Aberrantly elevated microRNA-34a in obesity attenuates hepatic responses to FGF19 by targeting a membrane coreceptor β-Klotho

Abstract

MicroRNA-34a (miR-34a) is the most highly elevated hepatic miR in obese mice and is also substantially elevated in patients who have steatosis, but its role in obesity and metabolic dysfunction remains unclear. After a meal, FGF19 is secreted from the ileum; binds to a hepatic membrane receptor complex, FGF19 receptor 4 and coreceptor β-Klotho (βKL); and mediates postprandial responses under physiological conditions, but hepatic responses to FGF19 signaling were shown to be impaired in patients with steatosis. Here, we show an unexpected functional link between aberrantly elevated miR-34a and impaired βKL/FGF19 signaling in obesity. In vitro studies show that miR-34a down-regulates βKL by binding to the 3′ UTR of βKL mRNA. Adenoviral-mediated overexpression of miR-34a in mice decreased hepatic βKL levels, impaired FGF19-activated ERK and glycogen synthase kinase signaling, and altered expression of FGF19 metabolic target genes. Consistent with these results, βKL levels were decreased and hepatic responses to FGF19 were severely impaired in dietary obese mice that have elevated miR-34a. Remarkably, in vivo antisense inhibition of miR-34a in obese mice partially restored βKL levels and improved FGF19 target gene expression and metabolic outcomes, including decreased liver fat. Further, anti–miR-34a treatment in primary hepatocytes of obese mice restored FGF19-activated ERK and glycogen synthase kinase signaling in a βKL-dependent manner. These results indicate that aberrantly elevated miR-34a in obesity attenuates hepatic FGF19 signaling by directly targeting βKL. The miR-34a/βKL/FGF19 axis may present unique therapeutic targets for FGF19-related human diseases, including metabolic disorders and cancer.

Metabolic disorders, such as fatty liver, obesity, and type II diabetes, due to abnormally regulated lipid and glucose levels are serious medical problems worldwide (1). The roles of pancreatic insulin in the regulation of fed-state metabolism and development of such metabolic disorders are well known, but recently discovered and relatively less understood is the role of an intestinal hormone, FGF19 (or mouse FGF15) (2). FGF19 constitutes a unique endocrine metabolic regulatory axis. After a meal, expression of FGF19 is induced by the bile acid-activated nuclear receptor, farnesoid X receptor (FXR), in the small intestine (2). Secreted FGF19 binds to a hepatic membrane receptor complex, FGF19 receptor 4 (FGFR4), and its coreceptor β-Klotho (βKL) (3–6), and it then triggers the activation of cellular kinases, including ERK and glycogen synthase kinase (GSK), to mediate postprandial metabolic responses (7, 8). Interestingly, a recent study showed that the hepatic response to FGF19 is impaired in patients with nonalcoholic fatty liver disease (NAFLD) and insulin resistance (9). Despite the functional importance of βKL in transmitting FGF19 signaling, little is known about how the expression of βKL is regulated and why FGF19 signaling is impaired in patients who have fatty liver.

MicroRNAs (miRs) are small, noncoding RNAs and function as negative gene regulators (10). miRs directly bind to the 3′UTR of target mRNAs and inhibit translation and/or destabilize target mRNAs (11). Consistent with their critical biological functions, miRs are aberrantly expressed in human diseases, such as metabolic disease and cancer (10, 12). We have shown that miR-34a, which targets SIRT1 deacetylase, is aberrantly elevated in fatty livers of high-fat (HF) diet-induced obese mice and leptin-deficient ob/ob mice and that miR-34a is the most highly elevated hepatic miR in metabolic disease-prone FXR-null mice (13, 14). Consistent with these findings, a recent study has shown that miR-34a was the most highly elevated hepatic miR in dietary and genetic obese mice (15). Importantly, hepatic miR-34a levels are substantially elevated in NAFLD and in patients with type 2 diabetes (16–19). The role of miR-34a in obesity and metabolic dysfunction remains unclear, however.

Here, we present evidence demonstrating a surprising functional link between aberrantly elevated miR-34a and impaired βKL/FGF19 signaling in obesity. We further present the exciting therapeutic possibility of using in vivo antisense inhibition of elevated hepatic miR-34a for treating metabolic disorders.

Results

miR-34a and βKL Levels Are Inversely Correlated.

The 3′ UTRs of mouse and human βKL mRNAs contain imperfect complementary nucleotide sequences to the miR-34a seed sequence (SI Appendix, Fig. S1). Down-regulation of miR-34a by antisense miR-34a in mouse Hepa1c1c7 cells resulted in a dose-dependent increase in expression of βKL, as well as a known miR-34a target, SIRT1 (14, 20) (Fig. 1 A–D and SI Appendix, Fig. S2). Conversely, overexpression of miR-34a resulted in decreased βKL protein levels (Fig. 1 E and F and SI Appendix, Fig. S3). In mouse liver in vivo, adenoviral-mediated overexpression of miR-34a resulted in substantial decreases in βKL protein levels (Fig. 1 G and H). These results indicate that miR-34a and βKL levels are inversely correlated, suggesting that miR-34a may target βKL.

Fig. 1.

Levels of miR-34a and βKL are inversely correlated in vivo and in vitro. (A and B) Hepa1c1c7 cells were transfected with control scrambled RNA or anti–miR-34a, and miR-34a and βKL mRNA levels were detected by quantitative PCR (qPCR). Rel, Relative. (C and D) Protein levels of βKL and tubulin, as a control (Con), were detected by Western blot (WB) analysis, and the band intensities were quantified; the ratio of βKL to tubulin is plotted. (E and F) Hepa1c1c7 cells were infected with control Ad-empty or Ad–miR-34a, and the levels of miR-34a or βKL protein were detected. (G and H) Mice were injected via the tail vein with Ad-empty or Ad–miR-34a at a dose of 1.0 × 10⁹ active viral particles, and 6 d later, hepatic miR-34a and βKL protein levels were measured. Statistical significance was determined by the Student t test (SEM, n = 3). *P < 0.05; **P < 0.01.

miR-34a Directly Targets βKL.

To examine whether miR-34a directly targets the βKL 3′UTR, the WT or a mutated miR-34a binding site in the βKL 3′UTR was inserted into a luciferase reporter (Fig. 2A). Down-regulation of miR-34a with anti–miR-34a increased luciferase activity in a dose-dependent manner in Hepa1c1c7 cells transfected with the WT luciferase βKL reporter but not with the mutated βKL reporter or with the control miR oligonucleotide (Fig. 2 B–D). Conversely, overexpression of miR-34a inhibited the luciferase activity of the WT βKL reporter in a dose-dependent manner but not that of the mutant βKL reporter (Fig. 2 E–G). These results indicate that miR-34a negatively regulates βKL and that the inhibition is likely mediated by direct binding of miR-34a to the 3′UTR of βKL mRNA.

Fig. 2.

miR-34a directly binds to the 3′UTR of βKL mRNA. (A) Schematic of the βKL 3′UTR with the WT or mutated miR-34a site inserted into a luciferase (Luc) reporter. (B–D) Cos-1 cells were transfected with control or miR-34a antisense RNA as indicated, along with a luciferase reporter plasmid containing the WT (wt) or mutated (mut) 3′UTR of the βKL gene. The miR-34a levels were measured, and the values for luciferase activities were normalized to β-gal activities. Rel, relative. (E–G) Hepa1c1c7 cells were transfected with expression plasmid as indicated (Tf plasmids), the miR-34a levels were measured, and the values for luciferase activities were normalized by dividing them by β-gal activities. Statistical significance was determined by the Student t test (SEM, n = 6). *P < 0.05; **P < 0.01; NS, statistically nonsignificant.

Hepatic Overexpression of miR-34a Impairs FGF19 Signaling in Vivo.

FGF19 triggers activation of ERK and GSK in hepatocytes and regulates postprandial responses (3, 4, 6, 7). Because our in vitro studies suggest that miR-34a targets βKL, the coreceptor for FGF19 (Figs. 1 and 2), we examined whether overexpression of hepatic miR-34a would down-regulate βKL and affect expression of FGF19 metabolic target genes. Adenoviral-mediated hepatic overexpression of miR-34a in mice significantly decreased expression of βKL, as well as a known miR-34a target, SIRT1 (14, 20) (Fig. 3 A–C and SI Appendix, Fig. S4). Expression of a well-known FGF19 downstream target, c-Fos (3, 4), was also decreased, whereas expression of the hepatic FGF19 receptor, FGFR4, was not significantly altered (Fig. 3C). Consistent with the metabolic action of FGF19 (7, 21), mRNA levels of neutral bile acid biosynthetic genes Cyp7a1 and Cyp8b1 (Fig. 3D) and of a gluconeogenic gene, Pepck, were substantially elevated, whereas that of G-6-pase was slightly but not significantly increased (Fig. 3F). In contrast, mRNAs of acidic bile acid synthetic genes Cyp27a1 and Cyp7b1 were decreased as were those of fatty acid oxidation genes Cpt and Mcad and the bile acid transporter genes Bsep and Ntcp (Fig. 3 D–G and SI Appendix, Fig. S4). These results indicate that hepatic overexpression of miR-34a leads to decreased βKL levels, resulting in altered expression of FGF19 metabolic target genes.

Fig. 3.

Hepatic overexpression of miR-34a impairs FGF19 signaling in vivo. (A–C) Mice were injected with PBS, Ad-Empty, or Ad–miR-34a in the tail vein at a dose of 10⁸ active viral particles, and 7 d later, hepatic miR-34a levels and expression levels of βKL and FGFR4 were measured. Rel, relative; WB, Western blot. (D–G) mRNA levels of FGF19 signaling targets were measured by q-RT-PCR. Statistical significance was determined by the Student t test (SEM, n = 9). *P < 0.05; **P < 0.01. BA, bile acid; FA, fatty acid. (H and I) For determination of in vivo FGF19 signaling, mice injected with Ad-Empty or Ad–miR-34a were treated with PBS or FGF19; 30 min later, levels of hepatic p-ERK (P-Erk), p-GSK (P-Gsk), t-ERK (T-Erk), and t-GSK (T-Gsk) were detected by Western blot analysis. The band intensities of p-ERK (or p-GSK) and t-ERK (or t-GSK) were quantified, and the ratio of p-ERK (or p-GSK) to t-ERK (or t-GSK) was calculated. The ratio in normal mice expressing Ad-empty treated with PBS was then set to 1.

To define the effect of miR-34a on FGF19 signaling in vivo, mice overexpressing miR-34a were treated with PBS or FGF19. Treatment with FGF19 resulted in increased levels of phosphorylated ERK and GSK in mice injected with Ad-empty virus, but little increase was observed in mice overexpressing miR-34a (Fig. 3 H and I). Similar effects were observed in primary mouse hepatocytes (SI Appendix, Fig. S5). These results suggest that elevated hepatic miR-34a is associated with impaired βKL/FGF19 signaling in vivo.

Abnormal miR-34a/βKL Axis in Obesity Correlates with Impaired FGF19 Signaling.

Hepatic miR-34a levels are substantially elevated in human patients with fatty liver (16, 17) and in obese mice (14, 15). Moreover, hepatic responses to FGF19 are impaired in patients with fatty liver disease (9). Therefore, to test if aberrantly elevated miR-34a in obesity correlates inversely with βKL levels, we determined the miR-34a and βKL levels in two mouse models of obesity. Mice fed an HF chow for 16 wk showed marked fat accumulation in the liver (SI Appendix, Fig. S6), whereas hepatic miR-34a levels were increased and the expression of βKL and FGFR4 was significantly decreased (Fig. 4 A–C). Similarly, decreased hepatic βKL protein levels and increased miR-34a levels were observed in the leptin gene-deficient ob/ob mice (SI Appendix, Fig. S7). In vivo treatment of normal mice with FGF19 resulted in a substantial increase in phosphorylated ERK levels but not in phosphorylated AKT levels (Fig. 4D and SI Appendix, Fig. S8). In contrast, FGF19 had little effect on phosphorylated ERK levels in dietary obese mice (Fig. 4D), suggesting that FGF19 signaling is impaired in obese mice. These results suggest that an abnormal miR-34a/βKL pathway in obesity contributes to impaired FGF19 signaling.

Fig. 4.

Elevated hepatic miR-34a in obesity impairs FGF19 signaling. (A–C) Hepatic miR-34a and βKL expression levels from mice fed a normal diet (ND) or HF diet (HFD) for 16 wk were measured. Rel, relative. (D) Normal or HF dietary obese mice were injected with FGF19 or PBS. Thirty minutes later, p-ERK (P-Erk) and p-AKT (P-Akt) and t-ERK (T-Erk) and t-AKT (T-Akt) levels were measured. (E–H) Anti–miR-34a experiments in primary mouse hepatocytes. (E and F) miR-34a and βKL levels were measured in primary hepatocytes transfected with control or anti–miR-34a RNA. (G and H) Effects of anti–miR-34a on FGF19-mediated ERK and GSK phosphorylation were measured. p-GSK (P-Gsk), t-GSK (T-Gsk), Sc, Scramble. *P < 0.05; **P < 0.01.

To define the role of elevated miR-34a in impaired FGF19 signaling directly, we examined the effects of antisense inhibition of miR-34a on FGF19 signaling in primary hepatocytes. Anti–miR-34a treatment decreased miR-34a levels and increased βKL mRNA as expected (Fig. 4 E and F). Consistent with the role of βKL as a coreceptor for FGF19, when βKL was down-regulated, FGF19 treatment, even at superphysiological doses, did not activate FGF19 signaling (SI Appendix, Fig. S9). In FGF19 signaling experiments, FGF19-mediated ERK and GSK activation was impaired in obese mice but anti–miR-34a treatment restored the ERK and GSK activation similar to that observed in normal mice (Fig. 4 G and H and SI Appendix, Fig. S10). These results suggest that elevated hepatic miR-34a plays a causative role in impaired FGF19 signaling in obesity.

In Vivo Antisense Inhibition of miR-34a Restores Expression of βKL and FGF19 Metabolic Target Genes.

Because impaired βKL/FGF19 signaling in hepatocytes from obese mice results from the aberrantly elevated miR34a levels (Fig. 4), we tested whether the defective FGF19 signaling in obesity might be improved by down-regulating the miR-34a by injecting with antisense miR-34a (Fig. 5A). Levels of miR-34a were markedly increased, and βKL and Fos expression was decreased in mice fed HF chow (Fig. 5 B and C). Treatment of obese mice with anti–miR-34a partially but significantly reduced miR-34a levels toward those in normal mice (Fig. 5B) and significantly restored expression of both βKL and a known miR-34a target, SIRT1 (14, 20) (Fig. 5 B–D and SI Appendix, Fig. S11). In addition, expression of metabolic target genes involved in bile acid, glucose, and fat metabolism was altered in mice fed HF chow (Fig. 5E). The changes in expression of these genes were significantly reversed in mice treated with anti–miR-34a (Fig. 5E and SI Appendix, Fig. S11). Notably, the responses of these genes to antisense miR-34a in obese mice were the opposite of the effects of overexpression of miR-34a in normal mice (Fig. 3 D–G). We also examined the effect of anti–miR-34a on βKL and FGFR4 protein levels and their membrane localization. Expression of βKL and FGFR4 was dramatically decreased in obese mice, and anti–miR-34a treatment markedly, although partially, restored their expression levels and membrane localization (Fig. 5F). Liver fat levels were decreased, glycogen levels were partially restored, and insulin sensitivity was improved by anti–miR-34a treatment of obese mice (Fig. 5 G and H).

Fig. 5.

In vivo antisense miR-34a experiments. (A) Experimental outline. HFD, HF diet; ND, normal diet. (B–D) Effects of anti–miR-34a on hepatic miR-34a and βKL levels were measured. Rel, relative. (E) Hepatic mRNA levels of FGF19 metabolic target genes were measured. BA, bile acid; FA, fatty acid. (F) Hepatic βKL and FGFR4 proteins were detected by immunohistochemistry. (Magnification, 1,000×.) Sc, Scramble. (G and H) Liver fat, liver glycogen, and insulin sensitivity were measured. (Magnification, 250×.) GTT, glucose tolerance test; ITT, insulin tolerance test; PAS, periodic acid–Schiff. [B–H: SEM, n = 5; *P < 0.05; **P < 0.01; NS, statistically nonsignificant.] (I–M) FGF19 signaling experiments in PMH. (I) Experimental outline. Tf, transfected. (J and K) Effects of βKL siRNA on FGF19-mediated ERK phosphorylation in normal mice are shown. IP, immunoprecipitation; WB, Western blot. (L and M) Effects of βKL siRNA on p-ERK (P-Erk) and p-GSK (P-Gsk) in normal and obese mice were examined. The band intensities of p-ERK (or p-GSK) and t-ERK (T-Erk) [or t-GSK (T-Gsk)] were quantified, and the ratio of p-ERK (or p-GSK) to t-ERK (or t-GSK) was calculated. The ratio in normal mice expressing Ad-empty treated with PBS was then set to 1 (SEM, n = 3).

βKL Is Important for Improved FGF19 Signaling by Anti–miR-34a in Obesity.

To determine whether restored βKL levels are important for improved FGF19 signaling by anti–miR-34a treatment in obesity (Figs. 4 and 5), we examined FGF19 signaling in primary hepatocytes of obese mice in which miR-34a and βKL levels were down-regulated by anti–miR-34a and siRNA, respectively (Fig. 5I). In control experiments, transfection with siRNA for βKL substantially decreased endogenous βKL levels, which resulted in impaired ERK signaling in hepatocytes from normal mice (Fig. 5 J and K). FGF19 treatment increased phosphorylation of ERK in hepatocytes from normal mice, although little increase was observed in hepatocytes of obese mice (Fig. 5L and SI Appendix, Fig. S12). Anti–miR-34a treatment in hepatocytes of obese mice restored the FGF19-mediated ERK or GSK phosphorylation (Fig. 5 L and M, lanes 5 and 6), and this improved FGF19 response was largely blocked by down-regulation of βKL (Fig. 5 L and M, lanes 9 and 10, and SI Appendix, Fig. S13). These results indicate that aberrantly elevated hepatic miR-34a in obesity impairs FGF19 signaling largely by inhibiting expression of βKL, which contributes to resistance to hepatic FGF19 signaling in obesity.

Discussion

In this paper, we show that miR-34a directly targets βKL by binding to the 3′UTR of βKL mRNA. This miR-34a/βKL regulatory axis is abnormal in obese mice, which contributes to impaired hepatic responses to FGF19. In vivo antisense inhibition of hepatic miR-34a in obese mice improved metabolic gene expression and metabolic outcomes. Further, anti–miR-34a treatment in primary hepatocytes of obese mice restored FGF19-activated ERK and GSK signaling in a βKL-dependent manner.

We have shown that hepatic miR-34a levels were highly elevated in fatty livers of dietary and genetic obese mice and exhibited the greatest fold increase in FXR-null mice compared with WT mice (14, 15). Consistent with our initial findings, miR-34a was recently shown in a microarray study to be the most highly elevated miR in fatty livers of HF dietary and genetic ob/ob mice (15). Importantly, miR-34a levels are aberrantly elevated in human patients with NAFLD and patients with type 2 diabetes (16–19). All these recent findings indicate that there is a strong link between elevated hepatic miR-34a levels and metabolic abnormalities in the liver. However, it was not clear whether miR-34a was a cause or consequence of the metabolic abnormalities in obesity. Our studies provide evidence that abnormally elevated miR-34a in obesity plays a causative role in metabolic dysregulation, at least in part, by targeting the hepatic βKL/FGF19 axis.

FGF19 plays an important role in the late fed-state metabolic responses (7, 8, 22). Binding of FGF19 to the hepatic membrane receptor complex, FGFR4 and βKL, triggers activation of downstream cellular kinases, such as ERK and GSK, resulting in inhibition of hepatic bile acid and glucose synthesis and stimulation of glycogen and protein synthesis (3, 4, 7). Interestingly, the hepatic response to FGF19 signaling is impaired in patients with NAFLD, despite relatively normal FGF19 production in these patients (9). Our current study suggests that aberrant miR-34a/FGF19 signaling may explain FGF19 resistance in patients with patients with fatty liver. Moreover, the miR-34a/βKL/FGF19 axis is likely more relevant in pathological conditions in which miR-34a levels are aberrantly elevated, because there was no significant correlation between miR-34a and βKL levels in response to fasting and feeding under physiological conditions (SI Appendix, Fig. S14). Further, we unexpectedly found that expression of FGFR4, the hepatic membrane receptor for FGF19, is also decreased in obese mice and in mice overexpressing miR-34a. However, FGFR4 is not likely a direct target of miR-34a because there is no miR-34a site in the FGFR4 transcript. The present data are consistent with the idea that the protein levels and localization of FGFR4 are affected by the levels of its coreceptor βKL, but additional experiments will be needed to establish this.

In vivo antisense inhibition of elevated miR-34a resulted in beneficial metabolic outcomes, such as decreased liver fat and increased liver glycogen, which is consistent with expression of glucose and fatty acid metabolic genes (Fig. 5). Although this study focuses on the βKL/FGF19 pathway, βKL also functions as a coreceptor for FGF21 in adipose tissue during prolonged fasting (5, 6, 23). FGF21 is induced by peroxisome proliferator-activated receptor α in the liver in response to prolonged fasting (22, 24). Secreted FGF21 activates lipolysis in adipose tissue and β-oxidation and ketogenesis in the liver to meet energy demands during starvation. Therefore, our finding that βKL is an in vivo target of miR-34a suggests that aberrantly elevated miR-34a in obesity might also result in abnormal βKL/FGF21 signaling and contribute to unfavorable energy homeostasis. Further, it has been shown that miR-34a directly inhibits other genes involved in metabolic regulation, including SIRT1 in the liver (14) and VAMP2 in pancreatic β-cells (25). SIRT1 is a NAD⁺-dependent deacetylase that mediates homeostatic responses to nutrient limitation by deacetylating and modulating the activity of metabolic regulators, resulting in increased β-oxidation and decreased lipogenesis (26–29). VAMP2 plays a key role in insulin secretion by regulating exocytosis (25). Therefore, it is possible that the βKL/FGF21 axis, SIRT1, and VAMP2 regulatory pathways in the liver and other metabolic tissues, in addition to the βKL/FGF19 axis in the liver, contribute to the beneficial metabolic outcomes observed from the global in vivo anti–miR-34a experiments.

In addition to the role of FGF19 in fed-state metabolic regulation, abnormal activation of FGF19 signaling has been associated with colon and liver cancer (30). Interestingly, miR-34a is a well-known tumor suppressor, and absence of miR-34a expression due to deletion of its gene was associated with liver and colon cancer (31, 32). Consistent with these reports, adenoviral-mediated overexpression of miR-34a dramatically inhibited tumorigenesis in the liver (33). Therefore, our studies suggest a possible molecular mechanism by which miR-34a suppresses the development of liver cancer through targeting βKL/FGF19 signaling. Targeting the βKL/FGF19 signaling may thus be of value in the treatment of cancer as well as metabolic disease, although the signaling would need to be carefully modulated because excess miR-34a is associated with metabolic disease and miR-34a deficiency is associated with liver cancer.

Methods

Reagents and Materials.

Antisense–miR-34a and quantitative (q)-RT-PCR primers for miR-34a were purchased from Applied Biosystems. The siRNA for βKL was purchased from Ambion. βKL antibodies for Western blotting and immunohistochemistry were purchased from R&D Systems (AF2619) and Life Span Biosciences (LS-B3568), respectively. Antibodies to lamin (sc-20680), tubulin (sc-8085), and FGFR4 (sc-136988) were purchased from Santa Cruz Biotechnology, and antibodies for phosphorylated (p) ERK (no. 9101), total (t) ERK (no. 4695), p-AKT (no. 9271), t-AKT (no. 9272), p-GSK (no. 9327), and t-GSK (no. 5676) were purchased from Cell Signaling.

Animal Experiments.

Six-week-old male BALB/c mice were fed normal chow or HF chow (60% fat, wt/wt) for 14–20 wk. Control Ad-empty or Ad–miR-34a, at a dose of 0.1–1.0 × 10⁹ active viral particles in 200 μL of PBS, was injected via the tail vein of mice as previously described (27, 28, 34, 35). Adenoviral vectors are useful for hepatic studies because adenoviral-mediated expression of proteins, miRs, and shRNA is largely confined to the liver (36). In vivo anti–miR-34a experiments were performed as in the previous miR-122 studies in liver (37). All animal use and adenoviral/anti–miR-34a protocols were approved by the Institutional Animal Care and Use and Institutional Biosafety Committees at University of Illinois at Urbana–Champaign and were in accordance with National Institutes of Health guidelines (38).

Primary Mouse Hepatocytes.

Hepatocytes were isolated by collagenase (0.8 mg/mL; Sigma–Aldrich) perfusion through the portal vein of mice anesthetized with isoflurane. The hepatocyte suspension was filtered through a cell strainer (100 μm of nylon, Becton Dickinson), washed with M199 medium (M4530; Sigma), resuspended in M199 medium, and centrifuged through 45% Percoll (Sigma–Aldrich).

FGF19 Signaling Experiments in Primary Mouse Hepatocytes.

Hepatocytes were transfected with 100 nM anti–miR-34a or control RNA using lipofectamine. Thirty-six to 48 hours later, hepatocytes were further transfected with siRNA for βKL or control siRNA (5 nmol), and 36–48 h later, they were incubated with serum-free media for 3 h and then further treated with FGF19 (100 ng/mL) for 15–30 min and harvested for further analyses.

Construction of βKL 3′UTR-Luciferase Reporter.

A SpeI/HindIII fragment containing the 3′UTR of βKL was inserted into the pMIR plasmid (Invitrogen). Mutations in the 3′UTR were made using site-directed mutagenesis (Stratagene). Positive clones were identified by DNA sequencing. Construction of Ad–miR-34a was as described before (14).

Transfection Reporter Assay.

Hepa1c1c7 cells were used for up-regulation of miR-34a because they have relatively low levels of miR-34a, whereas Cos-1 cells, which have high levels of miR-34a, were used for down-regulation of miR-34a. Cells were transfected using lipofectamine, and reporter assays were performed as described before (27, 28, 34, 35).

q-RT-PCR Analysis.

Total RNA was isolated by TRIzol reagent (Invitrogen), and the levels of mRNAs or miR-34a were determined by q-RT-PCR. Primer sequences are available in SI Appendix, Fig. S15.