Farnesoid X receptor is essential for normal glucose homeostasis (original) (raw)
Abnormal glucose metabolism in FXR–/– mice. Mice deficient in FXR display elevated plasma cholesterol and triglyceride levels due to the loss of FXR regulation of hepatic bile acid and lipid metabolism (12). In accord with previous results, we observed increased plasma cholesterol and triglyceride levels in FXR–/– mice (Figure 1, A and B). The FXR–/– mice also exhibited increased hepatic triglyceride accumulation (Figure 1D) accompanied by marked induction of several genes involved in lipogenesis, such as SREBP-1c, stearoyl-CoA desmutase, and fatty acid synthase (Figure 1E). In addition, the level of circulating FFAs was approximately 2-fold higher in the FXR–/– mice (Figure 1C). These elevated FFA levels were comparable to those of WT mice fed a high-fat diet for 3 months and were not further increased by feeding the FXR–/– mice a high-fat diet.
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.
Due to the well-established role of high FFA levels in the pathogenesis of insulin resistance, we investigated glucose homeostasis in the FXR–/– mice. The serum glucose levels of random-fed FXR–/– mice were approximately 30% higher than those of the WT mice, whereas fasting glucose levels showed an age-dependent increase, with significantly higher levels in 12-week-old mice (Figure 1F). In the glucose tolerance test (GTT), glucose levels were elevated at all time points after loading in the FXR–/– mice, reaching almost twice the WT levels at 2 hours (Figure 2A). The failure of the FXR–/– glucose levels to return to baseline suggests markedly impaired peripheral glucose uptake. Insulin levels during the GTT also deviated from the normal response in WT mice. The insulin response to glucose in FXR–/– mice was diminished, with levels persistently elevated at 2 hours when those of the WT had returned to baseline (Figure 2B). The insulin tolerance test (ITT) confirmed the impaired insulin sensitivity in FXR–/– mice (Figure 2C). Fifteen minutes after i.p. insulin challenge, WT glucose levels dropped to about 50% of baseline, while the FXR–/– levels decreased by only 23%. The FXR–/– mice also showed faster recovery to about 55% of baseline at 90 minutes, compared with 33% in the WT mice.
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.
To examine the possible contribution of nonhepatic insulin sensitizing factors to the dysregulation of glucose metabolism in the FXR–/– mice, serum leptin and adiponectin were measured. The levels of these 2 major adipokines were comparable between FXR–/– and WT mice (data not shown).
The hyperinsulinemic euglycemic clamp was used to characterize glucose metabolic abnormalities in the absence of FXR in more detail. The contribution of liver glucose output to overall glucose metabolism was examined under both basal and low-dose insulin clamp conditions. FXR–/– and WT mice exhibited similar basal glucose production (Figure 3A) in the absence of insulin infusion. However, under the low-dose insulin clamp (3 U/kg/min), glucose output from the liver was completely suppressed in WT but not FXR–/– mice (Figure 3B). Note that the negative value of glucose production rate shown for WT mice was a result of the calculation used when the radioactive tracer was enriched instead of being diluted in plasma. In addition, the markedly lower rates for both glucose infusion and glucose disposal under low- and high-dose clamp conditions demonstrate a substantial defect in peripheral insulin sensitivity (Figure 3, C and D). Taken together, these data show that both failure of suppression of glucose production by the liver and attenuated peripheral disposal led to overall glucose intolerance in FXR–/– mice.
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.
Impaired insulin signaling in FXR–/– skeletal muscle and liver. A major consequence of elevated circulating FFA is interference with the insulin signaling cascade, which could account for the peripheral insulin resistance. Thus we investigated insulin action in skeletal muscle of WT and FXR–/– mice. After insulin administration, IR tyrosine phosphorylation was attenuated in the FXR–/– muscle (Figure 4A), and the level of insulin receptor substrate–1 (IRS-1) associated with PI3K was also markedly decreased (Figure 4B). Insulin-stimulated PI3K activity, measured in anti-phosphotyrosine antibody IPs from muscle homogenates of FXR–/– mice, was decreased to approximately 50% that of WT mice (Figure 4C). Similarly, insulin failed to stimulate serine phosphorylation of the Akt kinase in FXR–/– mice, though it led to a robust response in WT mice (Figure 4D). The higher basal activation of Akt in the absence of FXR could be the result of the elevated level of serum bile acid in these mice, which is known to activate both the JNK and MAPK pathways (20–22).
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.
The effect on insulin signaling cannot be due to any direct consequences of loss of FXR function in skeletal muscle, since sensitive RT-PCR did not detect expression in that tissue (Figure 4E). Due to the well-documented inverse correlation between insulin sensitivity and the amount of intramuscular fat in skeletal muscle, we analyzed intramuscular triglyceride and FFA content, which were both markedly elevated in the FXR–/– tissue (Figure 4, F and G). Consistent with this, we observed an increased level of IRS-1 serine 307 phosphorylation (Figure 4H), which has previously been linked to triglyceride-induced insulin resistance (23). Interestingly, expression of several PPARα target genes involved in fatty acid metabolism, including the FFA transporter CD36 and the mitochondrial and peroxisomal oxidation enzymes acyl-CoA oxidase and long-chain fatty acid dehydrogenase, was induced in FXR–/– muscle (Figure 4I), suggesting that lipid accumulation and substantial defects in insulin signaling occur despite augmented FFA transport and oxidation.
The major effect of insulin in liver is suppression of gluconeogenesis. Since FXR deficiency led to defective insulin inhibition of hepatic glucose output (Figure 3B), the insulin response was examined in FXR–/– livers. Insulin-dependent IR phosphorylation (Figure 5A) and IRS-2 association with PI3K (Figure 5B) were markedly blunted, suggesting substantial insulin resistance. The basis for the increased basal expression of IRS-2 and association with PI3K in untreated FXR–/– mice is unclear, but it could be secondary to elevated hepatic bile acid levels. Furthermore, insulin-induced PI3K activity in the FXR–/– livers was only approximately 30% that of WT mice in anti-phosphotyrosine antibody IPs (Figure 5C). As in the muscle, increased expression of PPARα target genes, including liver type FFA–binding protein, CD36, and acyl-CoA oxidase (Figure 5D), suggested increased FFA load. Interestingly — and in accord with a recent report (19) — the fasting response of key gluconeogenic factors, including PPARγ coactivator-1α (PGC-1α) and PEPCK, was diminished in the FXR–/– mice (Figure 5E). This defect could be due to either elevated lipid storage or elevated bile acids.
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.
FXR regulation of gluconeogenesis. To explore the possibility of direct regulation of gluconeogenesis by FXR, we tested the effect of CA on hepatic expression of several key genes involved in gluconeogenesis in WT and FXR–/– mice. As expected, no FXR mRNA was detected in FXR–/– mice with a cDNA probe corresponding to the deleted last exon from the targeting construct (11), and SHP expression was markedly lower. Feeding a 1% CA diet for 5 days increased SHP expression in WT mice and strongly decreased CYP7A1 expression, as expected (4, 11, 24). Both responses were lost in FXR–/– mice, indicating that FXR-SHP–independent mechanisms for repression observed in some circumstances (5, 6) were not active over this time course. Dietary CA also decreased expression of multiple gluconeogenic genes in WT mice, including PGC-1α, PEPCK, and G-6-Pase, and this response was completely absent in FXR–/– mice (Figure 6A). Hepatocyte nuclear factor 4α (HNF-4α) expression showed a similar response to CA treatment, and its basal expression was modestly increased in FXR–/– mice compared with WT mice.
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.
Interestingly, not all genes involved in gluconeogenesis were affected in the same fashion. Pyruvate carboxylase expression (Figure 6B) was unaffected by CA treatment in either the WT or FXR–/– mice, whereas forkhead transcription factor FOXO1 mRNA levels were decreased in the FXR–/– mice compared with WT in the presence and absence of CA. We also studied the effect of CA treatment on 2 rate-limiting enzyme genes involved in the glycolytic pathway, glucokinase and pyruvate kinase. Pyruvate kinase expression was higher in FXR–/– livers but was not affected by CA. Glucokinase transcripts were increased by CA feeding in both the WT and FXR–/– mice, suggesting this effect might be due to CA stimulation of signaling pathways independent of FXR.
We then investigated whether the suppressed gluconeogenic gene expression affected serum glucose levels. Indeed, CA feeding decreased fasting glucose by approximately 50% in WT mice but did not have a statistically significant effect in FXR–/– mice (Figure 6C). Consistent with previous results (25), CA feeding also reduced serum triglyceride levels in the WT mice but not the FXR–/– mice (Figure 6D).
The increased expression of SHP provides 1 potential mechanism for these FXR-dependent negative effects, as observed in the bile acid negative feedback regulation of CYP7A1 expression (4–6, 24). We tested this hypothesis by studying the effects of CA feeding on gluconeogenic gene expression in SHP–/– mice. On the chow diet, SHP–/– mice responded in a manner similar to FXR–/– mice, showing somewhat higher serum glucose in both fed (152 ± 13 versus 135 ± 9 mg/dl in WT) and fasted conditions (139 ± 15 versus 105 ± 12 mg/dl in WT) without significant changes in insulin. As observed in the FXR–/– mice, the SHP–/– mice also did not show repression of PEPCK, PGC-1α, or G-6-Pase expression in response to CA feeding (Figure 6E). On a chow diet, they exhibited modestly elevated serum glucose in both fasted and fed conditions (Figure 6F) without significant changes in insulin. However, they did not decrease serum glucose in response to dietary CA, suggesting that this effect of CA is mediated through the induction of SHP by FXR. Taken together, these results allow us to conclude that the previously identified pathways of bile acid feedback regulation by the FXR-SHP nuclear receptor cascade (4–6, 24) also target glucose metabolism.