Monosaccharide-induced lipogenesis regulates the human hepatic sex hormone-binding globulin gene

Abstract

The liver produces plasma sex hormone-binding globulin (SHBG), which transports sex steroids and regulates their access to tissues. In overweight children and adults, low plasma SHBG levels are a biomarker of the metabolic syndrome and its associated pathologies. Here, we showed in transgenic mice and HepG2 hepatoblastoma cells that monosaccharides (glucose and fructose) reduce human SHBG production by hepatocytes. This occurred via a downregulation of hepatocyte nuclear factor-4alpha (HNF-4alpha) and replacement of HNF-4alpha by the chicken OVA upstream promoter-transcription factor 1 at a cis-element within the human SHBG promoter, coincident with repression of its transcriptional activity. The dose-dependent reduction of HNF-4alpha levels in HepG2 cells after treatment with glucose or fructose occurred in concert with parallel increases in cellular palmitate levels and could be mimicked by treatment with palmitoyl-CoA. Moreover, inhibition of lipogenesis prevented monosaccharide-induced downregulation of HNF-4alpha and reduced SHBG expression in HepG2 cells. Thus, monosaccharide-induced lipogenesis reduced hepatic HNF-4alpha levels, which in turn attenuated SHBG expression. This provides a biological explanation for why SHBG is a sensitive biomarker of the metabolic syndrome and the metabolic disturbances associated with increased fructose consumption.

Figure 1. Hepatic production of SHBG in mice expressing human SHBG transgenes (21) is reduced after feeding diets with high monosaccharide content or increasing blood glucose levels by streptozotocin treatment.

(A and B) Mice were fed high-sucrose or isocaloric basal diets for 7 days (3 per group), and the diets were then reversed in two 7-day cycles. Serum SHBG levels are expressed as mean ± SEM relative to pretreatment levels to compensate for between-animal variability (A). At day 21, human SHBG mRNA abundance was determined in relation to 18S RNA (mean ± SEM) in liver and kidney; **P < 0.01 compared with basal diet values (B). (C) Serum SHBG levels in mice expressing a human SHBG transgene lacking a USF-binding site in the promoter (24) were reduced by feeding a high-sucrose diet. Animals (3 per group) were fed a basal diet (squares) or a high-sucrose diet (diamonds) for 7 days, and diets were then reversed for 7 days. Serum SHBG measurements (mean ± SEM) are expressed relative to pretreatment levels. (D) Serum SHBG levels (mean ± SEM) were reduced relative to pretreatment levels in human SHBG transgenic mice (3–4 per group) fed equicaloric diets containing high glucose, sucrose, or fructose, when compared with a basal diet. (E) Human SHBG transgenic mice treated with streptozotocin were maintained on a basal diet for 11 days, followed by a high-sucrose diet (phase I) and the basal diet (phase II). Serum SHBG levels (mean ± SEM) are expressed relative to pretreatment values.

Figure 2. Insulin does not influence SHBG production by HepG2 cells.

(A–D) HepG2 cells were cultured in serum-free medium, inactivated FBS, or untreated FBS for 5 days and treated daily with insulin at different concentrations. Accumulation of human SHBG in the medium was measured at timed intervals using an immunofluorometric assay (46) over a 5-day treatment period. Data points are shown as mean ± SD of triplicates; *P < 0.05, **P < 0.01 compared with no insulin treatment (A–C). On day 5 cells were taken for human SHBG mRNA and cyclophilin A mRNA measurements by RT-PCR analyses (D). (E) The effect of insulin (12 μg/ml) on human SHBG promoter activity was analyzed in HepG2 cells in the context of a luciferase reporter gene assay. Data points are mean ± SD of triplicate measurements.

Figure 3. Monosaccharides decrease human SHBG production by HepG2 cells in concert with reduced cellular HNF-4α levels.

(A) Human SHBG accumulation in the medium of HepG2 cells treated daily with 10 mM of glucose or fructose was substantially reduced. Data points are shown as mean ± SD of triplicates; **P < 0.01 compared with no glucose/fructose supplementation. (B) Human SHBG and HNF-4α mRNA levels determined by RT-PCR in HepG2 cells cultured for 5 days as in A. Cyclophilin A (CypA) mRNA was amplified as in internal control. (C) HNF-4α and cyclophilin A levels measured by Western blotting in HepG2 cells cultured for 5 days as in B. (D) Human SHBG promoter activity was measured in the context of a luciferase reporter gene assay in HepG2 cells cultured for 4 days in the presence or absence of 10 mM glucose or fructose. Data points are shown as mean ± SD of triplicates; **P < 0.01 compared with no glucose/fructose supplementation.

Figure 4. Insulin does not alter human SHBG production or HNF-4α protein levels in HepG2 cells cultured in the presence of high glucose.

(A) Human SHBG accumulation in the medium is decreased in HepG2 cells treated daily with 1 mM or 10 mM glucose in the absence or presence of 6 μg insulin/ml for 5 days. Data points are shown as mean ± SD of triplicates; **P < 0.01 compared with no glucose supplementation. (B) Western blots of HNF-4α and cyclophilin A in HepG2 cells treated as in A for 5 days. (C) Western blots of phospho-S6 and cyclophilin A in HepG2 cells treated as in A.

Figure 5. Competition between HNF-4α and COUP-TF1 at a cis-element within the human SHBG promoter regulates its activity.

(A) HNF-4α levels were reduced in HepG2 cells after transient transfection of an HNF-4α siRNA versus a control siRNA oligonucleotide (left), and siRNA-mediated downregulation of HNF-4α reduced human SHBG promoter activity in a luciferase reporter gene assay (right). Data points are shown as mean ± SD of triplicates; **P < 0.01 compared with cells treated with an siRNA control. (B) ChIP assays of HNF-4α and COUP-TF1 binding to the human SHBG promoter (top). As a control for the ChIP, anti–RNA polymerase (RNApol) antibodies were used with human-specific oligonucleotide primers to PCR amplify the GAPDH promoter (bottom). A nonspecific mouse IgG was used in ChIP reactions to control for nonspecific immunoprecipitation. Positive PCR controls of sheared genomic DNA templates indicated the integrity of the input DNA used in the ChIP reactions. (C) Reduction of COUP-TF1 mRNA in HepG2 cells after siCOUP-TF1 treatment (left) did not influence the activity of the human SHBG promoter under basal conditions (right). Data points are shown as mean ± SD of triplicates. (D) Reduction of human SHBG promoter activity after treatment with siHNF-4α was mitigated by cotreatment with siCOUP-TF1. Data points are shown as mean ± SD of triplicates; *P < 0.05, **P < 0.01 compared with cells treated with an siRNA control.

Figure 6. Increased palmitate levels in HepG2 cells as a result of monosaccharide or palmitoyl-CoA supplementation reduce HNF-4α levels and SHBG production.

(A) Concentrations of human SHBG in the medium from untreated HepG2 cells and HepG2 cells after 5 days supplementation with 0.1–10 mM glucose or fructose. Data points are shown as mean ± SD of triplicates; *P < 0.05, **P < 0.01 compared with no glucose/fructose supplementation. In addition, the cellular content of palmitate was measured in cells after 5-day treatment with monosaccharides as indicated; **P < 0.01 compared with no glucose/fructose supplementation. (B) Concentrations of SHBG in medium from untreated HepG2 cells and HepG2 cells treated for 5 days with 1 μM or 10 μM palmitoyl-CoA. **P < 0.01 compared with no treatment. (C) HNF-4α and SHBG mRNA levels determined by RT-PCR in untreated HepG2 cells and HepG2 cells treated for 5 days with 1 μM or 10 μM palmitoyl-CoA. Cyclophilin A mRNA was amplified as an internal control. (D) Western blots of HNF-4α and actin in extracts of untreated HepG2 cells and HepG2 cells treated for 5 days with 1 μM or 10 μM palmitoyl-CoA. (E) The effects of 1–10 μM palmitoyl-CoA on human SHBG promoter activity was analyzed in HepG2 cells in the context of a luciferase reporter gene. Data points are mean ± SD of triplicate measurements; **P < 0.01 compared with no treatment.

Figure 7. Monosaccharide-induced reductions of SHBG production by HepG2 cells are blocked by inhibiting fatty acid synthase.

(A) Reduction of human SHBG accumulation in HepG2 medium treated daily with 10 mM glucose or fructose was blocked by cotreatment of the cells with cerulenin (10 μg/ml) over 5 days. Data points are shown as mean ± SD of triplicates; **P < 0.01 compared with no glucose/fructose supplementation. (B) Reduction of HNF-4α levels in HepG2 grown for 5 days in the presence of 10 mM glucose or fructose was also blocked by cerulenin cotreatment.