LRH-1-dependent glucose sensing determines intermediary metabolism in liver

Abstract

Liver receptor homolog 1 (LRH-1), an established regulator of cholesterol and bile acid homeostasis, has recently emerged as a potential drug target for liver disease. Although LRH-1 activation may protect the liver against diet-induced steatosis and insulin resistance, little is known about how LRH-1 controls hepatic glucose and fatty acid metabolism under physiological conditions. We therefore assessed the role of LRH-1 in hepatic intermediary metabolism. In mice with conditional deletion of Lrh1 in liver, analysis of hepatic glucose fluxes revealed reduced glucokinase (GCK) and glycogen synthase fluxes as compared with those of wild-type littermates. These changes were attributed to direct transcriptional regulation of Gck by LRH-1. Impaired glucokinase-mediated glucose phosphorylation in LRH-1-deficient livers was also associated with reduced glycogen synthesis, glycolysis, and de novo lipogenesis in response to acute and prolonged glucose exposure. Accordingly, hepatic carbohydrate response element-binding protein activity was reduced in these animals. Cumulatively, these data identify LRH-1 as a key regulatory component of the hepatic glucose-sensing system required for proper integration of postprandial glucose and lipid metabolism.

Figure 1. Reduced hepatic glucokinase and glycogen synthase fluxes in Alb-Cre;Lrh1^fl/fl mice.

(A) Schematic representation of the model used for mass isotopomer distribution analysis. GP, glycogen phosphorylase; GS, glycogen synthase; G6Pase, glucose-6-phosphatase. (B–D) Glucose fluxes in Lrh1^fl/fl mice (white bars) and Alb-Cre;Lrh1^fl/fl mice (black bars) under normoglycemic (NG) and hyperglycemic (HG) conditions. (B) Glucokinase and (C) glucose-6-phosphatase flux and (D) glucose balance. (E–G) Glycogen fluxes in Alb-Cre;Lrh1^fl/fl and Lrh1^fl/fl mice under normoglycemic and hyperglycemic conditions. (E) Glycogen synthase and (F) glycogen phosphorylase flux and (G) glycogen balance. Data represent mean ± SEM for n = 5–9 per genotype. *P < 0.05 Alb-Cre;Lrh1^fl/fl versus Lrh1^fl/fl; ^#P < 0.05 hyperglycemic versus normoglycemic.

Figure 2. Reduced glucokinase expression in Alb-Cre;Lrh1^fl/fl mice.

(A) Blood glucose and (B) plasma insulin concentrations (n = 10 per genotype) and (C and D) hepatic mRNA levels of Lrh1, Shp, Gck, and Hk1 during an oral glucose tolerance test (n = 4–8 per genotype) in Lrh1^fl/fl mice (white boxes) and Alb-Cre;Lrh1^fl/fl mice (black boxes). (E and F) Hepatic mRNA levels in fed, 24-hour–fasted or 6-hour–refed Lrh1^fl/fl mice (white bars) and Alb-Cre;Lrh1^fl/fl mice (black bars) (n = 7–9 per genotype). (G) LRH-1 transcriptional activity in HeLa cells transfected with a heterologous (4x LRH-1 response element–Luc [4xLRE-Luc]) or an endogenous LRH-1 reporter driven by the Shp promoter (SHP-Luc). Luciferase activity was determined in the absence (empty; white bars) or presence (LRH-1; black bars) of LRH-1 after exposure to 5 or 25 mM glucose for 24 hours. Data are expressed as relative light units (RLUs) compared with empty reporter (pGL3). Data represent mean ± SEM. *P < 0.05 versus Lrh1^fl/fl or versus empty vector (pCMX).

Figure 3. Gck is a direct transcriptional target of LRH-1.

(A) Hepatic mRNA levels in Alb-Cre;Lrh1^fl/fl mice 5 weeks after in vivo transduction of the liver using AAV8-SHP virus (gray bars) in comparison with those in Lrh1^fl/fl mice (white bars) and Alb-Cre;Lrh1^fl/fl mice (black bars) (n = 4–5 per group). (B) Hepatic GCK protein expression in refed Lrh1^fl/fl and Alb-Cre;Lrh1^fl/fl mice. (C and D) Expression levels of LRH-1 and its targets in (C) wild-type primary hepatocytes and (D) Hepa 1.6 mouse hepatoma cells transduced with AdGFP (white bars) or AdLRH-1 (black bars) viruses (n = 3 per condition). (E) 2-deoxyglucose (2-DG) uptake and 2-deoxyglucose-6-phosphate (2-DG6P) production in Hepa 1.6 cells transduced with AdGFP (white bars) or AdLRH-1 (black bars) viruses (n = 6 per condition). (F) Schematic presentation of the 6 putative LRH-1 response elements in the mouse Gck promoter. (G) Assessment of LRH-1 recruitment to these sites, as depicted in F, determined by ChIP analysis using genomic DNA from livers of Lrh1^fl/fl and Alb-Cre;Lrh1^fl/fl mice. (H) Luciferase activities in HeLa cells transfected with empty luciferase reporter (pGL3; white bar) or long and short Gck promoter constructs (black bars). Data are expressed as fold induction in luciferase activity upon LRH-1 cotransfection. Data represent mean ± SEM. *P < 0.05 versus Lrh1^fl/fl, versus GFP, or versus empty reporter (pGL3); ^#P < 0.05 versus Alb-Cre;Lrh1^fl/fl.

Figure 4. Delayed glycogen synthesis and reduced glycolysis in Alb-Cre;Lrh1^fl/fl mice.

(A) Representative glycogen stainings during oral glucose tolerance test (n = 3–8 per genotype). Scale bar: 200 μm. (B) Quantification of hepatic glycogen content in Lrh1^fl/fl mice (white boxes) and Alb-Cre;Lrh1^fl/fl mice (black boxes) during oral glucose tolerance test. (C) Hepatic glycogen content in fed, 24-hour–fasted, or 6-hour–refed Lrh1^fl/fl mice (white bars) and Alb-Cre;Lrh1^fl/fl mice (black bars) (n = 7 per genotype). (D) Extracellular acidification rates (ECARs) in Lrh1^fl/fl (white boxes) and Alb-Cre;Lrh1^fl/fl (black boxes) primary hepatocytes (n = 5–6 per genotype). Data represent mean ± SEM. *P < 0.05 versus Lrh1^fl/fl.

Figure 5. Reduced de novo lipogenesis in Alb-Cre;Lrh1^fl/fl mice.

(A) Hepatic lipogenic gene expression and (B) de novo synthesis of hepatic palmitate, stearate, and oleate in fed, fasted, and refed Lrh1^fl/fl mice (white bars) and Alb-Cre;Lrh1^fl/fl mice (black bars) (n = 7–9 per genotype). Data represent mean ± SEM. *P < 0.05 versus Lrh1^fl/fl.

Figure 6. Impaired GCK activity in Alb-Cre;Lrh1^fl/fl mice reduces ChREBP expression and activity.

(A) Hepatic Chrebp, Pklr, Srebp-1c, and G6pd1 expression in fed, 24-hour–fasted, or 6-hour–refed Lrh1^fl/fl mice (white bars) and Alb-Cre;Lrh1^fl/fl mice (black bars) (n = 7–9 per genotype). (B) Nuclear ChREBP protein expression in 24-hour–fasted and 6-hour–refed Lrh1^fl/fl and Alb-Cre;Lrh1^fl/fl mice. (C) Hepatic G6P content in fasted and refed Lrh1^fl/fl mice (white bars) and Alb-Cre;Lrh1^fl/fl mice (black bars) (n = 6–7 per genotype). (D) Hepatic Chrebp and Pklr expression in Alb-Cre;Lrh1^fl/fl mice 5 weeks after in vivo transduction of the liver using AAV8-SHP virus (gray bars) in comparison with that in Lrh1^fl/fl mice (white bars) and Alb-Cre;Lrh1^fl/fl mice (black bars) (n = 4–5 per group). (E) Luciferase activities in HeLa cells transfected with Chrebp (ChREBP-Luc) or SHP (SHP-Luc) promoter (black bars) constructs in the absence (empty; white bars) or presence (LRH-1; black bars) of LRH-1. Data are expressed as relative light units and normalized to empty reporter (pGL3). (F) Hepatic mRNA levels in Alb-Cre;Lrh1^fl/fl mice 5 weeks after in vivo transduction of the liver using 2 different titers of AAV8-GCK virus (low, 10¹¹ particles per mouse [light gray bars], and high, 10¹² particles per mouse [dark gray bars]) in comparison with those in Lrh1^fl/fl mice (white bars) and Alb-Cre;Lrh1^fl/fl mice (black bars) (n = 4–5 per group). Data represent mean ± SEM. *P < 0.05 versus Lrh1^fl/fl or versus empty vector (pCMX); ^#P < 0.05 versus Alb-Cre;Lrh1^fl/fl.