Am J Physiol Endocrinol Metab 2014 June 1; 306 (11): E1225-38

Glucotoxicity targets hepatic glucokinase in Zucker diabetic fatty rats, a model of type 2 diabetes associated with obesity

Kiichiro Ueta1, Tracy P. O’Brien1, Gregory A. McCoy1, Kuikwon Kim1, Erin C. Healey1, Tiffany D. Farmer2, E Patrick Donahue2, Audree B. Condren1, Richard L. Printz1, 2, and Masakazu Shiota1, 2

1Department of Molecular Physiology and Biophysics, 2Diabetes Research and Training Center, Vanderbilt University School of Medicine, Nashville, TN 37232-0615

 

Abstract:

A loss of glucose effectiveness to suppress hepatic glucose production as well as increase hepatic glucose uptake and storage as glycogen is associated with a defective increase in glucose phosphorylation catalyzed by glucokinase (GK) in Zucker diabetic fatty (ZDF) rats. We extended these observations by investigating the role of persistent hyperglycemia (glucotoxicity) in the development of impaired hepatic GK activity in ZDF rats. We measured expression and localization of GK and GK-regulatory protein (GKRP), translocation of GK, and hepatic glucose flux in response to a gastric mixed meal load (MMT) and hyperglycemic-hyperinsulinemic clamp after 1 or 6 wk of treatment with the sodium-glucose transporter 2 inhibitor (canaglifrozin) that was used to correct the persistent hyperglycemia of ZDF rats. Defective augmentation of glucose phosphorylation in response to a rise in plasma glucose in ZDF rats was associated with the coresidency of GKRP with GK in the cytoplasm in the midstage of diabetes, which was followed by a decrease of GK protein levels due to impaired post-transcriptional processing in the late stage of diabetes. Correcting hyperglycemia from the middle diabetic stage normalized the rate of glucose phosphorylation by maintaining GK protein levels, restoring normal nuclear residency of GK and GKRP under basal conditions and normalizing translocation of GK from the nucleus to the cytoplasm with GKRP remaining in the nucleus in response to a rise in plasma glucose. This improved the liver’s metabolic ability to respond to hyperglycemic-hyperinsulinemia. Glucotoxicity is responsible for loss of glucose effectiveness and is associated with altered GK regulation in the ZDF rats.

 

Supplement:  

Type 2 diabetes mellitus (T2DM) is a disease characterized by persistent and progressive deterioration of glucose tolerance that is associated with loss of glucose effectiveness and development of insulin resistance. The effectiveness of glucose refers to the ability of glucose per se to function as a negative feedback regulator in determination of blood glucose levels. In non-diabetic subjects, an acute rise in plasma glucose, via an increased phosphorylation of glucose per se, increases insulin secretion from pancreatic beta cells and exerts an inhibitory effect on glucose production while stimulating glucose uptake. On the other hand, patients with established T2DM fail to respond normally to elevated plasma glucose and/or insulin, resulting in impaired glucose uptake in peripheral tissues and a failure to suppress net hepatic glucose production, both of which contribute to fasting hyperglycemia. In addition, a defect in splanchnic glucose uptake, accompanied by reduced hepatic glycogen synthesis, contributes to excessive postprandial hyperglycemia. With a progressive loss of the ability of insulin and glucose to effectively regulate hepatic glucose flux, glycemic control deteriorates and the severity of diabetes increases.

GK catalyzes glucose phosphorylation and plays a critical role in glucose effectiveness in the liver. The glucose-induced suppression of net hepatic glucose production and increase in glycogen synthesis are associated with an augmentation of flux through GK. Under low glucose concentrations, the binding of GKRP to GK inhibits GK activity by decreasing the affinity of the enzyme for glucose and sequesters GK in the nucleus. An increase in glucose concentration dissociates GK from GKRP in a competitive manner and accelerates the translocation of GK from the nucleus to the cytoplasm where glucose phosphorylation is catalyzed. This provides a mechanism for the acute effectiveness of glucose in regulating hepatic glucose flux. Insulin accelerates the glucose effect and in addition regulates GK protein levels at the transcriptional level. In studies of patients with T2DM, defective splanchnic glucose uptake and hepatic glycogen synthesis in response to hyperglycemia were associated with defective glucose phosphorylation.

In ZDF rats, a widely used genetic model of obese-T2DM, defective suppression of net hepatic glucose production and failure to increase flux from glucose to glycogen in response to a rise in plasma glucose and insulin is associated with defective augmentation of glucose phosphorylation resulting from impaired dissociation of GK from GKRP and subsequent decreased translocation of the enzyme from the nucleus to the cytoplasm in the early diabetic stage (1, 2), abnormal relocation of GKRP to the cytoplasm in the presence of hyperglycemia during the middle stage (3, 4) and a progressive reduction of hepatic GK protein levels in the later stage of the disease (5). Therefore, defective glucose effectiveness on hepatic glucose flux in T2DM is likely associated with impaired GK activity. The present study provides the mechanism for the progressive deterioration of hepatic GK activity in ZDF rats. We demonstrated that correction of persistent hyperglycemia by initiating treatment with the renal sodium-glucose transporter 2 (SGLT2) inhibitor, which inhibits the reabsorption of glucose in the kidney, in the middle stage of diabetes normalized all three different types of impairments of GK activity that occur in the liver during progression of the disease. These are a defect of glucose-induced GK translocation from the nucleus to the cytoplasm, abnormal level of intracellular residency of GKRP in the cytoplasm, and an eventual reduction of GK protein level. Reducing hyperglycemia corrected the defect in the suppression of net hepatic glucose production and the rate of hepatic glycogen synthesis in response to hyperglycemic-hyperinsulinemia. This was associated with restored augmentation of glucose phosphorylation resulting from maintaining GK protein expression and the normalization of GK translocation in response to hyperglycemia. These results suggest that glucotoxicity alters GK regulation and expression, which leads to an inability of liver to detect elevated plasma glucose and thereby a loss in the effectiveness of glucose to regulate liver glucose flux in T2DM.

Since hyperinsulinemia and insulin resistance precede and predict the subsequent development of T2DM, chronic hyperglycemia is unlikely to be the cause of the initial insulin resistance. However, once diabetes has developed, the chronic existence of hyperglycemia (glucotoxicity) may contribute to the vicious cycle, which worsens insulin resistance and glucose effectiveness, and may lead to diabetic complications. Therefore, breaking the vicious cycle by correcting hyperglycemia with a SGLT2 inhibitor is likely to be an attractive strategy to ameliorate diabetes. On the other hand, since in insulin-resistant diabetic patients and animals, hyperglycemia compensates for insulin resistance and glucose intolerance to allow utilization and storage of absorbed glucose at near normal levels, one might be concerned that the SGLT2 inhibitor strategy, which normalizes hyperglycemia by discarding glucose into urine, might impair carbohydrate storage in such patients. Interestingly, contrary to such expectations, ZDF rats treated with SGLT2 inhibitor did not exhibit a symptom of glucose deficiency, such as increased lipid oxidation or decreased glycogen storage, because of the concomitant improvement in glucose tolerance due to the elimination of glucotoxicity.

 

References:

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