Chronic glucokinase activator treatment activates liver Carbohydrate response element binding protein and improves hepatocyte ATP homeostasis during substrate challenge

Abstract

Aim: To test the hypothesis that glucokinase activators (GKAs) induce hepatic adaptations that alter intra-hepatocyte metabolite homeostasis. Methods: C57BL/6 mice on a standard rodent diet were treated with a GKA (AZD1656) acutely or chronically. Hepatocytes were isolated from the mice after 4 or 8 weeks of treatment for analysis of cellular metabolites and gene expression in response to substrate challenge. Results: Acute exposure of mice to AZD1656 or a liver-selective GKA (PF-04991532), before a glucose tolerance test, or challenge of mouse hepatocytes with GKAs ex vivo induced various Carbohydrate response element binding protein (ChREBP) target genes, including Carbohydrate response element binding protein beta isoform (ChREBP-β), Gckr and G6pc. Both glucokinase activation and ChREBP target gene induction by PF-04991532 were dependent on the chirality of the molecule, confirming a mechanism linked to glucokinase activation. Hepatocytes from mice treated with AZD1656 for 4 or 8 weeks had lower basal glucose 6-phosphate levels and improved ATP homeostasis during high substrate challenge. They also had raised basal ChREBP-β mRNA and AMPK-α mRNA (Prkaa1, Prkaa2) and progressively attenuated substrate induction of some ChREBP target genes and Prkaa1 and Prkaa2. Conclusions: Chronic GKA treatment of C57BL/6 mice for 8 weeks activates liver ChREBP and improves the resilience of hepatocytes to compromised ATP homeostasis during high-substrate challenge. These changes are associated with raised mRNA levels of ChREBP-β and both catalytic subunits of AMP-activated protein kinase.

The Protective Role of the Carbohydrate Response Element Binding Protein in the Liver: The Metabolite Perspective

“…In mouse adipose tissue ChREBP-β was more responsive to overnight fasting and refeeding and to overexpression or downregulation of Glut4 ( 15 ). In mouse liver, dietary glucose or fructose and glucokinase activator (GKA) drugs caused 4-8-fold elevation in ChREBP-β mRNA with little change in ChREBP-α mRNA, with similar responses in mouse hepatocytes challenged with sugars and GKAs ( 23 , 24 ). Interestingly in rats, fasting and refeeding caused a 40-fold increase in ChREBP-β ( 25 ).…”

“…Interestingly in rats, fasting and refeeding caused a 40-fold increase in ChREBP-β ( 25 ). It is noteworthy that Gck mRNA also shows several-fold larger changes in rat compared with mouse hepatocytes ( 24 , 26 , 27 ) but whether this is due to intrinsic species differences or to differences in the severity of nutritional state remains unclear.…”

“…Acetylation is dependent on the level of substrate, acetyl-CoA, and deacetylation on the level of NAD + and thereby on the NAD + /NADH redox state. Raised acetyl-CoA is expected in conditions of substrate overload with high glucose, fructose, xylitol or ethanol, all of which are known to cause ChREBP activation ( 24 , 47 , 48 ). Depletion of NAD + , occurs with reduced substrates such as xylitol and ethanol and is further enhanced when the malate aspartate shuttle which transfers NADH reducing equivalents from the cytoplasm to the mitochondria is inhibited with amino-oxyacetate ( 42 ).…”

“…Depletion of NAD + , occurs with reduced substrates such as xylitol and ethanol and is further enhanced when the malate aspartate shuttle which transfers NADH reducing equivalents from the cytoplasm to the mitochondria is inhibited with amino-oxyacetate ( 42 ). In hepatocytes ChREBP is very strongly activated by xylitol in combination with inhibition of malate aspartate shuttle despite modest elevation in hexose phosphates ( 24 ). The represents an analogous metabolic state as occurs with ethanol ( 47 , 48 ) supporting a potential role for acetylation in mediating the effects of other reduced substrates like xylitol.…”

“…In this context glucokinase activators (GKA) mimic the effect of micromolar fructose ( 85 ). Recent work exploring the chronic effects of a glucokinase activator on the liver provided evidence for activation of ChREBP as determined from raised levels of ChREBP-β and for improved ATP homeostasis in the isolated hepatocytes from the mice when challenged ex vivo with either xylitol or high glucose in combination with metabolic inhibitors ( 24 ). Paradoxically, improved ATP homeostasis in conditions of high substrate challenge, occurred despite sustained elevation in phosphate esters ( 24 ).…”