ChREBP Regulates Fructose-induced Glucose Production Independently of Insulin Signaling

In a study published in the Journal of Clinical Investigation (see attached), researchers suggest that ChREBP, a transcriptional activator of glycolytic and lipogenic genes, modulates selective liver insulin sensitivity.   Researchers believe that in insulin resistant states, where glucose is not readily taken up by peripheral tissues, glucose shunting to the liver activates ChREBP and promotes de novo lipogenesis (DNL). Furthermore, it has also been observed that ChREBP stimulates glycolysis through transactivation of glycolytic genes and also contributes to glucose production through transactivation of G6pc, the gene encoding for glucose-6-phosphatase. Below are the findings and summaries of experiments conducted to understand how ChREBP is involved in regulating the response to high carbohydrate intake.

High-fructose feeding activated hepatic ChREBP and induces metabolic disease

Researchers fed mice either a 60% dextrose (HDD), 60% fructose (HFrD), or chow diet for 9 weeks. Weight gain in the HFrD group was significantly higher than that in the control group but was not significantly different from weight gain seen in the HDD group. Adiposity was significantly higher in both the HDD and HFrD groups compared to the control group following the 9 week intervention. Both HDD and HFrD mice showed signs of hepatic steatosis as evidenced by triglyceride levels and hepatocyte histology.  Blood glucose levels were comparable among all three groups. Interestingly, insulin levels were significantly higher in the HFrD group compared to the chow group but were not significantly greater than the HDD group. Researchers suggest this indicates that fructose-fed mice are insulin resistant since mice fed the HFrD experienced a significantly increased “glycemic excursion” when compared to both HDD and chow fed mice. Taken together, researchers conclude that fructose consumption impairs glucose and lipid homeostasis independent of weight, adiposity, and hepatic steatosis.

To test for the involvement of ChREBP in hepatic DNL, researchers measured expression of ChREBP-α, ChREBP-β, and Srebp1c, another transcriptional factor involved in DNL.  Researchers found that ChREBP-α and Srebp1c were unaffected by any of the experimental diets. ChREBP-β, however, was upregulated in both the HDD and HFrD groups with the HFrD showing significantly more expression of ChREBP-β than both the HDD and chow groups. Glycolytic genes (Pklr, Gpi1, and Eno1), lipogenic genes (Fasn, Acly, and Acaca), and fructolytic genes (Aldob, Khk, and Dak) were similarly upregulated with HFrD showing the greatest impact on expression. Interestingly, HFrD but not HDD induced increased expression of gluconeogenic genes G6pc and Slc37a4.

Carbohydrates metabolized by the liver acutely activated ChREBP

Researchers then sought to determine if ChREBP is involved in the observed fructose-induced changes in gene expression. To test this, researchers utilized ChREBP knockout mice (ChKO) that will not voluntarily consume fructose diets; therefore, all experimental carbohydrates were force fed. In these mice, fructose but not glucose increased the expression of ChREBP-β, Fasn, Pklr, Dak, Aldob, and G6pc. Researchers note that ChREBP-α was not affected by fructose feeding and that the nuclear to cytosolic ChREBP-α ratio actually decreased with fructose feeding.

Next the researchers assessed whether activation of ChREBP was responsive to fructose-specific metabolites or whether other carbohydrate metabolites would elicit the same response. Glucokinase activation alone was enough to produce an increase in G6pc expression. Glucokinase activation in conjunction with glucose feeding resulted in enhanced expression of G6pc and ChREBP-β. Similarly, when challenged with glycerol, G6pc and ChREBP-β expression was enhanced. Together, these data suggest that any carbohydrate which can increase the amount of hepatic hexo- and triose-phosphate is likely to activate ChREBP.

ChREBP is necessary for fructose-induced hepatic gene expression and conversion of fructose to glucose

To determine if ChREBP was responsible for fructose-induced gene expression, both wild type (WT) and ChKO mice were force fed water or fructose and changes in hepatic gene expression 100 minutes after consumption were reported. At baseline, ChKO mice had lower levels of G6pc and lipogenic and fructolytic enzymes when compared to their WT counterparts. These were not increased when fed fructose. While the WT had a significantly greater expression of G6pc following fructose consumption, the activity of G6pc was only 50% that of the activity measured in ChKO mice. It was also documented that the ChKO mice had more circulating glucose-6-phosphate (G6P) than WT mice. Authors concluded that this data “suggests a homeostatic model whereby increased hepatocellular hexose-phosphates, and possibly G6P itself, might activate ChREBP to enhance hexose and triose-phosphate disposal.”

Researchers then measured the conversion of fructose to glucose in WT mice. Interestingly, they found that WT mice fed a HFrD for 1 week produced 20% more glucose from fructose than WT mice controls. Conversely, the conversion rate in ChKO mice was reduced 60%. Researchers suggest this is indicative of hepatic adaptive responses to a high fructose diet.

Hexose- and triose-phosphates are globally increased in ChKO mice

Using harvested hepatocytes from ChKO mice who were force fed with either water, glucose, or fructose, researchers report that glycolytic metabolites hexose and triose-phosphates, and fructolytic metabolite fructose-1-phosphate levels were elevated and suggest these data demonstrate that “increased in fructose-specific metabolites are not critical for activation of ChREBP.”

Fructose-induced hepatic G6pc activity and glucose production in vivo

To determine if a HFrD would enhance HPG in vivo, WT mice were fed a either a HFrD or chow for 2 weeks. Glycogen, blood glucose, and insulin levels were not significantly different. The HFrD demonstrated greater HGP with increased G6pc activity. Consistent with the homeostatic model, G6P concentrations decreased with increasing G6pc activity. Researchers were able to conclude that “the increasing HGP and G6pc activity, and decreased G6P levels in high-fructose-fed mice do not appear to be due to overt differences in hepatic insulin sensitivity.”

In ChKO mice, hepatic glycogen and G6P levels were strongly correlated and ChKO mice tended to have greater glycogen storage; independent of glucagon treatment. Low G6P levels caused by elevated G6pc activity could limit insulin’s ability for glycogenolysis, further reducing G6P levels and limiting HGP.

ChREBP regulates G6pc independently of insulin signaling, the ChREBP-G6pc signaling axis in conserved in humans

Lastly, researchers concluded that fructose’s ability to increase G6pc expression is dominant over the ability of insulin and glucose to suppress it. Furthermore, fructose-mediated activation of ChREBP “may contribute to lipogenic gene expression and apparent hepatic insulin resistance at the level of G6pc expression in humans as it does in mice.”

In summary, researchers believe that their evidence suggests that fructose, but not glucose, ingestion increases expression of ChREBP and enzymes involved in glycolysis and fructolysis. Moreover, fructose, but not glucose ingestion, promotes DNL and HGP via G6pc activity in mice.

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