Activation of nuclear receptor CAR ameliorates diabetes and fatty liver disease

Abstract

Constitutive androstane receptor CAR (NR1I3) has been identified as a central mediator of coordinate responses to xenobiotic and endobiotic stress. Here we use leptin-deficient mice (ob/ob) and ob/ob, CAR(-/-) double mutant mice to identify a metabolic role of CAR in type 2 diabetes. Activation of CAR significantly reduces serum glucose levels and improves glucose tolerance and insulin sensitivity. Gene expression analyses and hyperinsulinemic euglycemic clamp results suggest that CAR activation ameliorates hyperglycemia by suppressing glucose production and stimulating glucose uptake and usage in the liver. In addition, CAR activation dramatically improves fatty liver by both inhibition of hepatic lipogenesis and induction of beta-oxidation. We conclude that CAR activation improves type 2 diabetes, and that these actions of CAR suggest therapeutic approaches to the disease.

Fig. 1.

Activation of CAR represses serum glucose level and improves glucose tolerance after 1-week treatment in ob/ob mice. (A) Fed and Fasted blood glucose levels were measured in each group. (n = 5, *P < 0.01) (B and C) After 16 h of fasting, mice were performed glucose tolerance test. Blood glucose and plasma insulin values were assessed. (n = 6, *P < 0.01)

Fig. 2.

Activation of CAR improves glucose tolerance and insulin sensitivity after 1-month treatment in ob/ob mice and DIO mice. (A) ob/ob and ob/ob CAR^−/− mice were treated with TC or corn oil control for 1 month. After 16 h of fasting, glucose tolerance tests were performed. Blood glucose values were assessed. (n = 6, *P < 0.01) (B) Serum insulin level was measured after 6 h fasting in each treatment groups. (n = 4, *P < 0.01) (C) ob/ob mice from 1-month treatment were performed hyperinsulinemic-euglycemic clamp studies. Basal glucose production (CI), glucose infusion rate (CII), hepatic glucose production (CIII) and glucose uptake from peripheral tissue (CIV) were assessed during clamp. (n = 4, *P < 0.05) (D) Wild-type and CAR^−/− mice (n = 6) were fed on 45 Kcal% diet or control for 2 months and treated with TC or control for 1 month. After 16 h of fasting, mice were performed glucose tolerance test. Glucose levels at each time point were measured. (n = 6, *P < 0.01)

Fig. 3.

CAR regulates glucose metabolizing genes expression. Ob/ob and ob/ob CAR^−/− mice were treated with TC or corn oil control for 1 month. Liver total RNA was isolated from mice of different treatments and equal amounts of RNA were pooled from two individual mice and loaded on one lane. Northern blot was performed with Pepck and G6p probes. Hexokinase and Pgd were analyzed by quantitative RT-PCR. (n = 4, *P < 0.01)

Fig. 4.

CAR activation reduces liver lipids deposition and lipogenic gene expression in ob/ob mice. (A) Liver samples from ob/ob and ob/ob CAR^−/− mice of 1-month TC or control treatment were assessed for oil red O staining. (B) Hepatic triglyceride and NFFA were measured for quantitative liver lipids. (n = 4, *P < 0.05) (C) Liver RNA was extracted and gene expression analyzed by quantitative RT-PCR. Gene names were shown on top of each figure. (n = 4, **P < 0.01, *P < 0.05).

Fig. 5.

SULT2B1 plays an important role in CAR mediated suppression of lipogenic gene expression. (A) Ob/ob and ob/ob CAR^−/− mice were treated with TC or corn oil control for 1 month. Liver RNA was extracted and gene expression analyzed by quantitative RT-PCR. Gene names were shown on top of each figure. (n = 4, **P < 0.01, *P < 0.05) (B) Wild-type and SULT2B1 knockout mice were treated with TC or control for 3 days. Liver RNA was extracted and gene expression was assessed by quantitative RT-PCR. Lipogenic gene expressions were compared between WT and SULT2B1 KO groups. (n = 4, **P < 0.01, *P < 0.05)

Fig. 6.

CAR activation induces β-oxidation in the liver. (A) Ob/ob and ob/ob CAR^−/− mice were treated with TC or corn oil control for 1 month. Liver samples were used for measurement of a series of acylcarnitines with fatty acyl side chains of the indicated lengths, and also the indicated organic acids by stable isotope dilution mass spectrometry. (n = 5, *P < 0.01) (B) Ob/ob and ob/ob CAR^−/− mice were treated with TC or corn oil control for 1 month. Mice were fasted for 6 h and serum ketone bodies were measured. (n = 6, *P < 0.05) WT and CAR^−/− mice were treated with TC or control for 3 days. Primary hepatocytes were measured and performed β-oxidation assay. (n = 3, *P < 0.01) (C) Liver total RNA from 1 month treated ob/ob and ob/ob CAR^−/− mice were examined for ACC1 and ACC2 expression. (n = 4, *P < 0.01)