, such as acetyl-coenzyme A carboxylase (ACC) in the liver of obese persons. PARTICIPANTS AND METHODS: SHBG and ACC mRNA levels, as well as triglyceride content, were analyzed in 41 liver samples from nondiabetic obese patients with NAFLD who had undergone bariatric surgery. We also studied the effect of SHBG overexpression in HepG2 cells cultured under high-glucose conditions. RESULTS: SHBG mRNA and protein levels were lower in patients with metabolic syndrome than in those without metabolic syndrome; however, these differences were significant only for mRNA level. SHBG mRNA levels correlated positively with SHBG protein levels and hepatic triglyceride content. In addition, SHBG mRNA and protein levels correlated negatively with ACC mRNA levels and triglyceride content. Furthermore, SHBG overexpression abrogated the increase in ACC expression induced by high-glucose treatment in HepG2 cells. CONCLUSIONS: Our findings suggest that SHBG plays a role in regulating hepatic lipogenesis by reducing ACC levels. These results suggest a strategy for the treatment of NAFLD.

Resveratrol Increases Hepatic SHBG Expression through Human Constitutive Androstane Receptor: a new Contribution to the French Paradox.

Sex hormone-binding globulin (SHBG) carries sex steroids in blood regulating their bioavailability. Red wine consumption increases plasma SHBG levels, and we have discovered that resveratrol, a polyphenol enriched in red wine, acts specifically through the human constitutive androstane receptor (CAR), a drug/xenobiotic detoxification gene regulator, to increase hepatic SHBG production. Chromatin immunoprecipitation and luciferase reporter gene assays show that human CAR binds to a typical direct repeat 1 nuclear hormone receptor-binding element in the human SHBG proximal promoter. Resveratrol also increased hepatic SHBG production in humanized SHBG/CAR transgenic mice. Moreover, SHBG expression correlated significantly with CAR mRNA levels in human liver biopsies. We conclude that the beneficial effects of red wine on the metabolic syndrome and it associated co-morbidities, including cardiovascular disease and type 2 diabetes, may be mediated in part by resveratrol acting via CAR to increase plasma SHBG levels.

Sex Hormone-Binding Globulin Reduction in Metabolic Disorders May Play a Role in NAFLD Development.

Low plasma sex hormone-binding globulin (SHBG) levels are a hallmark in chronic metabolic diseases, including nonalcoholic fatty liver disease (NAFLD), which represents a spectrum of disease ranging from hepatocellular steatosis through steatohepatitis to fibrosis and irreversible cirrhosis. The functional link between altered SHBG production and NAFLD development and progression remains unclear. We investigated the effects of overexpressing human SHBG in 2 mouse models of NAFLD: a genetically induced double transgenic mouse and a diet-induced model. Remarkably, SHBG overexpression in both NAFLD models significantly reduced liver fat accumulation by reducing key lipogenic enzymes. These findings were corroborated by modulating SHBG expression and by adding exogenous SHBG in HepG2 cells, suggesting the cell autonomous nature of the mechanism. Mechanistically, exogenous SHBG treatment downregulated key lipogenic enzymes by reducing PPARγ messenger RNA and protein levels through activation of extracellular signal-regulated kinase-1/2 mitogen-activated protein kinase pathway. Taking together, we found that SHBG modulates hepatic lipogenesis. This is of importance because reduction of SHBG plasma levels in obese and type 2 diabetic subjects could be directly associated with NAFLD development through an increase in hepatic lipogenesis. Our results point to SHBG as a therapeutic target for preventing or arresting NAFLD development.

SHBG-C57BL/ksJ-db/db: A New Mouse Model to Study SHBG Expression and Regulation During Obesity Development.

Low plasma sex hormone-binding globulin (SHBG) levels in overweight individuals are a biomarker for the metabolic syndrome and are predictive of type 2 diabetes and cardiovascular disease risk. There are no in vivo models to study SHBG expression and regulation during obesity development. The main reason for this is that the obesity-prone rodent models cannot be used to study this issue, because rodents, unlike humans, do not express the SHBG gene in their livers. We have developed a unique mouse model that expresses the human SHBG, and it develops obesity, by crossing the human SHBG transgenic mice with the C57BL/ksJ-db/db mice. The results obtained with the SHBG-C57BL/ksJ-db/db mouse model have allowed us to determine that the SHBG overexpression in the C57BL/ksJ-db/db reduced the body weight gain but did not change the metabolic profile of these mice. Moreover, we elucidated the molecular mechanisms and transcription factors causing the SHBG down-regulation during obesity development, which involved changes in liver hepatocyte nuclear factor 4α and peroxisome proliferator-activated receptor-γ mRNA and protein levels. Furthermore, these results were confirmed using human liver biopsies. Importantly, we also showed that this model resembles what occurs in human obese subjects, because plasma SHBG and total testosterone levels where reduced in obese mice when compared with lean mice. Future research using this unique mouse model will determine the role of SHBG in the development and progression of obesity, type 2 diabetes, or fatty liver disease.

Adiponectin upregulates SHBG production: molecular mechanisms and potential implications.

Epidemiological studies have shown that plasma SHBG levels correlate with plasma adiponectin levels, both in men and women. There are no reports describing any molecular mechanism by which adiponectin regulates hepatic SHBG production. The aim of the present study is to explore whether adiponectin regulates SHBG production by increasing HNF-4α levels through reducing hepatic lipid content. For this purpose, in vitro studies using human HepG2 cells, as well as human liver biopsies, were performed. Our results show that adiponectin treatment increased SHBG production via AMPK activation in HepG2 cells. Adiponectin treatment decreased the mRNA and protein levels of enzymes related to hepatic lipogenesis (ACC) and increased those related to fatty acid oxidation (ACOX and CPTI). These adiponectin-induced changes in hepatic enzymes resulted in a reduction of total TG and FFA and an increase of HNF-4α. When HNF-4α expression was silenced by using siRNA, adiponectin-induced SHBG overexpression was blocked. Furthermore, adiponectin-induced upregulation of SHBG production via HNF-4α overexpression was abrogated by the inhibition of fatty acid oxidation or by the induction of lipogenesis with a 30mM glucose treatment in HepG2 cells. Finally, adiponectin levels correlated positively and significantly with both HNF-4α and SHBG mRNA levels in human liver biopsies. Our results suggest for the first time that adiponectin increases SHBG production by activating AMPK, which reduces hepatic lipid content and increases HNF-4α levels.

Thyroid hormone upregulates zinc-α2-glycoprotein production in the liver but not in adipose tissue.

Overproduction of zinc-α2-glycoprotein by adipose tissue is crucial in accounting for the lipolysis occurring in cancer cachexia of certain malignant tumors. The main aim of this study was to explore whether thyroid hormone could enhance zinc-α2-glycoprotein production in adipose tissue. In addition, the regulation of zinc-α2-glycoprotein by thyroid hormone in the liver was investigated. We performed in vitro (HepG2 cells and primary human adipocytes) and in vivo (C57BL6/mice) experiments addressed to examine the effect of thyroid hormone on zinc-α2-glycoprotein production (mRNA and protein levels) in liver and visceral adipose tissue. We also measured the zinc-α2-glycoprotein serum levels in a cohort of patients before and after controlling their hyperthyroidism. Our results showed that thyroid hormone up-regulates zinc-α2-glycoprotein production in HepG2 cells in a dose-dependent manner. In addition, the zinc-α2-glycoprotein proximal promoter contains functional thyroid hormone receptor binding sites that respond to thyroid hormone treatment in luciferase reporter gene assays in HepG2 cells. Furthermore, zinc-α2-glycoprotein induced lipolysis in HepG2 in a dose-dependent manner. Our in vivo experiments in mice confirmed the up-regulation of zinc-α2-glycoprotein induced by thyroid hormone in the liver, thus leading to a significant increase in zinc-α2-glycoprotein circulating levels. However, thyroid hormone did not regulate zinc-α2-glycoprotein production in either human or mouse adipocytes. Finally, in patients with hyperthyroidism a significant reduction of zinc-α2-glycoprotein serum levels was detected after treatment but was unrelated to body weight changes. We conclude that thyroid hormone up-regulates the production of zinc-α2-glycoprotein in the liver but not in the adipose tissue. The neutral effect of thyroid hormones on zinc-α2-glycoprotein expression in adipose tissue could be the reason why zinc-α2-glycoprotein is not related to weight loss in hyperthyroidism.

Oleic acid increases hepatic sex hormone binding globulin production in men.

SCOPE: Low circulating sex hormone-binding globulin (SHBG) is an independent risk factor for cardiovascular disease. Mediterranean diet has been associated with a decreased risk of cardiovascular disease. We aimed to test the hypothesis that the increase of circulating MUFA associated with olive oil consumption (primary fat source in Mediterranean diet) increases SHBG serum levels. METHODS AND RESULTS: A total of 315 men were included. In these patients, nutrition data and plasma samples for SHBG assessment were obtained. In vitro studies to examine the effects of oleic and linoleic acid on SHBG production using HepG2 cells were performed. We provided evidence that SHBG serum levels were significantly higher in subjects using olive oil for cooking in comparison with subjects using sunflower oil. The SHBG levels correlated positively with MUFA (p < 0.001) and negatively with saturated fatty acids (p = 0.003). In the multiple regression analysis, MUFA were independently associated with SHBG levels and accounted for the 20.4% of SHBG variance. In vitro studies revealed that oleoyl-CoA increases SHBG production by downregulating PPAR-γ levels in HepG2 cells. CONCLUSION: Olive oil consumption is associated with elevated SHBG serum levels. PPAR-γ downregulation induced by oleoyl-CoA is an important underlying mechanism of such regulation.

Diabetes protects from prostate cancer by downregulating androgen receptor: new insights from LNCaP cells and PAC120 mouse model.

Type 2 diabetes has been associated with decreased risk of prostate cancer in observational studies, and this inverse association has been recently confirmed in several large cohort studies. However the mechanisms involved in this protective effect remain to be elucidated. The aim of the present study was to explore whether different features of type 2 diabetes (hyperinsulinemia, hyperglycemia and tumor necrosis factor alpha [TNF-α]) protect against the development of prostate cancer. For this purpose LNCaP cells were used for in vitro experiments and nude mice in which PAC120 (hormone-dependent human prostate cancer) xenografts had been implanted were used for in vivo examinations. We provide evidence that increasing glucose concentrations downregulate androgen receptor (AR) mRNA and protein levels through NF-κB activation in LNCaP cells. Moreover, there was a synergic effect of glucose and TNFα in downregulating the AR in LNCaP cells. By contrast, insulin had no effect on AR regulation. In vivo experiments showed that streptozotocin-induced diabetes (STZ-DM) produces tumor growth retardation and a significant reduction in AR expression in PAC120 prostate cancer mice. In conclusion, our results suggest that hyperglycemia and TNF-α play an important role in protecting against prostate cancer by reducing androgen receptor levels via NF-κB.

IL1ß down-regulation of sex hormone-binding globulin production by decreasing HNF-4α via MEK-1/2 and JNK MAPK pathways.

Patients suffering from low-grade chronic inflammatory diseases, such as rheumatoid arthritis, osteoarthritis, diabetes, and obesity, have low plasma sex hormone-binding globulin (SHBG) levels. These diseases are characterized among other features by high plasma IL1ß levels. The aim of the present study is to explore whether IL1ß could regulate hepatic SHBG production to account for low SHBG levels in these diseases. We provide evidence that daily IL1ß treatment reduces SHBG production in HepG2 cells by the down-regulation of HNF-4A via the MAPK kinase (MEK)-1/2 and c-Jun N-terminal kinase (JNK) MAPK signaling pathways through the activation c-Jun transcription factors. The human SHBG promoter sequence contains two putative activator protein 1 (AP1) binding sites recognized by c-Jun transcription factors, but they are not necessary for the IL1ß-induced down-regulation of SHBG promoter activity in luciferase reporter gene assays. Daily treatment with IL1ß reduces hepatic nuclear factor (HNF)-4α mRNA and protein levels via the MEK-1/2 and JNK MAPK signaling pathways. Moreover, IL1ß rapidly decreased HNF-4α mRNA and protein levels while increased phospho-c-Jun protein levels after the treatment. Finally, daily IL1ß treatment of human SHBG transgenic mice reduced plasma SHBG and SHBG mRNA levels. Moreover, IL1ß treatment also reduced HNF-4α mRNA and protein levels while increased hepatic phospho-c-Jun protein levels. Our results show that IL1ß reduces hepatic SHBG production by decreasing HNF-4α via MEK-1/2 and JNK MAPK pathways. In addition, our findings suggest that IL1ß could be involved the low plasma SHBG levels reported in chronic low-grade inflammatory diseases.

Molecular Mechanism of TNFα-Induced Down-Regulation of SHBG Expression.

The reason why obesity (a chronic low-grade inflammatory disease) is associated with low levels of sex hormone-binding globulin (SHBG) remains to be elucidated. The present study provides evidence that TNFα (a proinflammatory cytokine increased in obesity) reduces SHBG production by human HepG2 hepatoblastoma cells. Although the human SHBG promoter contains one nuclear factor-κB (NF-κB) binding site, the human SHBG promoter activity did not change after TNFα treatment or transfection with either small interfering RNA against p65 or a p65 expression vector in luciferase reporter gene assays. The effect of TNFα on human SHBG expression was indirect, and it was mediated by NF-κB through the down-regulation of hepatocyte nuclear factor (HNF)-4A: a key SHBG transcriptional regulator. Furthermore, the HNF-4A proximal promoter contains three putative NF-κB binding sites. The HNF-4A promoter activity was decreased by the treatment with TNFα or the transfection of a p65 expression vector, and it was increased by the treatment with small interfering RNA against NF-κB in luciferase reporter gene assays. Finally, the TNFα treatment promotes the NF-κB binding to the HNF-4A promoter in chromatin immunoprecipitation assays. We conclude that sustained exposition to elevated levels of TNFα decreases SHBG production by reducing hepatic HNF-4α levels via NF-κB activation in HepG2 cells.

Potential role of tumor necrosis factor-α in downregulating sex hormone-binding globulin.

Low plasma sex hormone-binding globulin (SHBG) levels are associated with obesity and predict the development of type 2 diabetes. The reason why obese individuals have low circulating SHBG has been attributed to hyperinsulinemia, but no mechanistic evidence has been described. The aim of the current study is to explore whether tumor necrosis factor-α (TNF-α) rather than insulin could be the main factor accounting for low SHBG levels in obesity. We performed in vitro and in vivo studies using human HepG2 cells and human SHBG transgenic mice. In addition, a cross-sectional study to explore the relationship between TNF-α and SHBG in obese patients and an interventional study to examine the effect of insulin administration on circulating SHBG in type 2 diabetic patients were performed. We provide evidence that TNF-α, but not insulin, is the main factor by which SHBG is reduced in obesity. Plasma SHBG was significantly increased rather than decreased after insulin treatment in diabetic patients. TNF-α-induced reduction of SHBG expression was mediated by downregulating HNF4A. Finally, a negative and independent correlation was found between plasma TNF-α receptor 1 and SHBG levels in obese patients. Our results suggest that TNF-α plays an important role downregulating SHBG in chronic low-grade inflammatory diseases such as obesity and type 2 diabetes.

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 ß-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.

APOH is increased in the plasma and liver of type 2 diabetic patients with metabolic syndrome.

OBJECTIVE: To assess the association of APOH with metabolic and cardiovascular risk markers in type 2 diabetic patients. METHODS: In a cohort of 169 type 2 diabetic subjects, plasma levels of APOH, antibodies anti-APOH, lipoprotein subfractions, oxidation, inflammatory and insulin resistance markers and the Trp316Ser and Val247Leu variations in the APOH gene were analyzed. Apo H mRNA levels and protein content were measured in hepatic and adipose tissue (subcutaneous and visceral) samples obtained during bariatric surgery from three diabetics who fulfilled metabolic syndrome (MS) criteria and three non-diabetic, non-MS. RESULTS: APOH plasma levels were significantly associated with triglycerides (p<0.001), all the components of triglyceride-rich lipoproteins (p<0.001) and RBP4 (p<0.001) levels. APOH was higher in type 2 diabetic patients with MS (p=0.003) and with clinical evidence of macrovascular disease (p=0.012). The Trp316Ser and Val247Leu APOH gene variants did not modulate APOH plasma values. Neither Apo H mRNA nor protein was detected in the adipose tissue. Liver from patients with diabetes and MS showed a significant increase of both Apo H mRNA and protein respect to the non-diabetic, non-MS patients. CONCLUSION: APOH plasma concentrations are strongly associated to MS alterations and vascular disease in type 2 diabetic patients and could be considered as a clinical marker of cardiovascular risk. The enhanced APOH levels in these patients are due to an increased liver synthesis. If APOH plays a major causal role in macrovascular lesions associated to diabetes and MS need further studies.

Lower zinc-alpha2-glycoprotein production by adipose tissue and liver in obese patients unrelated to insulin resistance.

CONTEXT: Zinc-alpha2 glycoprotein (ZAG) has been proposed as a new candidate in the pathogenesis of obesity, but most of the information stems from studies performed in rodents and in vitro assays. OBJECTIVE: The main aim of the study was to compare serum levels of ZAG and its expression (mRNA levels and protein) in adipose tissue and the liver between obese and nonobese subjects. The relationship between ZAG and insulin resistance was also explored. DESIGN: This was a case-control study. SETTING: The study was conducted at a university referral center. PATIENTS AND METHODS: Samples of serum, sc adipose tissue (SAT), visceral adipose tissue (VAT), and liver were obtained from 20 obese subjects during bariatric surgery. Samples from 10 nonobese patients matched by age and gender were used as a control group. Serum ZAG levels were determined by ELISA. ZAG mRNA levels were measured by real-time PCR and protein content by Western blot. The effect of insulin on liver production of ZAG was assessed using HepG2 cultures. RESULTS: Serum concentration of ZAG (micrograms per milliliter) was significantly lower in obese subjects (40.87 +/- 10.45 vs. 63.26 +/- 16.40; P = 0.002). ZAG expression was significantly lower in the adipose tissue (SAT and VAT) and liver of obese patients than in control subjects. Significant negative correlations between body mass index and circulating ZAG (r = -0.65, P < 0.001) as well as between body mass index and mRNA ZAG levels in SAT (r = -0.68, P < 0.001) and VAT were detected (r = -0.64, P < 0.001). No relationship was found between ZAG and homeostasis model assessment for insulin resistance and insulin had no effect on ZAG production in vitro. CONCLUSION: A down-regulation of ZAG in SAT, VAT, and liver exists in obese patients but seems unrelated to insulin resistance.

Thyroid hormones act indirectly to increase sex hormone-binding globulin production by liver via hepatocyte nuclear factor-4alpha.

Thyroid hormones increase hepatic sex hormone-binding globulin (SHBG) production, which is also regulated by hepatocyte nuclear factor-4alpha (HNF-4alpha) in response to changes in the metabolic state of the liver. Since the human SHBG promoter lacks a typical thyroid hormone response element, and because thyroid hormones influence metabolic state, we set out to determine whether thyroid hormones mediate SHBG expression indirectly via changes in HNF-4alpha levels in HepG2 human hepatoblastoma cells, and in the livers of transgenic mice that express a 4.3 kb human SHBG transgene under the control of its own 0.8 kb promoter sequence. Thyroid hormones (triiodothyronine (T(3)) and thyroxine (T(4))) increase SHBG accumulation in HepG2 cell culture medium over 5 days, and increase cellular SHBG mRNA levels. In addition, T(4) treatment of HepG2 cells for 5 days increased HNF-4alpha mRNA and HNF-4alpha levels in concert with decreased cellular palmitate levels. Plasma SHBG levels were also increased in mice expressing a human SHBG transgene after 5 days treatment with T(3) along with increased hepatic HNF-4alpha levels. In HepG2 cells, the human SHBG promoter failed to respond acutely (within 24 h) to T(4) treatment, but a 4-day pre-treatment with T(4) resulted in a robust response that was prevented by co-treatment with HNF-4alpha siRNA, or by blocking the beta-oxidation of palmitate through co-treatment with the carnitine palmitoyltransferase I inhibitor, etomoxir. These data lead us to conclude that thyroid hormones increase SHBG production indirectly by increasing HNF-4alpha gene expression, and by reducing cellular palmitate levels that further contribute to increased HNF-4alpha levels in hepatocytes.

Peroxisome-proliferator receptor gamma represses hepatic sex hormone-binding globulin expression.

Plasma SHBG production by the liver is influenced by its metabolic state, and hepatocyte nuclear factor-4alpha regulates SHBG expression in response to changes in lipogenesis. Peroxisome-proliferator receptors (PPARs) also regulate glucose homeostasis and fatty acid metabolism. The human SHBG promoter contains a PPAR-response element (PPAR-RE), and plasma SHBG levels increase in polycystic ovarian syndrome patients treated with the PPARgamma agonist, rosiglitazone. In addition, plasma SHBG levels are associated with a genetic polymorphism in the PPARgamma-2 coding sequence that alters its transcriptional activity. Therefore, we set out to determine whether PPARgamma influences hepatic production of SHBG by using human HepG2 hepatoblastoma cells as an in vitro model. Surprisingly, treatment of HepG2 cells with rosiglitazone reduced SHBG production and SHBG promoter activity (as assessed in a luciferase reporter gene assay) by 20-25%, whereas the PPARgamma antagonist, GW9662, increased both by 2- to 3-fold. The effects of PPARgamma agonists and antagonists on SHBG promoter activity were substantially diminished when the PPAR-RE in the SHBG promoter was mutated. A PPARgamma small interfering RNA also increased SHBG production by HepG2 cells as well as SHBG promoter activity, and the latter was accentuated by cotreatment with GW9662. Importantly, overexpression of a PPARgamma-2 Pro12 variant in HepG2 cells was more effective at reducing SHBG promoter activity, when compared with PPARgamma-2 Ala12, consistent with its superior PPAR-RE binding activity. We conclude that PPARgamma represses human SHBG expression in liver cells, and that differences in PPARgamma levels and activity contribute directly to variations in plasma SHBG levels.