Glucokinase links Krüppel-like factor 6 to the regulation of hepatic insulin sensitivity in nonalcoholic fatty liver disease

Abstract

The polymorphism, KLF6-IVS1-27A, in the Krüppel-like factor 6 (KLF6) transcription factor gene enhances its splicing into antagonistic isoforms and is associated with delayed histological progression of nonalcoholic fatty liver disease (NAFLD). To explore a potential role for KLF6 in the development of insulin resistance, central to NAFLD pathogenesis, we genotyped KLF6-IVS1-27 in healthy subjects and assayed fasting plasma glucose (FPG) and insulin sensitivities. Furthermore, we quantified messenger RNA (mRNA) expression of KLF6 and glucokinase (GCK), as an important mediator of insulin sensitivity, in human livers and in liver tissues derived from a murine Klf6 knockdown model (DeltaKlf6). Klf6 overexpression studies in a mouse hepatocyte line were utilized to mechanistically link KLF6 with Gck promoter activity. KLF6-IVS1-27Gwt (i.e., less KLF6 splicing) was associated with stepwise increases in FPG and insulin and reduced hepatic insulin sensitivity. KLF6 binds to the liver-specific Gck promoter and activates a GCK promoter-reporter, identifying GCK as a KLF6 direct transcriptional target. Accordingly, in DeltaKlf6 hepatocytes Gck expression was reduced and stable transfection of Klf6 led to up-regulation of Gck. GCK and KLF6 mRNAs correlate directly in human NAFLD tissues and immunohistochemistry studies confirm falling levels of both KLF6 and GCK in fat-laden hepatocytes. In contrast to full-length KLF6, splice variant KLF6-SV1 increases in NAFLD hepatocytes and inversely correlates with glucokinase regulatory protein, which negatively regulates GCK activity.

Conclusion: KLF6 regulation of GCK contributes to the development of hepatic insulin resistance. The KLF6-IVS1-27A polymorphism, which generates more KLF6-SV1, combats this, lowering hepatic insulin resistance and blood glucose.

Figure 1. KLF6_IVS1-27AA individuals have a normal glucose profile during the OGTT, despite lower plasma insulin levels

The profile of plasma glucose levels during the oral glucose tolerance test (OGTT) was unchanged between the three KLF6_IVS1-27G>A genotype groups, depicted as homozygous variant (-○- AA), heterozygous variant (-●- GA) and wild type (-●- GG) (A). In contrast, the plasma insulin levels were lower, significantly so at 30 minutes, in the homozygous variant AA individuals (* = p<0.05) (B).

Figure 2. Reduced Klf6 and glucokinase expression in vivo DeltaKlf6 mice

Haematoxylin and Eosin (H&E) stains for wt and DeltaKlf6 mice were similar with no apparent phenotypic differences at 3 months of age. KLF6 immunohistochemistry revealed a decreased expression of KLF6 within hepatocytes (4.1 fold fewer positive nuclei by manual counting; p=0.002) rather than non-parenchymal cells. GCK immunofluorescent staining (1300ms exposure time, DAPI blue, GCK red) confirmed a relative abundance (1.92 fold by integrated density quantification, p=0.038) of hepatocyte Gck expression in wt versus DeltaKlf6 mice.

Figure 3. Reduced Klf6 and glucokinase expression in DeltaKlf6 mice

While expression of Klf6, Gck and Gckr mRNA was not significantly suppressed in whole liver tissues in DeltaKlf6 mice compared to wild type littermates (n=7) (n=7) (A), levels were markedly suppressed in primary hepatocytes isolated from DeltaKlf6 mice (n=3) (D). Data are presented as fold change (FC) relative to wt littermates. Known transcriptional activators of Gck were not reduced in the presence of reduced Gck in deltaKlf6 mice (B & E). Western blot of both whole tissues and isolated hepatocytes confirmed decreased Gck protein in association with reduced Klf6. (* denotes p-value<0.05; **p-value<0.005)

Figure 4. Increased Klf6 and glucokinase expression in vitro in Klf6 transfected AML12 cells

Increased Klf6 and Gck expression were present in the murine hepatoma cell line, AML12 after transfection with a plasmid vector expressing Klf6 (A, C). Known transcriptional activators of Gck remained unchanged. Hnf4a mRNA was downregulated indicating counter-regulatory mechanisms (B). (* = p<0.05).

Figure 5. KLF6-FL and GCK are reduced with advanced steatosis, while elevated KLF6-SV1 is associated with suppressed GCKR

Expression of the KLF6 full-length isoform (KLF6-FL) correlated positively with GCK expression (A). In contrast, expression of KLF6-SV1 was associated with a dramatic reduction in expression of GCKR (B). KLF6-FL expression was significantly suppressed with more advanced steatosis (grade 2+3) while KLF6-SV1 was significantly upregulated (C). Both GCK and GCKR were significantly downregulated in patients with advanced steatosis (grade 2+3) (D). (* = p<0.05; ** = p<0.01).

Figure 6. KLF6-FL falls in hepatocytes with advanced steatosis, but increases in inflammatory and sinusoidal cells

KLF6 expression detected by the 1A9 monoclonal (all isoforms) is evident in hepatocytes and non-parenchymal cells in normal liver (Ai). A fall in hepatocytes in the presence of steatosis is contrary to an increase in inflammatory and sinusoidal cells (Bi+Ci). In contrast, KLF6-SV1 increases preferentially in hepatocytes in the presence of more advanced steatosis (Aii–Cii). These changes in representative sections parallel mRNA data in Figure 5, as do changes in GCK and GCKR. Both are present in normal liver (Aiii+iv), with notable nuclear localisation in this fasted state. The pattern is similar but less dramatic in the presence of fat grade 1 simple steatosis (Biii+iv). In the presence of more severe steatosis, expression of both is notably reduced (Ciii+iv). Quantification of nuclear expressions have been performed using imaging software and these additional data are included as Supplementary Figures.

Figure 7. Proposed Mechanisms for KLF6 Regulation of Hepatic Glucose Control

KLF6 is a ubiquitously expressed immediate early gene expressed in response to stressful stimuli. It is regulated by alternative splicing to dominant negative splice forms. The KLF6-IVS1-27A polymorphism favours alternative splicing. In the obesity related metabolic syndrome, fat accumulates in hepatocytes. While KLF6 expression increases in inflammatory and non-parenchymal cells in NASH, it falls in fat laden hepatocytes. Consequently expression of its transcriptional target GCK falls, leading to reduced GCK protein in hepatocytes, an increase in hepatic insulin resistance and an elevated plasma glucose. Importantly, however, the activity of the GCK that is expressed is determined by its localisation – being active in the cytoplasm, but inactive in the nucleus. GCKR is its negative regulator, responsible for its nuclear sequestration. A relative increase in KLF6-SV1 expression in steatotic hepatocytes is associated with a fall in GCKR, which we propose facilitates GCK activity, antagonising the KLF6-FL (or wt genotype) association with elevated serum glucose and HepIR.