Post-transcriptional activation of PPAR alpha by KLF6 in hepatic steatosis

Background & Aims

Dysregulated glucose homeostasis and lipid accumulation characterize non-alcoholic fatty liver disease (NAFLD), but underlying mechanisms are obscure. We report here that Krüppel-like factor 6 (KLF6), a ubiquitous transcription factor that promotes adipocyte differentiation, also provokes the metabolic abnormalities of NAFLD by post-transcriptionally activating PPARα-signaling.

Methods

Mice with either hepatocyte-specific depletion of KLF6 (‘ΔHepKlf6’) or global KLF6 heterozygosity (Klf6+/−) were fed a high fat diet (HFD) or chow for 8 or 16 weeks. Glucose and insulin tolerance tests were performed to assess insulin sensitivity. Overexpression and knockdown of KLF6 in cultured cells enabled the elucidation of underlying mechanisms. In liver samples from a cohort of 28 NAFLD patients, the expression of KLF6-related target genes was quantified.

Results

Mice with global- or hepatocyte-depletion of KLF6 have reduced body fat content and improved glucose and insulin tolerance, and are protected from HFD-induced steatosis. In hepatocytes, KLF6 deficiency reduces PPARα-regulated genes (Trb3, Pepck) with diminished PPARα protein but no change in Pparα mRNA, which is explained by the discovery that KLF6 represses miRNA 10b, which leads to induction of PPARα. In NAFLD patients with advanced disease and inflammation, the expression of miRNA 10b is significantly downregulated, while PEPCK mRNA is upregulated; KLF6 mRNA expression also correlates with TRB3 as well as PEPCK gene expression.

Conclusions

KLF6 increases PPARα activity, whereas KLF6 loss leads to PPARα repression and attenuation of lipid and glucose abnormalities associated with a high fat diet. The findings establish KLF6 as a novel regulator of hepatic glucose and lipid metabolism in fatty liver.

Introduction

Non-alcoholic fatty liver disease (NAFLD) prevalence in the US and Europe is rapidly rising. NAFLD is characterized by hepatic steatosis, which can progress to non-alcoholic steatohepatitis (NASH). Subsequent progression from NASH to cirrhosis may precipitate liver failure and hepatocellular carcinoma (HCC) [1]. The mechanisms underlying the pathogenesis of NAFLD and its progression to NASH are poorly understood.

Krüppel-like factor 6 (KLF6) is a ubiquitously expressed, multifunctional transcription factor and tumor suppressor gene with emerging roles in glucose and lipid homeostasis [2], [3], [4]. KLF6 promotes adipogenesis in preadipocytes and fibroblasts, and interferes with peroxisome proliferator activated receptor γ (PPARγ) signaling [5], [6]. A functional polymorphism in the KLF6 gene that increases the generation of antagonistic KLF6 alternative splice forms protects patients from advanced NAFLD [7]. KLF6 directly transactivates hepatic glucokinase, and modulates hepatic insulin sensitivity in NAFLD [8].

Insulin resistance (IR) is a sentinel feature of the metabolic syndrome and is associated with to NASH progression. IR is mediated in part by the actions of peroxisome proliferator activated receptor alpha (PPARα), a xenobiotic and lipid sensor that regulates hepatic steatosis, lipoprotein synthesis, and hepatic gluconeogenesis. PPARα is activated by excess fatty acids (FA), thereby inducing expression of downstream genes that metabolize FA [9], including fatty acid transport proteins [10].

Effects of PPARα activation on IR are multifaceted and incompletely understood. While fibrates, which act as PPARα ligands, improve IR, the PPARα co-activator peroxisome proliferator-activated receptor gamma co-activator 1-α (PGC1α) has a paradoxical effect on IR, since PGC1α knockout mice are protected from high fat diet-induced steatosis and IR [11]. A similar observation was made for farnesoid X receptor (FXR), which also induces PPARα signaling, but FXR agonists increase hepatic gluconeogenesis via PPARα-mediated phosphoenolpyruvate carboxykinase (PEPCK) upregulation and therefore worsen hepatic IR [12]. In contrast, micro-RNA 10b (miRNA 10b) opposes the actions of PPARα signaling by repressing PPARα protein expression [13], [14]. Downregulation of the PPARα target tribbles homologue 3 (TRB3) improves IR via increased AKT phosphorylation and PPARγ activation [15], [16].

Here, we describe a functional link between KLF6 expression and hepatic PPARα signaling, in part via miRNA 10b repression. We have uncovered this link by characterizing two mouse models, one with global KLF6 heterozygosity, the second with hepatocyte KLF6 deficiency, as well as by quantifying KLF6 mRNA in a cohort of 28 NAFLD patients. Together these data reveal a regulatory role of KLF6 in insulin signaling and lipid metabolism.