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Comparative Study
, 116 (4), 1102-9

Farnesoid X Receptor Is Essential for Normal Glucose Homeostasis

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Comparative Study

Farnesoid X Receptor Is Essential for Normal Glucose Homeostasis

Ke Ma et al. J Clin Invest.

Abstract

The bile acid receptor farnesoid X receptor (FXR; NR1H4) is a central regulator of bile acid and lipid metabolism. We show here that FXR plays a key regulatory role in glucose homeostasis. FXR-null mice developed severe fatty liver and elevated circulating FFAs, which was associated with elevated serum glucose and impaired glucose and insulin tolerance. Their insulin resistance was confirmed by the hyperinsulinemic euglycemic clamp, which showed attenuated inhibition of hepatic glucose production by insulin and reduced peripheral glucose disposal. In FXR-/- skeletal muscle and liver, multiple steps in the insulin signaling pathway were markedly blunted. In skeletal muscle, which does not express FXR, triglyceride and FFA levels were increased, and we propose that their inhibitory effects account for insulin resistance in that tissue. In contrast to the results in FXR-/- mice, bile acid activation of FXR in WT mice repressed expression of gluconeogenic genes and decreased serum glucose. The absence of this repression in both FXR-/- and small heterodimer partner-null (SHP-/-) mice demonstrated that the previously described FXR-SHP nuclear receptor cascade also targets glucose metabolism. Taken together, our results identify a link between lipid and glucose metabolism mediated by the FXR-SHP cascade.

Figures

Figure 1
Figure 1. Lipid abnormalities in FXR–/– mice.
Elevated plasma triglyceride (A), cholesterol (B), and FFA levels (C) were observed in FXR–/– mice compared with WT mice (n = 8–11 per group) after overnight fasting. (D) Elevated liver triglyceride content was seen in FXR–/– mice. (E) Induction of genes involved in lipogenesis in the liver in FXR–/– mice at random-fed state. RNA samples were pooled from 5 mice in each group and loaded in duplicates. FAS, fatty acid synthase; SCD-1, stearoyl-CoA desmutase 1. (F) Plasma glucose levels in random-fed and fasting states. **P < 0.01 versus WT.
Figure 2
Figure 2. Impaired insulin sensitivity in FXR–/– mice.
Glucose (A) and insulin (B) levels during 2 g/kg i.p. GTT in 8-week-old mice (n = 8–10 per group) after overnight fasting. (C) Glucose level during i.p. ITT in the fed state (n = 8–10 mice per group). *P < 0.05, **P < 0.01 versus WT.
Figure 3
Figure 3. Low-dose and high-dose hyperinsulinemic-euglycemic clamp in 8- to 10-week-old WT and FXR–/– mice (n = 6 per group).
(A and B) Glucose production rate under basal (before insulin infusion; A) and low-dose clamp (3 mU/kg/min; B) conditions. (C) Glucose infusion rate during low- (3 mU/kg/min) and high-dose clamp (10 mU/kg/min) conditions. (D) Glucose disposal rate during low- and high-dose clamp conditions. *P < 0.05, **P < 0.01 versus WT.
Figure 4
Figure 4. Impaired insulin signaling and upregulation of genes involved in fatty acid metabolism in muscle of FXR–/– mice.
Muscle tissue homogenate from 4–5 mice per group were pooled together and subjected to IP and IB using antibodies as indicated. Northern blot analysis was performed on individual mice. Results are representative of at least 3 independent experiments. (A) Phosphorylation of IR after insulin stimulation (1 U/kg). Muscle homogenates were subjected to IP by anti-phosphotyrosine (P-Y) antibody 4G10 and IB by IR antibody. Total IR level was analyzed by IP followed by IB using the IR antibody. Quantitation was derived from 3 independent experiments. (B) Level of PI3K-associated IR after insulin stimulation as analyzed by IP using PI3K antibody followed by IRS-1 IB. (C) PI3K activity assay using immunopricipitates by anti-phosphotyrosine antibody. Muscle homogenates from individual mice were subjected to IP by anti-phosphotyrosine antibody followed by PI3K assay. Quantitation was derived from individual mice. (D) Phosphorylation of Akt (serine 473; P-S Akt) after insulin stimulation. (E) FXR expression by RT-PCR. (F and G) Analysis of intramuscular triglyceride and FFA content (n = 8 per group). (H) Serine 307 phosphorylation (P-S) of IRS-1 in the muscle after IP using IRS-1 antibody. (I) Expression of genes involved in fatty acid transport and oxidation in WT and FXR–/– muscle. ACO, acyl-CoA oxidase; LCAD, long-chain acyl-CoA dehydrogenase. **P < 0.01.
Figure 5
Figure 5. Impaired hepatic insulin signaling and expression of fatty acid metabolism and gluconeogenesis in the livers of FXR–/– mice.
Liver tissue homogenates from 4–5 mice per group were pooled together and subjected to IP and IB using antibodies as indicated. Northern blot analysis was performed on individual mice. Results are representative of — and quantitation was derived from — at least 3 independent experiments. (A) Phosphorylation of IR after insulin stimulation (1U/kg). Liver homogenates were subjected to IP by anti-phosphotyrosine antibody 4G10 and IB by IR antibody. Total IR level was analyzed by IP followed by IB using the IR antibody. Quantitation was derived from 3 independent experiments. (B) PI3K-associated IRS-2 level. (C) PI3K activity assay using immunopricipitates by anti-phosphotyrosine antibody. Liver homogenates from individual mice were subjected to IP by anti-phosphotyrosine antibody followed by PI3K assay. Quantitation was derived from individual mice. (D) Hepatic expression of genes involved in fatty acid transport and oxidation. L-FABP, liver type FFA–binding protein. (E) Hepatic expression of genes involved in gluconeogenesis. **P < 0.01.
Figure 6
Figure 6. Effects of FXR agonists.
Expression of genes involved in gluconeogenesis and glucose and triglyceride levels, which were measured after a 1% CA diet for 5 days. Glucose and triglyceride levels were measured at fed state from 6–7 mice per group and RNA from each group was pooled from 3–4 mice after overnight fasting. (A) Suppression of genes involved in gluconeogenesis by CA feeding was observed in WT but not FXR–/– mice. FOXO1, forkhead transcription factor FOXO1. (B) Effect of CA diet on glycolytic genes. GK, glucokinase; PYC, pyruvate carboxylase; PYK, pyruvate kinase. (C) Reduced serum glucose was observed in CA-fed WT mice. (D) Reduced triglyceride levels were seen in CA-fed WT mice. (E) SHP was required for suppressed expression of gluconeogenic genes in response to CA feeding. (F) Plasma glucose levels in fasted and fed WT and SHP–/– mice (n = 8 per group). **P < 0.01 versus control diet; *P < 0.05, #P < 0.01 versus WT.
Figure 7
Figure 7. Dysregulation of hepatic and skeletal muscle glucose homeostasis in FXR–/– mice.
Loss of FXR function in the liver results in increased hepatic lipid accumulation and elevation of FFAs in the serum. Insulin resistance both in the liver, which fails to suppress gluconeogenesis, and in the skeletal muscle, which reduces glucose uptake, contributes to dysregulation of glucose homeostasis in FXR–/– mice.

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