Bárbara Maiztegui

Changes induced by a fructose-rich diet on hepatic metabolism and the antioxidant system

AIMS The effect of a three-week fructose-rich diet (FRD) upon gene expression, protein and activity levels of liver antioxidant system and carbohydrate metabolism was studied. MAIN METHODS Serum glucose (fasting and after a glucose load), triglyceride and insulin levels of normal male Wistar rats were measured. In liver, we measured gene/protein expression and enzyme activity of catalase (CAT), copper-zinc-superoxide dismutase (CuZnSOD) and glutathione peroxidase (GSHPx); reduced glutathione (GSH); protein carbonyl content; thiobarbituric acid reactive substances (TBARS) content and microsomal membrane susceptibility to lipid peroxidation; glucokinase (GK), glucose-6-phosphatase (G-6-Pase) and glucose-6-phosphate dehydrogenase (G-6-PDH) activity; and glycogen, pyruvate, lactate and triglyceride content. KEY FINDINGS Similar body weights and caloric intake were recorded in both groups. FRD rats had higher serum glucose, insulin and triglyceride levels, molar insulin:glucose ratio, HOMA-IR values and impaired glucose tolerance, whereas CAT, CuZnSOD and GSHPx relative gene expression levels were significantly lower. CAT and CuZnSOD protein expression, CAT activity and GSH content were also lower, while protein carbonyl content was higher. No differences were recorded in CuZnSOD, MnSOD and GSHPx activity, TBARS content and membrane susceptibility to lipid peroxidation. Glycogen, lactate and triglyceride content and GK, G-6-Pase and G-6-PDH activity were significantly higher in FRD rats. SIGNIFICANCE In the presence of oxidative stress, the liver exhibits changes in the carbohydrate and lipid metabolic pathways that would decrease reactive oxygen species production and their deleterious effect, thus inducing little impact on specific antioxidant mechanisms. This knowledge could facilitate the design and implementation of strategies to prevent oxidative stress-induced liver damage.

Islet NADPH oxidase activity modulates β-cell mass and endocrine function in rats with fructose-induced oxidative stress.

BACKGROUND Islet NADPH oxidase activity is modulated by glucose and other insulin secretagogues and it might be part of the regulatory mechanism of insulin secretion. We studied its modulatory role of islet NADPH oxidase upon β-cell function in rats with fructose-induced oxidative stress. METHODS Normal rats were fed for 3weeks with a standard diet, a fructose-rich diet or both diets plus apocynin. We measured plasma glucose, insulin, triacylglycerol and lipid peroxidation levels and the homeostasis model assessment-insulin resistance (HOMA-IR) and HOMA-β indexes, and performed an oral glucose tolerance test. β-cell volume density and the number of islets per mm(2) were determined by immunomorphometric analysis of the pancreas. Insulin secretion, glucose metabolism, glucokinase and NADPH oxidase activities were studied in islets isolated from each experimental group. RESULTS Fructose-fed rats had increased plasma triacylglycerol, insulin and lipid peroxidation levels associated with an insulin resistance state; the reactive higher secretion was unable to cope with the increased demand of insulin, leading to an impaired glucose tolerance. They also have a lower number of islets per area unit with a decreased β-cell volume density. All these alterations were prevented by blocking NADPH oxidase activity with apocynin. CONCLUSION Fructose-induced changes are partly mediated by modulation of NADPH oxidase activity. GENERAL SIGNIFICANCE The metabolic dysfunctions and enhanced oxidative stress measured in fructose-fed rats resemble those recorded in human prediabetes; thus, successful strategies employed in this model could be later used to prevent the progression of this state towards type 2 diabetes in human beings.

Role Of Islet Glucokinase, Glucose Metabolism, And Insulin Pathway In The Enhancing Effect Of Islet Neogenesis-associated Protein On Glucose-induced Insulin Secretion

Objective To demonstrate the role of islet glucokinase, glucose metabolism, and intracellular insulin mediators in the enhancing effect of islet neogenesis-associated protein pentadecapeptide (INGAP-PP) on glucose-induced insulin secretion. Methods Islets from normal rats were cultured for 4 days in the absence or presence of 10 &mgr;g/mL INGAP-PP, with/without Wortmannin or LY294002. Islets were incubated with different glucose concentrations to measure insulin secretion and content, hexokinase and glucokinase activity, glucose oxidation and utilization, glucokinase, insulin receptor, insulin receptor substrate (IRS)-1/2, and PI3K concentration and phosphorylation. Results The INGAP-PP significantly increased insulin release at high but not at low glucose concentration, glucokinase activity, glucose metabolism, glucokinase, insulin receptor, IRS-2 and PI3K protein concentration, insulin receptor and IRS-1/2 tyrosine phosphorylation, and the association of p85 with IRS-1. Wortmannin and LY294002 blocked INGAP-PP effect on insulin secretion and glucokinase protein levels in a dose-dependent manner. Conclusions The enhancing effect of INGAP-PP on glucose-induced insulin release could be partly ascribed to its effect on glucokinase activity and glucose metabolism and is mainly mediated by the PI3K/AKT pathway. These results, together with the low hypoglycemia risk associated with the use of INGAP-PP, offer a new alternative for diabetes prevention and treatment.

Islet neogenesis-associated protein (INGAP)-positive cell mass, β-cell mass, and insulin secretion: their relationship during the fetal and neonatal periods

Objectives To study the chronological appearance of pancreatic islet neogenesis–associated protein (INGAP)-positive cells and its correlation with the increase in &bgr;-cell mass and function in fetal and neonatal rats. Methods Normal Wistar rat embryos (E) at gestational days 15, 17, and 19 (E15, E17, E19) and 7-day-old postnatal rats (P7) were humanely killed to determine body and pancreas weight; blood glucose; glucose and arginine-induced insulin secretion; real-time polymerase chain reaction of Pdx1 and Ngn3; quantitative immunomorphometric analysis of &bgr;-cell replication and apoptosis rate, cytokeratin and INGAP cell mass, and Pdx-1– and Ngn3-positive cells. Results Body and pancreas weight increased with age (P7 > E19 > E17 > E15; P < 0.05). Neonates had higher blood glucose concentrations than embryos (P < 0.05). We recorded a simultaneous and significant age-dependent trend of increase in the number of &bgr;- and Pdx-1-positive cells, &bgr;- and cytokeratin-positive cell mass and &bgr;-cell capacity to release insulin in response to glucose and arginine, and decreased &bgr;-cell apoptotic rate. These changes closely paralleled the increase in INGAP-positive cell mass. Conclusions These findings suggest that INGAP exerts a positive modulatory effect on &bgr;-cell mass and its secretory function in fetal and neonatal rats, thus becoming a new component in the multifactorial regulation of such processes.

Liver carbohydrates metabolism: A new islet-neogenesis associated protein peptide (INGAP-PP) target

HighlightsINGAP‐PP significantly increases liver glucose metabolism.INGAP‐PP effects were possibly mediated by P‐Akt signaling pathway.INGAP‐PP might become an effective pharmacological tool to treat people with T2D. &NA; Islet‐Neogenesis Associated Protein‐Pentadecapeptide (INGAP‐PP) increases &bgr;‐cell mass and enhances glucose and amino acids‐induced insulin secretion. Our aim was to demonstrate its effect on liver metabolism. For that purpose, adult male Wistar rats were injected twice‐daily (10 days) with saline solution or INGAP‐PP (250 &mgr;g). Thereafter, serum glucose, triglyceride and insulin levels were measured and homeostasis model assessment (HOMA‐IR) and hepatic insulin sensitivity (HIS) were determined. Liver glucokinase and glucose‐6‐phosphatase (G‐6‐Pase) expression and activity, phosphoenolpyruvate carboxykinase (PEPCK) expression, phosphofructokinase‐2 (PFK‐2) protein content, P‐Akt/Akt and glycogen synthase kinase‐3&bgr; (P‐GSK3/GSK3) protein ratios and glycogen deposit were also determined. Additionally, glucokinase activity and G‐6‐Pase and PEPCK gene expression were also determined in isolated hepatocytes from normal rats incubated with INGAP‐PP (5 &mgr;g/ml). INGAP‐PP administration did not modify any of the serum parameters tested but significantly increased activity of liver glucokinase and the protein level of its cytosolic activator, PFK‐2. Conversely, INGAP‐PP treated rats decreased gene expression and enzyme activity of gluconeogenic enzymes, G‐6‐Pase and PEPCK. They also showed a higher glycogen deposit and P‐GSK3/GSK3 and P‐Akt/Akt ratio. In isolated hepatocytes, INGAP‐PP increased GK activity and decreased G‐6‐Pase and PEPCK expression. These results demonstrate a direct effect of INGAP‐PP on the liver acting through P‐Akt signaling pathway. INGAP‐PP enhances liver glucose metabolism and deposit and reduces its production/output, thereby contributing to maintain normal glucose homeostasis. These results reinforce the concept that INGAP‐PP might become a useful tool to treat people with impaired islet/liver glucose metabolism as it occurs in T2D.

Impaired endocrine-metabolic homeostasis: underlying mechanism of its induction by unbalanced diet

To characterize the intrinsic mechanism by which sucrose induces β-cell dysfunction. Normal rats received for 3 weeks a standard diet supplemented with 10% sucrose in the drinking water (high sucrose (HS)) with/out an antioxidant agent (R/S α-lipoic acid). We measured plasma glucose, insulin, triglyceride, leptin, and lipid peroxidation levels; homeostasis model assessment (HOMA)-insulin resistance (HOMA-IR) and HOMA for β-cell function (HOMA-β) indexes were also determined. Insulin secretion, β-cell apoptosis, intracellular insulin and leptin mediators, and oxidative stress (OS) markers were also measured in islets isolated from each experimental group. HS rats had increased plasma triglyceride, insulin, leptin, and lipid peroxidation (OS marker) levels associated with an insulin-resistant state. Their islets developed an initial compensatory increase in glucose-induced insulin secretion and mRNA and protein levels of β-cell apoptotic markers. They also showed a significant decrease in mRNA and protein levels of insulin and leptin signaling pathway mediators. Uncoupling protein 2 (UCP2), peroxisome proliferator-activated receptor (PPAR)-α and -δ mRNA and protein levels were increased whereas mRNA levels of Sirtuin-1 (Sirt-1), glutathione peroxidase, and catalase were significantly lower in these animals. Development of all these endocrine-metabolic abnormalities was prevented by co-administration of R/S α-lipoic acid together with sucrose. OS may be actively involved in the mechanism by which unbalanced/unhealthy diet induces β-cell dysfunction. Since metabolic-endocrine dysfunctions recorded in HS rats resembled those measured in human pre-diabetes, knowledge of its molecular mechanism could help to develop appropriate strategies to prevent the progression of this metabolic state toward type 2 diabetes (T2D).