Romina Bertinat

Broad expression of fructose-1,6-bisphosphatase and phosphoenolpyruvate carboxykinase provide evidence for gluconeogenesis in human tissues other than liver and kidney.

The importance of renal and hepatic gluconeogenesis in glucose homeostasis is well established, but the cellular localization of the key gluconeogenic enzymes liver fructose‐1,6‐bisphosphatase (FBPase) and cytosolic phosphoenolpyruvate carboxykinase (PEPCK) in these organs and the potential contribution of other tissues in this process has not been investigated in detail. Therefore, we analyzed the human tissue localization and cellular distribution of FBPase and PEPCK immunohistochemically. The localization analysis demonstrated that FBPase was expressed in many tissues that had not been previously reported to contain FBPase activity (e.g., prostate, ovary, suprarenal cortex, stomach, and heart). In some multicellular tissues, this enzyme was detected in specialized areas such as epithelial cells of the small intestine and prostate or lung pneumocytes II. Interestingly, FBPase was also present in pancreas and cortex cells of the adrenal gland, organs that are involved in the control of carbohydrate and lipid metabolism. Although similar results were obtained for PEPCK localization, different expression of this enzyme was observed in pancreas, adrenal gland, and pneumocytes type I. These results show that co‐expression of FBPase and PEPCK occurs not only in kidney and liver, but also in a variety of organs such as the small intestine, stomach, adrenal gland, testis, and prostate which might also contribute to gluconeogenesis. Our results are consistent with published data on the expression of glucose‐6‐phosphatase in the human small intestine, providing evidence that this organ may play an important role in the human glucose homeostasis. J. Cell. Physiol. 197: 189–197, 2003© 2003 Wiley‐Liss, Inc.

Different involvement for aldolase isoenzymes in kidney glucose metabolism: Aldolase B but not aldolase A colocalizes and forms a complex with FBPase

The expression of aldolase A and B isoenzyme transcripts was confirmed by RT‐PCR in rat kidney and their cell distribution was compared with characteristic enzymes of the gluconeogenic and glycolytic metabolic pathway: fructose‐1,6‐bisphosphatase (FBPase), phosphoenol pyruvate carboxykinase (PEPCK), and pyruvate kinase (PK). We detected aldolase A isoenzyme in the thin limb and collecting ducts of the medulla and in the distal tubules and glomerula of the cortex. The same pattern of distribution was found for PK, but not for aldolase B, PEPCK, and FBPase. In addition, co‐localization studies confirmed that aldolase B, FBPase, and PEPCK are expressed in the same proximal cells. This segregated cell distribution of aldolase A and B with key glycolytic and gluconeogenic enzymes, respectively, suggests that these aldolase isoenzymes participate in different metabolic pathways. In order to test if FBPase interacts with aldolase B, FBPase was immobilized on agarose and subjected to binding experiments. The results show that only aldolase B is specifically bound to FBPase and that this interaction was specifically disrupted by 60 μM Fru‐1,6‐P2. These data indicate the presence of a modulated enzyme–enzyme interaction between FBPase and isoenzyme B. They affirm that in kidney, aldolase B specifically participates, along the gluconeogenic pathway and aldolase A in glycolysis.

Altered expression and localization of insulin receptor in proximal tubule cells from human and rat diabetic kidney

Diabetes is the major cause of end stage renal disease, and tubular alterations are now considered to participate in the development and progression of diabetic nephropathy (DN). Here, we report for the first time that expression of the insulin receptor (IR) in human kidney is altered during diabetes. We detected a strong expression in proximal and distal tubules from human renal cortex, and a significant reduction in type 2 diabetic patients. Moreover, isolated proximal tubules from type 1 diabetic rat kidney showed a similar response, supporting its use as an excellent model for in vitro study of human DN. IR protein down‐regulation was paralleled in proximal and distal tubules from diabetic rats, but prominent in proximal tubules from diabetic patients. A target of renal insulin signaling, the gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK), showed increased expression and activity, and localization in compartments near the apical membrane of proximal tubules, which was correlated with activation of the GSK3β kinase in this specific renal structure in the diabetic condition. Thus, expression of IR protein in proximal tubules from type 1 and type 2 diabetic kidney indicates that this is a common regulatory mechanism which is altered in DN, triggering enhanced gluconeogenesis regardless the etiology of the disease. J. Cell. Biochem. 114: 639–649, 2013.

Nuclear localization of liver FBPase isoenzyme in kidney and liver

Nuclear localization has been observed for glycolytic enzymes but not for key gluconeogenic enzymes. We report our findings on the intracellular localization of liver FBPase in rat liver and kidney, the main organs in the endogenous glucose production. Immunofluorescence and confocal analysis revealed that FBPase was present in the cytosol and, unexpectedly, inside the nucleus of hepatocytes and proximal cells of the nephron. Additionally, FBPase was found in the plasma membrane area of adjacent hepatocytes where glycogen is synthesized and in the apical region of proximal kidney cells. This subcellular distribution in multiple compartments suggests the presence of different localization signals on FBPase for diverse metabolic functions.

Subcellular localization of liver FBPase is modulated by metabolic conditions.

In primary cultured hepatocytes, fructose‐1,6‐bisphosphatase (FBPase) localization is modulated by glucose, dihydroxyacetone (DHA) and insulin. In the absence of these substrates, FBPase was present in the cytoplasm, but the addition of glucose or DHA induced its translocation to the nucleus. As expected, we observed the opposite effect of glucose on glucokinase localization. The addition of insulin in the absence of glucose largely increased the amount of nuclear FBPase. Moreover, at high concentrations of glucose or DHA, FBPase shifted from the cytosol to the cell periphery and co‐localized with GS. Interestingly, the synthesis of Glu‐6‐P and glycogen induced by DHA was not inhibited by insulin. These results indicate that FBPase is involved in glycogen synthesis from gluconeogenic precursors. Overall, these findings show that translocation may be a new integrative mechanism for gluconeogenesis and glyconeogenesis.

Novel expression of liver FBPase in Langerhans islets of human and rat pancreas.

Several reports have indicated the absence of gluconeogenic enzymes in pancreatic islet cells. In contrast, here we demonstrate that liver fructose‐1,6‐bisphosphatase (FBPase) is highly expressed both in human and rat pancreas. Interestingly, pancreatic FBPase is active and functional, and is inhibited by AMP and fructose‐2,6‐bisphosphate (Fru‐2,6‐P2). These results suggest that FBPase may participate as a component of a metabolic sensing mechanism present in the pancreas. Immunolocalization analysis showed that FBPase is expressed both in human and rat Langerhans islets, specifically in β cells. In humans, FBPase was also located in the canaliculus and acinar cells. These results indicate that FBPase coupled with phosphofructokinase (PFK) plays a crucial role in the metabolism of pancreatic islet cells. The demonstration of gluconeogenic recycling of trioses as a new metabolic signaling pathway may contribute to our understanding of the differences between the insulin secretagogues trioses, fructose, and glucose in pancreas.

Over-expression of muscle glycogen synthase in human diabetic nephropathy.

Abstract Diabetic nephropathy (DN) is a major complication of diabetic patients and the leading cause of end-stage renal disease. Glomerular dysfunction plays a critical role in DN, but deterioration of renal function also correlates with tubular alterations. Human DN is characterized by glycogen accumulation in tubules. Although this pathological feature has long been recognized, little information exists about the triggering mechanism. In this study, we detected over-expression of muscle glycogen synthase (MGS) in diabetic human kidney. This enhanced expression suggests the participation of MGS in renal metabolic changes associated with diabetes. HK2 human renal cell line exhibited an intrinsic ability to synthesize glycogen, which was enhanced after over-expression of protein targeting to glycogen. A correlation between increased glycogen amount and cell death was observed. Based on a previous transcriptome study on human diabetic kidney disease, significant differences in the expression of genes involved in glycogen metabolism were analyzed. We propose that glucose, but not insulin, is the main modulator of MGS activity in HK2 cells, suggesting that blood glucose control is the best approach to modulate renal glycogen-induced damage during long-term diabetes.

Nuclear accumulation of fructose 1,6-bisphosphatase is impaired in diabetic rat liver

Using a streptozotocin‐induced type 1 diabetic rat model, we analyzed and separated the effects of hyperglycemia and hyperinsulinemia over the in vivo expression and subcellular localization of hepatic fructose 1,6‐bisphosphatase (FBPase) in the multicellular context of the liver. Our data showed that FBPase subcellular localization was modulated by the nutritional state in normal but not in diabetic rats. By contrast, the liver zonation was not affected in any condition. In healthy starved rats, FBPase was localized in the cytoplasm of hepatocytes, whereas in healthy re‐fed rats it was concentrated in the nucleus and the cell periphery. Interestingly, despite the hyperglycemia, FBPase was unable to accumulate in the nucleus in hepatocytes from streptozotocin‐induced diabetic rats, suggesting that insulin is a critical in vivo modulator. This idea was confirmed by exogenous insulin supplementation to diabetic rats, where insulin was able to induce the rapid accumulation of FBPase within the hepatocyte nucleus. Besides, hepatic FBPase was found phosphorylated only in the cytoplasm, suggesting that the phosphorylation state is involved in the nuclear translocation. In conclusion, insulin and not hyperglycemia plays a crucial role in the nuclear accumulation of FBPase in vivo and may be an important regulatory mechanism that could account for the increased endogenous glucose production of liver of diabetic rodents. J. Cell. Biochem. 113: 848–856, 2012.

Expression of key substrate cycle enzymes in rat spermatogenic cells: Fructose 1,6 bisphosphatase and 6 phosphofructose 1-kinase

A substrate cycle composed of phosphofructo 1‐kinase I (PFK) and fructose 1,6 bisphosphatase I (FBPase) has been proposed in rat spermatids. This substrate cycle can explain the ability of glucose to induce a decrease in intracellular ATP, a phenomenon that was related to regulation of [Ca2+]i in these cells. In spite of the importance of this metabolic cycle, the expression and activities of the enzymes that compose such cycle have not been systematically studied in spermatogenic cells. Here, we show that PFK and FBPase activities were present in pachytene spermatocytes and round spermatids extracts. Expression of PFK at the mRNA and protein levels showed a relatively similar expression in spermatogenic cells, but a stronger expression in Sertoli cells. Instead, expression of FBPase at the mRNA and protein levels was stronger in round and elongating spermatids as compared to other spermatogenic cells. A similar pattern was observed when evidencing FBPase activity by a NADPH‐nitroblue tetrazolium‐linked cytochemical assay in isolated pachytene spermatocytes and round spermatids. Rat spermatids also showed the ability to convert lactate to fructose‐ and glucose‐6‐P, indicating that both glycolytic and gluconeogenic fluxes are present in these cells. Our results indicate that a coordinated expression of key substrate cycle enzymes, at the level of PFK/FBPase, appear in the last stages of spermatogenic cell differentiation, suggesting that the co‐regulation of these enzymes are required for the ability of these cells to respond to glucose and induce metabolic and Ca2+ signals that can be important for sperm development and function. J. Cell. Physiol. 212:807–816, 2007.

Preclinical and Clinical Studies for Sodium Tungstate: Application in Humans

Diabetes is a complex metabolic disorder triggered by the deficient secretion of insulin by the pancreatic β-cell or the resistance of peripheral tissues to the action of the hormone. Chronic hyperglycemia is the major consequence of this failure, and also the main cause of diabetic problems. Indeed, several clinical trials have agreed in that tight glycemic control is the best way to stop progression of the disease. Many anti-diabetic drugs for treatment of type 2 diabetes are commercially available, but no ideal normoglycemic agent has been developed yet. Moreover, weight gain is the most common side effect of many oral anti-diabetic agents and insulin, and increased weight has been shown to worsen glycemic control and increase the risk of diabetes progression. In this sense, the inorganic salt sodium tungstate (NaW) has been studied in different animal models of metabolic syndrome and diabetes, proving to have a potent effect on normalizing blood glucose levels and reducing body weight, without any hypoglycemic action. Although the liver has been studied as the main site of NaW action, positive effects have been also addressed in muscle, pancreas, brain, adipose tissue and intestine, explaining the effective anti-diabetic action of this salt. Here, we review NaW research to date in these different target organs. We believe that NaW deserves more attention, since all available anti-diabetic treatments remain suboptimal and new therapeutics are urgently needed.