Glucokinase has a very high flux control coefficient (greater than unity) on glycogen synthesis from glucose in hepatocytes (Agius et al., J. Biol. Chem. 271, 30479 -30486, 1996). Hepatic glucokinase is inhibited by a 68-kDa glucokinase regulatory protein (GKRP) that is expressed in molar excess. To establish the relative control exerted by glucokinase and GKRP, we applied metabolic control analysis to determine the flux control coefficient of GKRP on glucose metabolism in hepatocytes. Adenovirus-mediated overexpression of GKRP (by up to 2-fold above endogenous levels) increased glucokinase binding and inhibited glucose phosphorylation, glycolysis, and glycogen synthesis over a wide range of concentrations of glucose and sorbitol. It decreased the affinity of glucokinase translocation for glucose and increased the control coefficient of glucokinase on glycogen synthesis. GKRP had a negative control coefficient of glycogen synthesis that is slightly greater than unity (؊1.2) and a control coefficient on glycolysis of ؊0.5. The control coefficient of GKRP on glycogen synthesis decreased with increasing glucokinase overexpression (4-fold) at elevated glucose concentration (35 mM), which favors dissociation of glucokinase from GKRP, but not at 7.5 mM glucose. Under the latter conditions, glucokinase and GKRP have large and inverse control coefficients on glycogen synthesis, suggesting that a large component of the positive control coefficient of glucokinase is counterbalanced by the negative coefficient of GKRP. It is concluded that glucokinase and GKRP exert reciprocal control; therefore, mutations in GKRP affecting the expression or function of the protein may impact the phenotype even in the heterozygote state, similar to glucokinase mutations in maturity onset diabetes of the young type 2. Our results show that the mechanism comprising glucokinase and GKRP confers a markedly extended responsiveness and sensitivity to changes in glucose concentration on the hepatocyte.Glucokinase (hexokinase IV) is the predominant glucose phosphorylating enzyme in hepatocytes and insulin-secreting and glucagon-secreting cells of the pancreas and has a major role in the control of blood glucose homeostasis (1, 2). Its importance has been confirmed by the finding that mutations in its gene cause a form of diabetes known as maturity onset diabetes of the young type 2 (MODY-2), 1 which is characterized by mild hyperglycemia, decreased glucose-induced insulin secretion, impaired hepatic glycogen storage, and dominant inheritance (3), and by studies on hepatic glucokinase knockout mice (4). Hepatic glucokinase is Regulated by a 68-kDa regulatory protein (GKRP) (5, 6). Expression of GKRP during development precedes expression of glucokinase (7), and no physiological situations are known in which hepatic glucokinase is expressed in the absence of its regulatory protein. GKRP is located mainly in the nucleus of hepatocytes, whereas glucokinase translocates between the nucleus and the cytoplasm (8). Glucokinase binds GKRP in the nucleus at...

Diabetic nephropathy (DN) is one of the most serious microvascular complications of diabetes. The study aims to evaluate the diagnostic value of serum neutrophil gelatinase-associated lipocalin (NGAL) and retinol-binding protein 4 (RBP4) as biomarkers for early detection of nephropathy in type 2 diabetic patients. The current study was performed on 150 type 2 diabetic patients. These patients were classified into three equal groups according to their albumin/creatinine ratio (ACR), including patients with normoalbuminuria (ACR <30 mg/g creatinine), patients with microalbuminuria (ACR = 30–300 mg/g creatinine), and patients with macroalbuminuria (ACR >300 mg/g creatinine). Fifty apparently healthy subjects matching the same age and socioeconomic status with diabetic subjects were selected as a control group. The plasma glucose, insulin, glycosylated hemoglobin (HbA1c), homeostasis model assessment of insulin resistance (HOMA-IR), lipid profile, urea, creatinine, cystatin C, glomerular filtration rate (GFR), NGAL, and RBP4 were measured in the studied groups. Significantly elevated NGAL and RBP4 levels were observed in micro- and macroalbuminuric diabetic groups when compared to the control and normoalbuminuric diabetic groups. NGAL and RBP4 were found to correlate positively with duration of diabetes, systolic and diastolic blood pressure, glucose, HbA1c, HOMA-IR, triacylglycerol, and ACR, but correlate inversely with GFR in DN groups. Receiver operating characteristic curves revealed that for early detection of DN, the best cutoff values to discriminate DN and diabetic without nephropathy groups were 91.5 ng/mL for NGAL with 87% sensitivity, 74% specificity, and area under the curve (AUC) = 0.881; 24.5 ng/mL for RBP4 with 84% sensitivity, 90% specificity, and AUC = 0.912; and 37.5 mg/g creatinine for ACR with 89% sensitivity, 72% specificity, and AUC = 0.819. RBP4 is more specific (90% specificity) than NGAL (74% specificity) and ACR (72% specificity). Therefore, RBP4 marker may serve as a tool to follow-up clinical monitoring of the development and progression of DN.

The role of glucose 6-P (glucose 6-phosphate) in regulating the activation state of glycogen synthase and its translocation is well documented. In the present study, we investigated the effects of glucose 6-P on the activation state and compartmentation of phosphorylase in hepatocytes. Glucose 6-P levels were modulated in hepatocytes by glucokinase overexpression or inhibition with 5-thioglucose and the effects of AMP were tested using AICAR (5-aminoimidazole-4-carboxamide 1-beta-D-ribofuranoside), which is metabolized to an AMP analogue. Inhibition of glucokinase partially counteracted the effect of glucose both on the inactivation of phosphorylase and on the translocation of phosphorylase a from a soluble to a particulate fraction. The increase in glucose 6-P caused by glucokinase overexpression caused translocation of phosphorylase a to the pellet and had additive effects with glucose on inactivation of phosphorylase. It decreased the glucose concentration that caused half-maximal inactivation from 20 to 11 mM, indicating that it acts synergistically with glucose. AICAR activated phosphorylase and counteracted the effect of glucose 6-P on phosphorylase inactivation. However, it did not counteract translocation of phosphorylase by glucose 6-P. Glucose 6-P and AICAR had opposite effects on the activation state of glycogen synthase, but they had additive effects on translocation of the enzyme to the pellet. There was a direct correlation between the translocation of phosphorylase a and of glycogen synthase to the pellet, suggesting that these enzymes translocate in tandem. In conclusion, glucose 6-P causes both translocation of phosphorylase and inactivation, indicating a more complex role in the regulation of glycogen metabolism than can be explained from regulation of glycogen synthase alone.

Glucokinase is rapidly exported from the nucleus of hepatocytes in response to a rise in glucose or fructose 1-P. We demonstrate using confocal microscopy and quantitative imaging that in contrast to previous findings, the regulatory protein of glucokinase (GKRP) also translocates from the nucleus during substrate-induced translocation of glucokinase. However, the fractional decrease in nuclear GKRP is smaller than for glucokinase and is determined by the metabolic state and not by the distribution of glucokinase. Translocation of glucokinase and GKRP is not inhibited by leptomycin B, an inhibitor of exportin-1 function. These findings highlight the importance of quantitative imaging for determining nuclear export of proteins and suggest that GKRP may have a role in nuclear export or import of glucokinase.z 1999 Federation of European Biochemical Societies.

The rate of glucose phosphorylation in hepatocytes is determined by the subcellular location of glucokinase and by its association with its regulatory protein (GKRP) in the nucleus. Elevated glucose concentrations and precursors of fructose 1-phosphate (e.g., sorbitol) cause dissociation of glucokinase from GKRP and translocation to the cytoplasm. In this study, we investigated the counter-regulation of substrate-induced translocation by AICAR (5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside), which is metabolized by hepatocytes to an AMP analog, and causes activation of AMP-activated protein kinase (AMPK) and depletion of ATP. During incubation of hepatocytes with 25 mM glucose, AICAR concentrations below 200 microM activated AMPK without depleting ATP and inhibited glucose phosphorylation and glucokinase translocation with half-maximal effect at 100-140 microM. Glucose phosphorylation and glucokinase translocation correlated inversely with AMPK activity. AICAR also counteracted translocation induced by a glucokinase activator and partially counteracted translocation by sorbitol. However, AICAR did not block the reversal of translocation (from cytoplasm to nucleus) after substrate withdrawal. Inhibition of glucose-induced translocation by AICAR was greater than inhibition by glucagon and was associated with phosphorylation of both GKRP and the cytoplasmic glucokinase binding protein, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK2) on ser-32. Expression of a kinase-active PFK2 variant lacking ser-32 partially reversed the inhibition of translocation by AICAR. Phosphorylation of GKRP by AMPK partially counteracted its inhibitory effect on glucokinase activity, suggesting altered interaction of glucokinase and GKRP. In summary, mechanisms downstream of AMPK activation, involving phosphorylation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase and GKRP are involved in the ATP-independent inhibition of glucose-induced glucokinase translocation by AICAR in hepatocytes.