Although NAFLD prevalence is increasing in Western societies, established predictors of disease progression to advanced stages are scarce (2
). We previously demonstrated that the functional KLF6-IVS1-27G>A
polymorphism correlates inversely with disease progression in NAFLD. Individuals with KLF6-IVS1-27G>A
were less likely to have significant fibrosis when compared to those with the wildtype allele (5
). In the work presented here, we have functionally linked KLF6 to components of the hepatic insulin response, which is central the progression of NAFLD.
The association between the KLF6 genotype groups and FPG is novel and striking. While the KLF6-IVS1-27A allele was also associated with a lower FPI, the euglycemic clamp data discount peripheral insulin resistance as the cause of the variation with genotype. Together, the OGIS and OGTT derived data and absence of a difference in pancreatic beta cell insulin secretion, support increased hepatic clearance of insulin and increased hepatic insulin sensitivity in KLF6-IVS1-27A individuals. This was subsequently confirmed by assessing endogenous glucose production relative to fasting insulin levels.
IR is an established key feature of the metabolic syndrome and increased resistance associated with fasting hyperglycaemia is believed to contribute to hepatic steatosis in obese individuals (29
). There is evidence, however, that insulin resistance is not essential for the development of steatosis. In hypobetalipoproteinaemia, for example, fatty liver develops because of defective VLDL export in the absence of IR (30
). Furthermore, while a fatty liver GIWAS study identified the rs738409 C>G single nucleotide polymorphism in PNPLA3 - which leads to a missense mutation (I148M) - as associated with increased liver fat, it is not associated with IR.(3
) Additional GWAS data in patients with fatty liver disease has further highlighted our incomplete understanding of the relationship between fatty liver disease and features of the metabolic syndrome. While some of the steatosis-associated variations of candidate genes are associated with high triglycerides and plasma LDL-cholesterol, for example, the opposite is true of other variants.(33
) Furthermore, the PP1R3B and GCKR polymorphisms are associated with a lower rather than a higher fasting glucose, in keeping with a relatively enhanced, rather than reduced, sensitivity to insulin. Therefore, while IR indisputably contributes to the progression of NAFLD to fibrosis and cirrhosis, its role in the development of steatosis is not as clear. Rather than being indicative of disease severity, in some individuals steatosis may instead be a biomarker of an enhanced ability to convert glucose to hepatic fat, conferring protection from an elevated blood glucose and reflecting enhanced insulin sensitivity.
Our own study has focused on GCK, whose expression in the liver is closely associated with hepatic insulin sensitivity (20
). In conjunction with GCKR it is a major determinant of insulin responsive hepatic glucose metabolism, catalyzing the production of glucose-6-phosphate (23
). While the hepatic expression of GCK protein is reduced in cirrhosis attributed to both alcohol excess and primary biliary cirrhosis (35
), its contribution to NAFLD has not previously been characterised. GCK’s vital role in determining blood glucose is underscored by the discoveries of over 600 hereditary mutations of GCK associated with glycemic disease (37
). Subjects with a single inactivating mutant allele have a mild form of diabetes, associated with elevated FPG (39
). Furthermore, recent data suggests a role for KLF6 in response to glucose-stimulation (41
). GCK activity is closely linked to the abundance of its regulator, GCKR, and small changes in the molar ratio of GCK/GCKR protein markedly impact hepatic glucose metabolism (42
). In the fasting state GCK is sequestered in an inactive state in the nucleus, bound to GCKR. However, after a meal, glucose and insulin act synergistically in causing rapid dissociation of GCK from GCKR and translocation to the cytoplasm (43
Our in vivo
and in vitro
murine data demonstrate that down-regulation of Klf6
in hepatocytes is associated with reduced Gck
, whereas Klf6
over-expression increases Gck
. ChIP and reporter studies confirm direct transactivation of the Gck
promoter by Klf6
, leading us to propose Klf6
as an additional mediator of glucose homeostasis. The normal phenotype of DeltaKlf6 mice is consistent with published data from liver specific Gck
knockout mice as well as Gckr
knockout mice, in which no differences in fasting glucose levels were detected (44
). Biopsy studies from patients with histologically scored NAFLD confirm a significant association between KLF6-FL
mRNA expression in human tissues. While, the significant reductions of KLF6-FL and GCK in individuals with more advanced versus mild steatosis may simply be representative of more advanced disease, they may also represent effectors of the development of resistance of hepatocytes to insulin.
In human tissues, several alternative splice forms of KLF6
have been identified, and the presence of the KLF6-IVS1-27G>A
allele promotes their generation (5
). To date Klf6
splicing in the mouse has not been described, precluding our ability to study the effects of murine dominant negative splice variants on Gck
in an in vivo
model. The mechanism underlying the enhanced hepatic insulin sensitivity in human individuals with the KLF6-SV1-promoting KLF6-IVS1-27G>A
SNP is presently unknown. However, our expression data identifying a highly significant negative correlation between KLF6-SV1 and GCKR suggests that antagonism of GCKR, the negative regulator of GCK, is one potential mechanism. Here we are able to draw a link to the recently published data on SNPs in GCKR. The GCKR rs780094 GWAS identified SNP (34
) was previously associated with higher triacylglycerol, reduced insulin levels and a reduced risk of type 2 diabetes.(48
) GCKR rs780094 is commonly inherited with a GCKR coding SNP, rs1260326 (Pro446Leu), which codes for a mutant and inactive form of GCKR.(50
) The consequences include enhanced GCK activity and reduced FPG, as well as increased de novo lipogenesis attributed to enhanced production of malonyl coA, the substrate for fatty acid synthesis.(51
) Similar to this phenotypically altered GCKR variant, we hypothesize that KLF6-SV1 contributes to the lowering of FPG as a result of increased GCK activity brought about by the antagonism of GCKR, as summarised in .
Proposed Mechanisms for KLF6 Regulation of Hepatic Glucose Control
In conclusion, we propose KLF6 as an additional regulator of fasting plasma glucose and hepatic insulin sensitivity. Understanding the interactions between KLF6 and KLF6-SV1, as well as their roles in regulation of expression of GCK and GCKR, may help us to clarify the role(s) of NAFLD in the metabolic syndrome, as well as its progression to more advanced disease.