ChREBP regulates fructose-induced glucose production independently of insulin signaling - PubMed (original) (raw)

. 2016 Nov 1;126(11):4372-4386.

doi: 10.1172/JCI81993. Epub 2016 Sep 26.

Sarah A Krawczyk, Ludivine Doridot, Alan J Fowler, Jennifer X Wang, Sunia A Trauger, Hye-Lim Noh, Hee Joon Kang, John K Meissen, Matthew Blatnik, Jason K Kim, Michelle Lai, Mark A Herman

ChREBP regulates fructose-induced glucose production independently of insulin signaling

Mi-Sung Kim et al. J Clin Invest. 2016.

Abstract

Obese, insulin-resistant states are characterized by a paradoxical pathogenic condition in which the liver appears to be selectively insulin resistant. Specifically, insulin fails to suppress glucose production, yet successfully stimulates de novo lipogenesis. The mechanisms underlying this dysregulation remain controversial. Here, we hypothesized that carbohydrate-responsive element-binding protein (ChREBP), a transcriptional activator of glycolytic and lipogenic genes, plays a central role in this paradox. Administration of fructose increased hepatic hexose-phosphate levels, activated ChREBP, and caused glucose intolerance, hyperinsulinemia, hypertriglyceridemia, and hepatic steatosis in mice. Activation of ChREBP was required for the increased expression of glycolytic and lipogenic genes as well as glucose-6-phosphatase (G6pc) that was associated with the effects of fructose administration. We found that fructose-induced G6PC activity is a major determinant of hepatic glucose production and reduces hepatic glucose-6-phosphate levels to complete a homeostatic loop. Moreover, fructose activated ChREBP and induced G6pc in the absence of Foxo1a, indicating that carbohydrate-induced activation of ChREBP and G6PC dominates over the suppressive effects of insulin to enhance glucose production. This ChREBP/G6PC signaling axis is conserved in humans. Together, these findings support a carbohydrate-mediated, ChREBP-driven mechanism that contributes to hepatic insulin resistance.

PubMed Disclaimer

Figures

Figure 1

Figure 1. High-fructose feeding induces metabolic disease.

(A) Body weight and (B) fat pad weights (PG, perigonadal; SC, subcutaneous; BAT, brown adipose tissue) were measured in WT male mice fed chow, high-dextrose, or high-fructose diet for 9 weeks (n = 8 per group). (C) Glycemia and (D) serum insulin were measured in the ad libitum–fed state. (E) Serum triglyceride (TG) levels were measured after an overnight fast followed by 3 hours refeeding with chow, glucose, or fructose diet. (F) Hepatic TG levels and representative H&E-stained liver sections. Images were obtained at ×20 magnification. (G) Glucose tolerance test and (H) glycerol tolerance test with incremental areas under the curve (Δ AUC) for this cohort. P values were obtained by 1-way ANOVA. *P < 0.05 versus chow; **P < 0.05 versus all others. Values are the mean ± SEM.

Figure 2

Figure 2. High-fructose feeding activates hepatic ChREBP and its metabolic gene targets.

WT male mice were ad libitum fed a chow, high-dextrose, or high-fructose diet for 9 weeks (n = 8 per group) and (A–E) hepatic gene expression and (F) hepatic G6PC activity were measured. P values were obtained by 1-way ANOVA. *P < 0.05 versus chow diet; **P < 0.05 versus all others. Values are the mean ± SEM.

Figure 3

Figure 3. Multiple different carbohydrates acutely activate hepatic ChREBP.

(A–C) Five-hour-fasted, 8-week-old C3H/HeJ male mice were gavaged with water, glucose (4 g/kg body weight), or fructose (4 g/kg body weight) and sacrificed 100 minutes later. (A) Hepatic gene expression was measured by qPCR. P values were obtained by 1-way ANOVA. *P < 0.05 compared with water; **P < 0.05 compared with all others (n = 6 per group). (B) Nuclear and cytosolic ChREBP-α and the ratio of nuclear to cytosolic ChREBP-α from water- versus fructose-gavaged mice were measured by Western blot and quantified (n = 4 per group). P values were obtained by Student’s t test. *P < 0.05 compared with water. (C) ChIP was performed from liver tissue with anti-ChREBP antibody or IgG control and qPCR was performed on immunoprecipitated chromatin with primers spanning the E-box in the ChREBP-β promoter, the carbohydrate response element in the G6pc promoter, and a nonspecific genomic region. P values were obtained by 1-way ANOVA. †P < 0.05 compared with water- and glucose-ChREBP groups (n = 4 per group). (D) Five-hour-fasted, 8-week-old C3H/HeJ male mice were gavaged with glucokinase activator (PF-04991532, 100 mg/kg body weight) and then gavaged with water or glucose (4 g/kg body weight) 30 minutes later. Mice were sacrificed 100 minutes later and hepatic gene expression was measured by qPCR. P values were obtained by 2-way ANOVA. *P < 0.05 compared with methylcellulose (MC) within water- or glucose-gavaged groups; #P < 0.05 compared with water within MC- or GKA-gavaged groups (n = 6 per group). (E) Five-hour-fasted, 8-week-old C3H/HeJ male mice were gavaged with water or glycerol (4 g/kg body weight) and sacrificed 100 minutes later. P values were obtained by Student’s t test. *P < 0.05 compared with water (n = 6 per group). Values are the mean ± SEM.

Figure 4

Figure 4. ChREBP is necessary for fructose-induced hepatic gene expression.

(A) Hepatic gene expression, (B) hepatic G6PC activity, and (C) hepatic G6P levels were measured in 5-hour-fasted, 8- to 12-week-old WT and ChKO male mice gavaged with water or fructose (4 g/kg body weight) and sacrificed 100 minutes later (n = 6–9 per group). P values were obtained by 2-way ANOVA. *P < 0.05 compared with water within genotype; #P < 0.05 compared with WT within gavage treatment; †P < 0.05 main effect of genotype. Values are the mean ± SEM.

Figure 5

Figure 5. ChREBP mediates the conversion of fructose to glucose.

We measured accumulation of glucose in the media after treatment with fructose or lactate+pyruvate from mouse primary hepatocytes obtained from (A) WT mice fed chow versus HFrD for 1 week (n = 3 per group) and (B) chow-fed WT versus ChKO mice (n = 4 per group). P values were obtained by 2-way ANOVA. *P < 0.05 compared with lactate+pyruvate within genotype or diet; #P < 0.05 compared with lactate+pyruvate condition within genotype or diet. (C) Serum fructose concentrations were determined by LC-MS at the indicated time points in 5-hour-fasted, 8- to 12-week-old WT and ChKO male mice after gavage with U13C-fructose (4 g/kg body weight). P values were obtained by Student’s t test. *P < 0.05 compared with WT (n = 5 per group). (D) A schematic diagram illustrating our working hypothesis regarding how ChREBP regulates intracellular hexose-phosphate homeostasis. Increased sugar consumption increases hepatic carbohydrate uptake, which activates ChREBP and increases glycolysis, fatty acid synthesis, and glucose production to dispose of hexose-phosphates such as G6P.

Figure 6

Figure 6. HFrD increases HGP in association with increased G6PC activity.

(A) Glycemia (B) HGP, and (C) serum insulin levels were measured after a 4-hour fast in 4-month-old live, conscious mice fed chow vs. HFrD for 2 weeks (n = 8 or 9 per group). (D) Hepatic G6PC activity, (E) hepatic glycogen, and (F) hepatic G6P levels were measured in liver obtained from mice euthanized immediately after blood was obtained for the turnover measurement. P values were obtained by Student’s t test. *P < 0.05 compared with chow. (G) Western blot of hepatic total AKT and phospho-AKT (Ser473), with quantification of the AKT/phospo-AKT ratio (n = 8 per group). (H) Western blot of hepatic cytosolic FOXO1A with quantification of FOXO1A normalized for MEK1 (n = 4 per group). Bars represent the mean ± SEM.

Figure 7

Figure 7. HGP is unchanged in chow-fed ChKO.

(A) Glycemia, (B) serum insulin levels, and (C) HGP were measured after a 4-hour fast in 5-month-old live, conscious, chow-fed WT and ChKO mice (n = 5 per group). (D) Hepatic G6PC activity, (E) hepatic G6P levels, and (F) hepatic glycogen levels were measured in liver obtained from mice euthanized immediately after blood was obtained for the turnover measurement. P values were obtained by Student’s t test. *P < 0.05 compared with chow. Bars represent the mean ± SEM.

Figure 8

Figure 8. G6PC activity predicts hepatic G6P levels and HGP 4 hours after food removal.

(A) Hepatic G6PC activity versus hepatic G6P levels (n = 44), (B) hepatic G6PC activity versus HGP (n = 44), (C) serum insulin levels versus HGP (n = 44), and (D) hepatic G6P levels versus hepatic glycogen (n = 43) in 3 mouse cohorts. Each data point represents an individual mouse. In A and B, black dashed lines represent exponential fits including all mice. Red dashed lines represent exponential fits excluding ChKO mice. In D, the black dashed line represents a linear fit excluding ChKO mice. The blue dashed line is the linear fit for ChKO mice only.

Figure 9

Figure 9. ChREBP is essential for glucagon-stimulated glucose production.

(A) Changes in blood glucose levels were measured after glucagon (20 μg/kg body weight, ip) administration in ad libitum–fed 8- to 13-week-old male WT and ChKO mice (n = 6–9 per group) after 1 week on HDD. P values were obtained by Student’s t test. *P < 0.05 compared with ChKO at the indicated time point. After 2 weeks on HDD, (B) hepatic glycogen levels, (C) hepatic G6P levels, and (D) serum insulin levels were measured 20 minutes after injection with either glucagon (20 μg/kg body weight) or water (n = 3–5 per group). P values were obtained by 2-way ANOVA. *P < 0.05 compared with WT within treatment; #P < 0.05 compared with water within genotype. †P < 0.05 main effect of genotype. Values are the mean ± SEM.

Figure 10

Figure 10. ChREBP upregulates G6pc despite activated hepatic insulin signaling.

(A) Schematic diagram illustrating the experimental design. Insulin (1.5 mU/kg/min) and glucose (20 mg/kg/min) were infused into 8-week-old C3H/HeJ mice after 5 hours of fasting and water or fructose (4 g/kg) was injected into the stomach. Mice were sacrificed 100 minutes later. P values were obtained by 1-way ANOVA. *P < 0.05 compared with saline; #P < 0.05 compared with insulin + glucose + water (n = 11 or 12 per group). (B) Glycemia was measured in tail vein blood throughout the duration of the experiment and (C) serum insulin was measured at termination. (D) Hepatic gene expression was measured by qPCR. (E) Representative Western blots of hepatic total AKT and phospho-AKT (Ser473), with quantification of the AKT/phospo-AKT ratio. (F) Nuclear and cytosolic FOXO1A and phospho-FOXO1A (Ser256) with quantification of total FOXO1A normalized for the saline group within each cellular compartment (n = 4 per group). Values are the mean ± SEM.

Figure 11

Figure 11. Fructose activates ChREBP and induces G6PC and glycerol intolerance independently of FOXO1A.

Five-hour-fasted, 8- to 10-week-old CTL and Foxo1a-LKO mice were gavaged with water or fructose (4 g/kg body weight) and sacrificed 100 minutes later. (A–C) Hepatic gene expression was measured by qPCR (n = 3 or 4 per group). *P < 0.05 compared with water; #P < 0.05 compared with WT. Values are the mean ± SEM. (D) Weight gain of male CTL and Foxo1a-LKO mice fed chow versus HFrD for 4 weeks (n = 6–8/group). (E) Glucose tolerance test and (F) glycerol tolerance test. *P < 0.05 for chow vs. HFrD within genotype with area under the curve (AUC) for this cohort. †P < 0.05 main effect of HFrD; #P < 0.05 vs. chow-LKO. (G) Hepatic G6PC activity from ad libitum–fed mice. n = 3–6/group, *P < 0.05 within genotype versus chow. Values and bars are means ± SEM. All P values were obtained by 2-way ANOVA.

Figure 12

Figure 12. A ChREBP-G6PC signaling axis is conserved in human liver.

The correlation between ChREBP-β gene expression and (A) Fasn, (B) Pklr, (C) G6pc, and (D) Pck1 were compared in liver biopsy samples from overnight-fasted human subjects with NAFLD. Each data point represents an individual person (n = 95). P values were obtained by linear regression.

Similar articles

Cited by

References

    1. Ford ES, Giles WH, Dietz WH. Prevalence of the metabolic syndrome among US adults: findings from the third National Health and Nutrition Examination Survey. JAMA. 2002;287(3):356–359. doi: 10.1001/jama.287.3.356. - DOI - PubMed
    1. Brown MS, Goldstein JL. Selective versus total insulin resistance: a pathogenic paradox. Cell Metab. 2008;7(2):95–96. doi: 10.1016/j.cmet.2007.12.009. - DOI - PubMed
    1. Shimomura I, Matsuda M, Hammer RE, Bashmakov Y, Brown MS, Goldstein JL. Decreased IRS-2 and increased SREBP-1c lead to mixed insulin resistance and sensitivity in livers of lipodystrophic and ob/ob mice. Mol Cell. 2000;6(1):77–86. doi: 10.1016/S1097-2765(05)00010-9. - DOI - PubMed
    1. Pajvani UB, Qiang L, Kangsamaksin T, Kitajewski J, Ginsberg HN, Accili D. Inhibition of Notch uncouples Akt activation from hepatic lipid accumulation by decreasing mTorc1 stability. Nat Med. 2013;19(8):1054–1060. doi: 10.1038/nm.3259. - DOI - PMC - PubMed
    1. Li S, Brown MS, Goldstein JL. Bifurcation of insulin signaling pathway in rat liver: mTORC1 required for stimulation of lipogenesis, but not inhibition of gluconeogenesis. Proc Natl Acad Sci U S A. 2010;107(8):3441–3446. doi: 10.1073/pnas.0914798107. - DOI - PMC - PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources