FGFR4 Prevents Hyperlipidemia and Insulin Resistance but Underlies High-Fat Diet–Induced Fatty Liver

  1. Xinqiang Huang,
  2. Chaofeng Yang,
  3. Yongde Luo,
  4. Chengliu Jin,
  5. Fen Wang and
  6. Wallace L. McKeehan
  1. From the Center for Cancer and Stem Cell Biology, Institute of Biosciences and Technology, Texas A&M Health Science Center, Houston, Texas
  1. Address correspondence and reprint requests to Wallace L. McKeehan, PhD, Center for Cancer and Stem Cell Biology, Institute of Biosciences and Technology, Texas A&M Health Science Center, 2121 W. Holcombe Blvd., Houston, TX 77030. E-mail: wmckeehan{at}ibt.tamhsc.edu

Abstract

OBJECTIVE—Fibroblast growth factor (FGF) family signaling largely controls cellular homeostasis through short-range intercell paracrine communication. Recently FGF15/19, 21, and 23 have been implicated in endocrine control of metabolic homeostasis. The identity and location of the FGF receptor isotypes that mediate these effects are unclear. The objective was to determine the role of FGFR4, an isotype that has been proposed to mediate an ileal FGF15/19 to hepatocyte FGFR4 axis in cholesterol homeostasis, in metabolic homeostasis in vivo.

RESEARCH DESIGN AND METHODS—FGFR4−/− mice—mice overexpressing constitutively active hepatic FGFR4—and FGFR4−/− with constitutively active hepatic FGFR4 restored in the liver were subjected to a normal and a chronic high-fat diet sufficient to result in obesity. Systemic and liver-specific metabolic phenotypes were then characterized.

RESULTS—FGFR4-deficient mice on a normal diet exhibited features of metabolic syndrome that include increased mass of white adipose tissue, hyperlipidemia, glucose intolerance, and insulin resistance, in addition to hypercholesterolemia. Surprisingly, the FGFR4 deficiency alleviated high-fat diet–induced fatty liver in obese mice, which is also a correlate of metabolic syndrome. Restoration of FGFR4, specifically in hepatocytes of FGFR4-deficient mice, decreased plasma lipid levels and restored the high-fat diet–induced fatty liver but failed to restore glucose tolerance and sensitivity to insulin.

CONCLUSIONS—FGFR4 plays essential roles in systemic lipid and glucose homeostasis. FGFR4 activity in hepatocytes that normally serves to prevent systemic hyperlipidemia paradoxically underlies the fatty liver disease associated with chronic high-fat intake and obesity.

Metabolic syndrome (also known as insulin resistance syndrome or syndrome X) is a multicomponent disorder characterized by central body obesity, dyslipidemia, insulin resistance, glucose intolerance, and hypertension, which are risk factors for numerous diseases including type 2 diabetes, cardiovascular diseases, neurodegenerative diseases, liver disease, and cancer (1,2). The hepatic manifestation of the metabolic syndrome is nonalcoholic fatty liver disease (NAFLD), which is evident from triglyceride accumulation in macroscopic fat droplets (35). NAFLD is the most common liver disease in developed countries. NAFLD may be an indicator of metabolic syndrome and risk for its associated diseases equal to body mass and shape, insulin resistance, blood triglycerides, and HDL/LDL cholesterol (6,7). The mechanisms underlying NAFLD and its relationship to the other components of metabolic syndrome are largely unknown.

The fibroblast growth factor (FGF) signaling system is a ubiquitous microenvironmental regulator of cell-to-cell communication in development and adult homeostasis (812). Recent developments indicate that specific members of the family may regulate metabolic homeostasis by endocrine mechanisms where FGF originates in one tissue and acts distally on FGFR in another. Administration of FGF19 (13,14) and FGF21 (15) or their expression in the liver of transgenic animals impacts metabolic rate and multiple parameters associated with metabolic syndrome. Circulating FGF23 that resides in the same FGF subgroup as FGF15/19 and FGF21 based on sequence homology and affinity for heparan sulfate (16) regulates vitamin D and phosphate homeostasis (17,18). The regulation of expression and the tissue and cellular origin of FGF15/19, FGF21, and FGF23, as well as the isotype and location of the FGFR isotype underlying the metabolic effects of the three factors, is not well resolved.

An ileal origin of FGF15/19 under control of the bile acid–activated farnesoid X receptor (FXR) (NR1H4) that activates hepatocyte FGFR4 has been proposed to regulate cholesterol–to–bile acid metabolism (19). Hepatocyte FGFR4 regulates cholesterol–to–bile acid synthesis in the liver by transcriptional downregulation of cholesterol 7α-hydroxylase (CYP7A1), the rate-limiting enzyme for classical bile acid synthesis (20,21). A gut-to-liver FGF15/19-to-FGFR4 axis explains why only intestinal, compared with portal or intravenous, administration of bile acids represses hepatic cyp7a1 expression and bile acid synthesis (19). In contrast to FGF15/19, which is not expressed in liver (19), low-level FGF21 expression that increases on liver perturbation is relatively restricted to hepatocytes (22). When expressed in the hepatocyte, FGF21 improved glucose clearance and insulin sensitivity similar to systemic treatment of animals with FGF21 (15). We have shown that targeted expression of FGF21 in hepatocytes delays the appearance of diethyluitrosamine-induced liver adenoma. However, it has no effect on hepatocellular carcinoma incidence and burden. Although hepatocytes are a candidate for the autocrine action of FGF21, the most dramatic effects are on adipose tissue where neither FGF21 nor FGFR4 are significantly expressed (15,23). In adipocytes in vitro, FGF21 synergizes with the peroxisome proliferator–activated receptor (PPAR)γ ligand and antidiabetes agent rogsiglitazone to increase insulin-independent glucose uptake (23).

Because of the strong evidence that hepatocyte FGFR4 controls cholesterol–to–bile acid metabolism, which occurs primarily in the liver, it is a strong candidate for the mediator either directly or indirectly of some of the effects of FGF15/19 on general metabolic homeostasis. In this report, we evaluated the consequences of a general ablation of FGFR4 and hepatocyte-specific FGFR4 restoration on features associated with metabolic syndrome. We show that FGFR4 plays a general role in the maintenance of both lipid and glucose metabolism under normal dietary conditions in addition to its established role in cholesterol metabolism. Hepatocyte FGFR4 appears to exert a primary control on lipid metabolism. Effects on glucose metabolism could not be explained by activity of hepatocyte FGFR4 alone, suggesting an additional role of FGFR4 at other organ sites. Ironically, hepatocyte FGFR4, which normally protects against hyperlipidemia and hypercholesterolemia, underlies the fatty liver induced by high-fat intake and obesity.

RESEARCH DESIGN AND METHODS

Animals and diets.

Mice lacking FGFR4 (FGFR4−/−) and expressing constitutively activated human FGFR4 (Alb-caFGFR4), specifically in hepatocytes, have been previously described (20,21). The FGFR4−/− mice were a mixed 129Sv-C57BL/6 background, and Alb-caFGFR4 mice were an FVB background. FGFR4−/− and wild-type mice were produced from an FGFR4+/− mating. Alb-caFGFR4 and wild-type FVB mice were produced by mating heterozygous Alb-caFGFR4 transgenic males with wild-type FVB females. FGFR4−/− mice were crossed with Alb-caFGFR4 transgenic mice to obtain hybrids from the two strain backgrounds expressing caFGFR4 in the hepatocytes. Mice were maintained in 12-h light/dark cycles with free access to food and water. Except where indicated, experimental animals were male.

The normal diet (Prolab Isopro RMH 3000; PMI Nutrition International, Brentwood, MO) contained 3.46 kcal/g, of which 60 and 14% of the kilocalories were from carbohydrate and fat, respectively. Animals on a normal diet were analyzed at 6 months of age. Where indicated, mice were presented with a high-fat diet beginning at weaning over a period of 4 months to induce obesity. The high-fat diet (D12451; Research Diets, New Brunswick, NJ) presented 4.73 kcal/g, of which 35 and 45% of kilocalories were from carbohydrate and fat, respectively. Animals were killed and weighed, body fat depots were examined, and tissue was excised, weighed, and then subjected to analysis. All animal work was performed in accordance with the institutional animal care and use committee at the Institute of Biosciences and Technology, Texas A&M Health Science Center.

Histochemistry.

Tissues were fixed with Histochoice Tissue Fixative MB (Amresco, Solon, OH), and paraffin-embedded serial sections were prepared and archived; then, sections were stained for general pathological examination with hematoxylin and eosin. Lipid droplets were revealed by staining with Oil Red O. Livers were frozen in Neg-50 frozen section medium (Richard-Allan Scientific, Kalamazoo, MI). Frozen sections (10 μm) were prepared on glass slides, which were then incubated with Oil Red O for 8 min at 60°C. After washing with 85% isopropanol, tissue was counterstained with hematoxylin.

Analysis of blood chemistries and tissue lipids.

Blood was collected by retro-orbital puncture after anesthetization with 2,2,2-Tribromoethanol (avertin) (Sigma, St. Louis, MO). Serum was prepared by centrifugation of the clotted blood at 2,000g for 10 min, frozen in aliquots, and stored at −70°C for future analysis. Lipids were extracted from ∼50 mg tissue after homogenization in 1 ml PBS and incubation with 1 ml chloroform/methanol (2:1) overnight at room temperature. After centrifugation of the homogenate at 12,000g for 15 min, the lower organic phase–containing lipid was collected and evaporated under a vacuum in a rotary evaporator. The lipid pellet was dissolved in 200 μl PBS containing 1% Triton X-100. Triglyceride, free fatty acids, and cholesterol were measured enzymatically (Wako Pure Chemicals, Richmond, VA). Serum glucose was determined with the Glucometer Elite system (Bayer, Elkhart, IN). Serum insulin, leptin, and adiponectin levels were measured by enzyme-linked immunosorbent assay (Linco Research, St. Charles, MO).

Glucose tolerance and insulin responsiveness.

Conventional glucose and insulin tolerance tests were performed on mice fasted for 12 and 4 h, respectively. Mice were injected intraperitoneally with either 1 g glucose/kg body wt or 0.4 or 0.6 units recombinant human insulin/kg body wt (Eli Lilly, Indianapolis, IN). Blood was collected from the tail immediately before and 30, 60, 90, and 120 min after injection. Plasma glucose was measured as described above.

Analysis of gene expression.

Steady-state mRNA levels were quantified by real-time PCR analysis. Total RNA was prepared from tissues using the Ultraspec RNA isolation system (Biotecx Laboratories, Houston, TX). Equal amounts of RNA from four to five mice were pooled and subjected to reverse transcription with Superscript II (Life Technologies, Grand Island, NY) and random primers according to protocols provided by the manufacturer. Oligonucleotide primer sequences are shown in supplemental Table 1 (available in an online appendix at http://dx.doi.org/10.2337/db07-0648). Real-time PCR was performed using the Stratagene Mx 3000P QPCR system and SYBR Green JumpStart Taq Ready Mix (Sigma). All reactions were done in triplicate, and relative amounts of mRNA were calculated using the comparative threshold (Ct) cycle method. Mouse β-actin was used as the internal control.

Fatty acid β-oxidation activity.

Fatty acid oxidation activity was measured as previously described (24). Briefly, fresh livers were homogenized in four volumes of 0.25 mol/l sucrose containing 1 mmol/l EDTA. About 1 mg homogenate was incubated in 0.2 ml assay medium (150 mmol/l KCl, 10 mmol/l HEPES [pH 7.2], 0.1 mmol/l EDTA, 1 mmol/l potassium phosphate buffer [pH 7.2], 5 mmol/l malonate, 10 mmol/l MgCl2, 1 mmol/l carnitine, 0.5% BSA, 5 mmol/l ATP, and palmitic acid containing [9,10 (n)-3H]palmitic acid). The reaction was run for 30 min at 25°C and stopped by the addition of 0.2 ml of 0.6 N perchloric acid. The mixture was centrifuged at 2,000g for 10 min, and the unreacted fatty acid in the supernatant was removed with three extractions with 2 ml n-hexane. Radioactive degradation products in the water phase were counted.

Liver triglyceride secretion.

Liver triglyceride secretion rate was measured as previously described (25). Mice were fasted 4 h before intraperitoneal injection with 1 mg/g body wt Poloxamer 407. Blood samples were collected retro-orbitally immediately before injection and at 1, 2, and 4 h following injection. The triglyceride accumulation was linear during this time period. Hepatic triglyceride secretion rate was calculated from the slope of the curve and assuming a value of 0.071 ml plasma vol/g body wt (26).

Statistical analysis.

Metabolic parameters were expressed as means ± SD from the numbers of replicates described in the text. Statistical significance was determined by Student's t test, and P < 0.05 was considered significant.

RESULTS

FGFR4−/− mice exhibit increased white adipose tissue and hyperlipidemia.

FGFR4−/− mice appeared normal with respect to feeding behavior and physical activity. The impact of ablation of FGFR4 on body, liver, and adipose tissue mass in mice of both sexes that were fed normal diets was examined over a 6-month period (Table 1). No significant changes in body mass between wild-type and FGFR4−/− males or females were noted. Liver mass was slightly higher in FGFR4−/− females and significantly higher in FGFR4−/− males. Despite a similar body weight, the absence of FGFR4 caused a 1.5- and 2-fold increase, respectively, in mass of reproductive white adipose tissue in males and females (Table 1 and Fig. 1A). The weight of subcutaneous and perirenal fat pads was also higher in the FGFR4−/− mice but less notable (data not shown). The mass of brown adipose tissue was similar between the two genotypes (Table 1). A histological analysis of the reproductive white adipose tissue showed that the increase in mass was associated with an increase in size of adipocytes in the FGFR4−/− mice (Fig. 1BE) and confirmed that there was no difference in brown adipose cell or tissue morphology (Fig. 1FI). Although plasma leptin and adiponectin did not differ between FGFR4−/− and wild-type mice, triglycerides, free fatty acids, and cholesterol were 30–40% higher in FGFR4−/− mice under normal dietary conditions (Fig. 2).

Both FGFR4−/− and wild-type mice exhibited the expected increases in body and white adipose tissue mass when presented with only a high-fat diet over a 4-month period after weaning. No significant differences in the two parameters were noted between the two genotypes (Table 1 and Fig. 1). Wild-type mice on the high-fat diet exhibited elevated levels of free fatty acids (P < 0.05), cholesterol (P < 0.001), and leptin (P < 0.001), while plasma triglycerides and adiponectin remained constant (Fig. 2). Plasma leptin, adiponectin, and free fatty acids did not differ between the two groups, but triglycerides and cholesterol were elevated by 1.4- (P < 0.05) and 1.25- (P < 0.001) fold, respectively, over wild-type levels in the FGFR4−/− mice. These results indicate that FGFR4 plays a key role in maintenance of systemic lipid homeostasis.

Hyperglycemia, glucose intolerance, and insulin resistance in FGFR4−/− mice.

To determine whether glucose metabolism was altered along with lipid metabolism and fat deposition in the FGFR4−/− mice, we examined fasting plasma glucose and insulin levels. Although insulin levels were similar, plasma glucose in fasting FGFR4−/− mice was about 1.3 times (P < 0.05) that observed in wild-type mice (Fig. 3A and B). When subjected to the glucose tolerance test by administration of 1 g glucose/kg body wt, FGFR4−/− mice exhibited elevated levels of glucose over wild-type mice at all times (183 ± 26 vs. 243 ± 33 mg/dl, wild-type vs. FGFR4−/−, respectively, P < 0.001) 30 min after the infusion, and levels were still elevated at 2 h when levels had almost returned to normal (121 ± 15 vs. 180 ± 42 mg/dl, wild-type vs. FGFR4−/−, respectively, P < 0.01) in wild-type mice (Fig. 3C). Administration of 0.4 units insulin/kg caused plasma glucose levels to drop to only 60% of normal in FGFR4−/− mice compared with 45% (P = 0.06) observed in wild-type mice after 1 h (Fig. 3D). At 90 and 120 min, glucose levels were at 87 and 113% of normal, respectively, in the FGFR4−/− mice, whereas they remained depressed at 51 and 57% of normal in wild-type mice (P < 0.01 and P < 0.001, respectively). These results show that FGFR4−/− mice exhibited reduced glucose tolerance concurrent with increased insulin resistance.

Wild-type mice subjected to chronic high-fat diet exhibited hyperinsulinemia (Fig. 3A) that was apparently sufficient to maintain similar fasting plasma glucose levels in animals on a normal diet in this strain of mouse (Fig. 3B). However, the high-fat diet caused a reduced glucose tolerance and increased insulin resistance, similar to the mice deficient in FGFR4 on the normal diet (Fig. 3C and D). No significant differences between wild-type and FGFR4−/− mice subjected to chronic high-fat diet were detected. This suggests that FGFR4 deficiency or high-fat diet causes glucose intolerance and insulin resistance. Any additional effects of the FGFR4 deficiency are overridden or masked by the high-fat diet.

The FGFR4 deficiency reduces high-fat diet–induced fatty liver.

Livers of FGFR4−/− mice on a normal diet exhibited no notable morphological differences coincident with the observed hyperlipidemia and insulin resistance. As expected, the chronic high-fat diet induced severe fatty liver in wild-type males and, to a lesser extent, in wild-type females (Fig. 4A and C). Surprisingly, fatty liver was dramatically reduced in FGFR4−/− males and undetectable in females (Fig. 4B and D). Oil Red O staining confirmed the reduction of lipid droplets caused by the absence of FGFR4 in livers of mice on the high-fat diet (Fig. 4EH). A direct analysis further confirmed the effect of the FGFR4 deficiency on the elevated hepatic lipid content under the high-fat dietary load (Fig. 4IK). No difference was observed between wild-type and FGFR4−/− mice on a normal diet. The 71% reduction (P < 0.001) in triglyceride content in FGFR4−/− mice was most dramatic (Fig. 4I). Cholesterol levels were also reduced by 36% (P < 0.01) (Fig. 4J), whereas a reduction in free fatty acids was less significant (18.4 ± 4.2 vs. 14.9 ± 1.5 μmol/g, P = 0.06) (Fig. 4K). Despite the reduction in lipid accumulation in the liver, no significant reduction of total liver weight was observed in the FGFR4−/− mice (Table 1) since lipid mass accounts for less than 10% of total mass (Fig. 4). Thus, while FGFR4 maintains systemic glucose homeostasis and prevents plasma hyperlipidemia and fat accumulation in the white adipose tissue under normal dietary conditions, it underlies hepatic accumulation of lipid and the fatty liver that results from a chronic high-fat dietary load.

The FGFR4 deficiency alters liver lipid metabolism but not glucose metabolism.

Liver plays a key role in metabolic homeostasis of organisms by hormone and metabolite-responsive transcriptional level regulation of rate-limiting enzymes in both synthetic and catabolic pathways in lipid and glucose metabolism (27,28). Therefore, steady-state levels of mRNA coding for key factors involved in hepatic lipid and glucose metabolism were examined in wild-type and FGFR4−/− mice subjected to normal and high-fat diets (Fig. 5A). No change in expression of sterol regulatory element–binding protein 1C, a major regulator of lipogenesis (29,30), was observed. However, expression of lipogenic transcription factor PPARγ (31,32) was elevated by 2.3- (P < 0.01) and 1.7- (P < 0.05) fold above wild-type levels in FGFR4−/− mice under normal and high-fat dietary conditions, respectively. Under normal dietary conditions, expression of lipogenic genes involved in fatty acid synthesis and uptake was generally higher in the FGFR4−/− livers compared with wild type. Most significant was the 2.5-fold increase (P < 0.05) in stearoyl-CoA desaturase (SCD)1 that converts saturated to monunsaturated fatty acids and a 2.6-fold increase (P < 0.01) in fatty acid translocase (CD36/FAT). Expression levels of fatty acid synthase, SCD1, and CD36, but not acetyl-CoA carboxylase (ACC1), increased 2- to 3.5-fold (P < 0.01) after administration of the high-fat diet in wild-type mice. However, no additional changes in the elevated levels were observed in the FGFR4−/− mice except a 2.6-fold increase of fatty acid synthase (P < 0.01). The increase in PPARγ, SCD1, and CD36 in the FGFR4−/− mice may contribute to the hyperlipidemia observed under normal dietary conditions.

In contrast to lipogenic transcripts PPARγ, SCD1, and CD36, expression of liver PPARα and its downstream targets, medium-chain acyl-CoA dehydrogenase that stimulates fatty acid oxidation (33) and microsomal triglyceride transfer protein that is required for the assembly and secretion of apoB-containing lipoproteins (34), were unaffected by FGFR4 deficiency or the high-fat dietary load (Fig. 5A). However levels of these genes were elevated by ∼40–60% (P < 0.05) in FGFR4−/− mice on the high-fat diet. As we previously reported (20), hepatic FGFR4 is a negative regulator of expression of CYP7A, the rate-limiting enzyme for the canonical pathway of cholesterol–to–bile acid synthesis, under normal dietary conditions. Figure 5A shows that the threefold elevation of CYP7A expression in FGFR4−/− mice relative to wild type was also apparent in the obese mice under the high-fat dietary load. Thus, the reduction in liver cholesterol may also contribute to the alleviation of fatty liver in mice devoid of FGFR4.

We then determined rates of liver fatty acid oxidation and triglyceride secretion. Fatty acid oxidation was 1.43 (P < 0.05) times higher in FGFR4−/− livers in mice on a high-fat diet, although they were similar to wild type under normal dietary conditions (Fig. 5B). FGFR4−/− mice on a normal diet exhibited an insignificant 20% (P = 0.08) increase in rate of hepatic triglyceride secretion. Similar to observations in obese ob/ob mice (35), the rate of secretion was reduced in the wild-type obese mice on the chronic high-fat diet. The absence of FGFR4 abolished the resultant reduction in obese mice (Fig. 5C). Obese FGFR4−/− mice exhibited an 82% (P < 0.01) increase in hepatic triglyceride secretion compared with wild-type littermates.

Lastly, we examined the effect of FGFR4 deficiency on expression of the two key transcriptionally regulated regulators of hepatic gluconeogenesis, PEPCK and glucose-6-phosphatase (G6Pase). The FGFR4 deficiency had no effect on expression of either PEPCK or G6Pase under normal dietary conditions. The high-fat dietary load caused an ∼1.8-fold increase in G6Pase mRNA (P < 0.05) that was reduced to normal levels in the FGFR4−/− mice (Fig. 5A). We then determined whether insulin responsiveness was altered in livers of the FGFR4-deficient mice. Although insulin-stimulated phosphorylation of the insulin receptor and Akt were significantly decreased in obese compared with normal animals, no significant difference was observed between livers of wild-type and FGFR4−/− mice under either condition (supplemental Fig. 1). This indicated normal insulin signaling in the FGFR4−/− mouse livers upstream through Akt. Together, these results suggest that lipid but not glucose metabolism in the liver is impaired by the absence of germline FGFR4.

Hepatocyte FGFR4 is the determinant of plasma lipid levels and fatty liver.

To determine the contribution of hepatocyte FGFR4 to lipid and glucose metabolism, we examined the impact of overexpression of FGFR4 in hepatocytes driven by the albumin promoter in FVB mice (21). A constitutively active FGFR4 (caFGFR4) mutant was used to ensure a sustained signal and to bypass the need for an activating ligand. No significant changes relative to the wild-type control in body, liver, or white or brown adipose tissue weight were observed in the Alb-caFGFR4 mice expressing hyperactive FGFR4 in hepatocytes (data not shown). Basal fasting levels of plasma triglycerides, free fatty acids, and cholesterol were higher in the wild-type FVB mice relative to the wild-type littermate control strain for the FGFR4−/− mice (Fig. 6A). However, in contrast to the elevation in FGFR4−/− mice relative to their wild-type control, the three parameters in Alb-caFGFR4 mice were decreased to about 80% (P < 0.01), 70% (P < 0.01), and 85% (P < 0.05), respectively, of the wild-type control (Fig. 6A).

To determine whether restoration of FGFR4 to the hepatocytes could rescue the metabolic phenotype of the FGFR4−/− mice, the two strains were crossed to produce a FGFR4−/−/Alb-caFGFR4 hybrid. Littermates from the cross were compared to minimize strain differences. The FGFR4−/−/Alb-caFGFR4 mice exhibited normal liver morphology, although their weight was reduced compared with FGFR4−/− littermates (1.51 ± 0.22 vs. 1.27 ± 0.18 g, P < 0.05). The hybrid mice exhibited a less significant 15% reduction in reproductive white adipose tissue. Plasma levels of triglyceride, free fatty acids, and cholesterol were also significantly reduced in the FGFR4−/−/Alb-caFGFR4 mice relative to their FGFR4−/− littermates (Fig. 6B). Except for a 30% reduction (P < 0.05) in blood triglyceride levels in Alb-caFGFR4 mice, no differences in other parameters measured were observed when the mice were administrated a high-fat diet (Fig. 6). This suggests an inability of FGFR4 activity to compensate for the increase in plasma lipids levels caused by the high-fat diet.

We then determined whether restoration of FGFR4 to hepatocytes of the germline FGFR4-deficient mice, which are resistant to high-fat diet–induced fatty liver (Fig. 4), would restore the fatty liver condition. A fatty liver similar to wild-type mice on the chronic high-fat dietary load was apparent in the FGFR4−/−/Alb-caFGFR4 hybrids (Fig. 7AD). Quantification revealed that restored hepatocyte FGFR4 expression increased liver triglyceride levels (35.9 ± 8.69 vs. 108.6 ± 33.8 mg/g, P < 0.01). Analysis of gene expression revealed that restoration of hepatic FGFR4 largely reversed the altered gene expression related to lipid metabolism caused by FGFR4 deficiency but as expected had no impact on genes related to glucose metabolism (supplemental Fig. 2). These results show that specifically hepatocyte FGFR4 plays a major role in lipid metabolism in the liver. Its activity directly impacts plasma lipid homeostasis and underlies the fatty liver disease that results from a chronic high-fat diet.

Hepatocyte FGFR4 does not directly affect glucose metabolism.

We then determined whether hyperactive FGFR4 in hepatocytes or the restoration of hepatocyte FGFR4 to deficient livers would restore defects in glucose metabolism induced by the global absence of FGFR4 (Fig. 3). In contrast to FGFR4−/− mice that exhibited hyperglycemia and hyperinsulinemia, neither Alb-caFGFR4 nor FGFR4−/−/Alb-caFGFR4 hybrids exhibited changes in these parameters relative to their appropriate wild-type or FGFR4−/− littermate controls (Fig. 8A and B). Glucose and insulin tolerance tests further confirmed that the transgenic mice exhibited similar glucose tolerance and insulin sensitivity as controls (supplemental Fig. 3). These results suggest that the absence of hepatocyte FGFR4 activity is insufficient to explain the abnormalities in glucose homeostasis observed in FGFR4−/− mice. They suggest a potential role of FGFR4 on glucose homeostasis at another organ site.

DISCUSSION

In this study, we showed that FGFR4−/− mice displayed multiple elements of metabolic syndrome that included increased white adipose tissue, hyperlipidemia, and insulin resistance. However, despite the beneficial effects of FGFR4 activity on plasma lipid and glucose homeostasis under normal dietary conditions, FGFR4 underlies the development of fatty liver with obesity that is caused by a chronic high-fat diet. The alleviation of fatty liver induced by the high-fat dietary load by ablation of FGFR4 was associated with elevated plasma triglycerides without effect on an increase in body mass, adiposity, glucose intolerance, and insulin resistance. In other words, the normal protection against hyperlipidemia mediated by hepatocyte FGFR4 is to the detriment of the liver under conditions of chronic high-fat diet and obesity. Our observations revealed the physiological importance of FGFR4 signaling in normal lipid and glucose homeostasis in addition to cholesterol and bile acid metabolism. Hyperlipidemia and fatty liver are clinically associated with hyperglycemia and insulin resistance—all of which are part of metabolic syndrome. Type 2 diabetic patients with fatty liver are substantially more insulin resistant and have higher levels of plasma free fatty acids than those without (6). Thus, reduced fatty liver in FGFR4−/− mice may explain the similar extent of insulin resistance of these mice under a high-fat diet, although FGFR4−/− mice on a normal diet were more insulin resistant.

Our findings that hepatocyte FGFR4 activity maintains systemic lipid homeostasis under normal dietary conditions but underlies fatty liver in obese mice on a high-fat diet indicate dramatically different roles of FGFR4 that are dependent on nutritional status. At the molecular level under normal dietary conditions, FGFR4 deficiency is associated with elevation of liver lipogenic genes PPARγ, SCD1, and CD36, with no change in catabolic factors, which is consistent with the hyperlipidemia observed in FGFR4−/− mice. In contrast, on a high-fat diet, FGFR4 deficiency caused a net increase in PPARα and its downstream target genes medium-chain acyl-CoA dehydrogenase and microsomal triglyceride transfer protein, which is accompanied by elevated levels of fatty acid oxidation and hepatic triglyceride secretion. How high-fat dietary overload causes the FGFR4 deficiency to increase gene expression associated with fatty acid oxidation and hepatic triglyceride secretion remains to be determined. Such dual and seemingly opposing effects dependent on nutritional status are not without precedent. When overexpressed in hepatocytes, transcriptional regulator liver X receptor α, which directly senses diverse lipid metabolites as ligands (38), elevates mouse serum lipid profiles on a normal diet but improves high blood lipid profiles and protects from atherosclerosis in mice on a Western diet (39). It should also be noted that bile acids reduce high-fat diet–induced hyperglycemia and triglyceride accumulation in liver (36). FGFR4−/− mice display increased bile acid levels (20).

An unresolved issue from our study is the contribution of other organs or tissues where FGFR4 is expressed other than liver for metabolic abnormalities in the FGFR4-deficient mice. We have shown that both overexpression and restoration of FGFR4 to, specifically, hepatocytes decreased plasma lipid levels but failed to improve glucose tolerance and insulin sensitivity. Consistent with this, FGFR4 deficiency neither affects liver gluconeogenic enzymes nor hepatic insulin signaling. This indicates that hepatocyte FGFR4 plays a major role in control of hyperlipidemia (but not hyperglycemia), glucose intolerance, and insulin resistance caused by the general deficiency of FGFR4. Cholesterol synthesis and conversion to bile acids are limited to mature liver hepatocytes where FGFR4 is the only FGFR isotype (40). In contrast, lipid and glucose homeostasis is a partnership between liver and peripheral organs, most significant of which are skeletal muscle and adipose tissue. This suggests that peripheral sites other than liver or the complex interaction of multiple sites are the determinant of hyperglycemia and insulin resistance in FGFR4-deficient mice. FGFR4 is not expressed in adipose tissue (13,15) but is functional in skeletal muscle and has been implicated in its cellular homeostasis during embryogenesis and regeneration (41,42). Compared with other obese mouse models that have a significantly increased body weight, FGFR4−/− mice displayed an increase in the mass of reproductive white adipose tissue without a change in overall body weight and levels of two major adipokines, leptin and adiponectin. Apparently neither an increase in caloric intake nor obesity is the primary factor in determining the basal FGFR4−/− metabolic phenotype. One candidate for the obesity-independent upset in both systemic lipid and glucose metabolism due to FGFR4 deficiency is FXR, which has been implicated in both systemic lipid and glucose metabolism (43,44,45). An enterohepatic FXR-FGF15-FGFR4 axis has been suggested for regulation of hepatic cholesterol–to–bile acid metabolism, and this may also extend to hepatic lipid metabolism. Since hepatocyte FGFR4 does not play a major role in glucose metabolism and FGFR4 is not at play in adipose tissue (13,15), an FXR-regulated FGF15–to–muscle FGFR4 axis that contributes to glucose metabolism is conceivable. Results not shown here indicated that skeletal muscle in FGFR4−/− mice exhibited elevated levels of lipid compared with wild-type mice. Elevation of skeletal muscle lipids is associated with metabolic syndrome and insulin resistance (46,47). Additional experiments using mice with muscle-specific alterations in FGFR4 should clarify the relative contributions of muscle FGFR4 to aberrant lipid and glucose metabolism.

The opposite phenotypes between FGFR4-deficient mice on a normal diet and those caused by systemic administration or overexpression of FGF19, the human ortholog of mouse FGF15 (13,14), further suggest FGF15/19 as a candidate activator of FGFR4. However, both systemic administration or overexpression of FGF19 (13,14) and the FGFR4 deficiency reduced triglyceride content in the liver in obese mice. It has also been reported that mice with an FGFR4 gene deletion are still metabolically responsive to systemic FGF19 (13). Although it has been argued that FGF19 may be a specific FGF ligand for FGFR4 (48,49), this specificity has yet to be confirmed, particularly in tissues where FGFR4 has impact. Another candidate FGF ligand for FGFR4 particularly in hepatocytes is FGF21. Systemic administration or forced expression of FGF21 in hepatocytes results in reduced adiposity, improved glucose clearance, and insulin sensitivity (15). FGF21 is expressed at low levels in hepatocytes and increases dramatically with liver perturbation (22,50). We have shown that constitutive expression of FGF21 in hepatocytes delays development of chemically induced early hepatic adenomas in mice (22). The FGF21-dependent delay of adenoma development may suggest an internal autocrine activation of hepatocyte FGFR4 since resident hepatocyte FGFR4 exerts a suppressive effect on chemically induced hepatomas (22, X.H., W.L.K., unpublished observations). However, the autocrine action of hepatocyte FGF21 on hepatocyte FGFR4 alone cannot explain the effects of systemic administration of FGF21 on systemic glucose metabolism. Hepatic glucose metabolism is insensitive to hepatic FGFR4 activity. From cell culture studies, Kharitonenkov and colleagues (15,23) have shown that FGF21 stimulates PPARγ agonist-enhanced glucose uptake and metabolism in adipocytes that express other isotypes of FGFR kinase isotypes than FGFR4. An endocrine activity of hepatocyte FGF21 on adipose tissue under control of FXR-FGF15/19–activated hepatocyte FGFR4 is an attractive adjunct to FGF signal-mediated four-way communication among ileum, liver, skeletal muscle, and adipose tissue in control of lipid and glucose homeostasis.

In summary, we have shown that in addition to cholesterol and bile acid homeostasis, FGFR4 plays an important role in systemic lipid and glucose homeostasis. Similar to bile acid metabolism, hepatocyte FGFR4 exerts a major impact on lipid metabolism, but it appears that FGFR4 at other organ sites affects glucose homeostasis. Ironically, hepatocyte FGFR4 is also responsible for the fatty liver associated with obesity induced by chronic high-fat intake. General agonists of FGFR4 may be beneficial in alleviation of elements and consequences of metabolic syndrome related to hyperglycemia and hyperlipidemia. However, in the obese, under conditions of high-caloric intake, the benefits may be at the expense of aggravating fatty liver. General antagonists of FGFR4 may relieve fatty liver in the obese but aggravate the other consequences of metabolic syndrome. A complete knowledge of FGF ligand and tissue context specificity of FGFR4 signaling will be essential to designing tissue-specific agonists and antagonists to alleviate all elements of metabolic syndrome in the obese.

FIG. 1.

Increase in mass of white adipose tissue (WAT) in FGFR4−/− mice. A: Reproductive white adipose tissue in representative 6-month-old wild-type and FGFR4−/− males. BI: Adipocyte size increases in white adipose tissue but not brown fat (BAT) in FGFR4−/− mice on a normal diet. Sections were prepared from the respective type of adipose tissue from representative 6-month-old males on a normal diet or males on a high-fat diet 4 months since weaning. Sections were stained with hematoxylin and eosin as described in research design and methods. (Please see http://dx.doi.org/10.2337/db07-0648 for a high-quality digital representation of this figure.)

FIG. 2.

Increased fasting plasma lipid levels in FGFR4−/− mice. Fasting plasma triglycerides (TG) (A), free fatty acids (FFA) (B), cholesterol (C), leptin (D), and adiponectin (E) levels of wild-type and FGFR4−/− mice on normal and high-fat diets were measured. Data are means ± SD (n = 8–18 mice). *P < 0.05; ***P < 0.001.

FIG. 3.

Glucose intolerance and insulin resistance in FGFR4−/− mice. A and B: Fasting plasma insulin and glucose levels were assessed in mice with the indicated genotype on a normal (N) and high-fat (HF) diet. C: Plasma glucose levels were measured at the indicated times after intraperitoneal administration of 1 g glucose/kg body wt to fasting mice. D: Plasma glucose levels were measured at the indicated times following administration of 0.4 units insulin/kg body wt. Data are means ± SD (n = 8–10 mice). *P < 0.05. WT, wild type.

FIG. 4.

FGFR4 deficiency alleviates high-fat diet–induced fatty liver. AD: Sections from livers of representative wild-type (WT) and FGFR4−/− mice of the indicated sex on the high-fat diet were prepared and fixed and stained with hematoxylin and eosin. E–H: Lipid in sections of livers from representative male wild-type and FGFR4−/− mice on a normal or high-fat diet was stained with Oil Red O. Intense red indicates lipid droplets. IK: Triglycerides (TG), cholesterol, and free fatty acids (FFA) were measured in lipid extracts of livers of wild-type and FGFR4−/− males on the indicated diet (HF, high fat; N, normal). Data are means ± SD (n = 7–12 mice). **P < 0.01; ***P < 0.001. (Please see http://dx.doi.org/10.2337/db07-0648 for a high-quality digital representation of this figure.)

FIG. 5.

FGFR4 deficiency alters liver lipid but not glucose metabolism. A: Expression of hepatic genes involved in lipid and glucose metabolism. Expression levels were determined in the indicated mice on the indicated diet by quantitative real-time PCR analysis of steady-state mRNA levels and normalized to β-actin expression. Values in wild-type (WT) mice on a normal diet were set to 1. Data are means ± SD of three independent experiments for each gene with four to five mice of each genotype on each dietary regimen. B: Hepatic fatty acid oxidation. C: Hepatic triglyceride secretion. Values are expressed relative to wild-type mice on a normal diet. Data are means ± SD (n = 6–9 mice). *P < 0.05; **P < 0.01 relative to the corresponding wild-type mice. ACC1, acetyl-CoA carboxylase; CYP7A, cholesterol 7α-hydroxylase; FAS, fatty acid synthase; MCAD, medium-chain acyl-CoA dehydrogenase; MTP, microsomal triglyceride transfer protein; SREBP1, sterol regulatory element-binding protein 1.

FIG. 6.

Hepatocyte-specific expression of activated FGFR4 reduces plasma lipid levels. A: Fasting plasma triglycerides (TG), free fatty acids (FFA), and cholesterol were determined in mice overexpressing constitutively active FGFR4 in hepatocytes (Alb-caFGFR4) and compared with the wild-type (WT) FVB strain on a normal (N) or high fat (HF) diet. B: Fasting plasma lipid levels were determined in the FGFR4−/− × Alb-caFGFR4 hybrid and compared with the FGFR4−/− littermates. Data are means ± SD (n = 8–11 mice). *P < 0.05; **P < 0.01; ***P < 0.001.

FIG. 7.

Hepatocyte-specific restoration of FGFR4 in FGFR4−/− mice restores high-fat diet–induced fatty liver. A and B: Representative livers from FGFR4−/− and hybrid FGFR4−/−/Alb-caFGFR4 mice on a high-fat diet were processed and stained with hematoxylin and eosin (H&E) as described in Fig. 4C and D. Sections from the same livers were stained with Oil Red O. (Please see http://dx.doi.org/10.2337/db07-0648 for a high-quality digital representation of this figure.)

FIG. 8.

Hepatocyte FGFR4 does not affect glucose metabolism. A and B: Fasting plasma insulin and glucose levels were determined in the caFGFR4 and FGFR4−/−/Alb-caFGFR4 hybrids as described in research design and methods. Data are means ± SD (n = 8–11 mice). HF, high-fat diet; N, normal diet.

TABLE 1

Body and tissue mass in wild-type and FGFR4−/− mice

Acknowledgments

This work was supported by Public Health Service grants R01DK35310 and R01CA59971 (to W.L.M.).

We thank Dr. Wemin He (Institute of Biosciences and Technology, Texas A&M Health Science Center) for helpful advice and sharing reagents.

Footnotes

  • Published ahead of print at http://diabetes.diabetesjournals.org on 30 July 2007. DOI: 10.2337/db07-0648.

  • Additional information for this article can be found in an online appendix at http://dx.doi.org/10.2337/db07-0648.

  • The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

    • Accepted July 18, 2007.
    • Received May 14, 2007.

REFERENCES

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