María Cecilia Castro

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.

Lipoic acid prevents liver metabolic changes induced by administration of a fructose-rich diet.

BACKGROUND To evaluate whether co-administration of R/S-α-lipoic acid can prevent the development of oxidative stress and metabolic changes induced by a fructose-rich diet (F). METHODS We assessed glycemia in the fasting state and during an oral glucose tolerance test, triglyceridemia and insulinemia in rats fed with standard diet (control) and fructose without or with R/S-α-lipoic acid. Insulin resistance and hepatic insulin sensitivity were also calculated. In liver, we measured reduced glutathione, protein carbonyl groups, antioxidant capacity by ABTS assay, antioxidant enzymes (catalase and superoxide dismutase 1 and 2), uncoupling protein 2, PPARδ and PPARγ protein expressions, SREBP-1c, fatty acid synthase and glycerol-3-phosphate acyltransferase-1 gene expression, and glucokinase activity. RESULTS R/S-α-lipoic acid co-administration to F-fed rats a) prevented hyperinsulinemia, hypertriglyceridemia and insulin resistance, b) improved hepatic insulin sensitivity and glucose tolerance, c) decreased liver oxidative stress and increased antioxidant capacity and antioxidant enzymes expression, d) decreased uncoupling protein 2 and PPARδ protein expression and increased PPARγ levels, e) restored the basal gene expression of PPARδ, SREBP-1c and the lipogenic genes fatty acid synthase and glycerol-3-phosphate acyltransferase, and f) decreased the fructose-mediated enhancement of glucokinase activity. CONCLUSIONS Our results suggest that fructose-induced oxidative stress is an early phenomenon associated with compensatory hepatic metabolic mechanisms, and that treatment with an antioxidant prevented the development of such changes. GENERAL SIGNIFICANCE This knowledge would help to better understand the mechanisms involved in liver adaptation to fructose-induced oxidative stress and to develop effective strategies to prevent and treat, at early stages, obesity and type 2 diabetes mellitus.

Apocynin administration prevents the changes induced by a fructose-rich diet on rat liver metabolism and the antioxidant system.

In the present study, we investigated the role of NADPH oxidase in F (fructose)-rich-diet-induced hepatic OS (oxidative stress) and metabolic changes, and their prevention by apocynin co-administration. Wistar rats were fed for 21 days on (i) a control diet, (ii) a control diet plus 10% F in the drinking water, (iii) a control diet with apocynin in the drinking water (CA) and (iv) F plus apocynin in the drinking water (FA). Glycaemia, triglyceridaemia, NEFAs (non-esterified fatty acids) and insulinaemia were determined. In the liver, we measured (i) NADPH oxidase activity, and gene and protein expression; (ii) protein carbonyl groups, GSH and TBARSs (thiobarbituric acid-reactive substances); (iii) catalase, CuZn-SOD (superoxide dismutase) and Mn-SOD expression; (iv) liver glycogen and lipid content; (v) GK (glucokinase), G6Pase (glucose-6-phosphatase) and G6PDH (glucose-6-phosphate dehydrogenase) activities; (vi) FAS (fatty acid synthase), GPAT (glycerol-3-phosphate acyltransferase), G6Pase and G6PDH, IL-1β (interleukin-1β), PAI-1 (plasminogen-activator inhibitor-1) and TNFα (tumour necrosis factor α) gene expression; and (vii) IκBα (inhibitor of nuclear factor κB α) protein expression. F-fed animals had high serum TAG (triacylglycerol), NEFA and insulin levels, high liver NADPH oxidase activity/expression, increased OS markers, reduced antioxidant enzyme expression, and increased glycogen, TAG storage and GK, G6Pase and G6PDH activities. They also had high G6Pase, G6PDH, FAS, GPAT, TNFα and IL-1β gene expression and decreased IκBα expression. Co-administration of apocynin to F-fed rats prevented the development of most of these abnormalities. In conclusion, NADPH oxidase plays a key role in F-induced hepatic OS production and probably also in the mechanism of liver steatosis, suggesting its potential usefulness for the prevention/treatment of T2DM (Type 2 diabetes mellitus).

Fructose-induced inflammation, insulin resistance and oxidative stress: A liver pathological triad effectively disrupted by lipoic acid.

AIMS Fructose administration induces hepatic oxidative stress, insulin resistance, inflammatory and metabolic changes. We tested their potential pathogenic relationship and whether these alterations can be prevented by R/S-α-lipoic acid. MAIN METHODS Wistar rats received during 21days a commercial diet or the same diet supplemented with 10% fructose in drinking water without/with R/S-α-lipoic acid injection. After this period, we measured a) serum glucose, triglyceride, insulin, homeostasis model assessment-insulin resistance (HOMA-IR), insulin glucose ratio (IGR) and Matsuda indexes and b) liver oxidative stress, inflammatory markers and insulin signaling pathway components. KEY FINDINGS Fructose fed rats had hyperinsulinemia, hypertriglyceridemia, higher HOMA-IR, IGR and lower Matsuda indices compared to control animals, together with increased oxidative stress markers, TNFα, IL1β and PAI-1 gene expression, and TNFα and COX-2 protein content. Whereas insulin receptor level was higher in fructose fed rats, their tyrosine-residue phosphorylation was lower. IRS1/IRS2 protein levels and IRS1 tyrosine-phosphorylation rate were lower in fructose fed rats. All changes were prevented by R/S-α-lipoic acid co-administration. SIGNIFICANCE Fructose-induced hepatic oxidative stress, insulin resistance and inflammation form a triad that constitutes a vicious pathogenic circle. This circle can be effectively disrupted by R/S-α-lipoic acid co-administration, thus suggesting mutual positive interaction among the triad components.

Lipoic acid prevents fructose-induced changes in liver carbohydrate metabolism: Role of oxidative stress

BACKGROUND Fructose administration rapidly induces oxidative stress that triggers compensatory hepatic metabolic changes. We evaluated the effect of an antioxidant, R/S-α-lipoic acid on fructose-induced oxidative stress and carbohydrate metabolism changes. METHODS Wistar rats were fed a standard commercial diet, the same diet plus 10% fructose in drinking water, or injected with R/S-α-lipoic acid (35mg/kg, i.p.) (control+L and fructose+L). Three weeks thereafter, blood samples were drawn to measure glucose, triglycerides, insulin, and the homeostasis model assessment-insulin resistance (HOMA-IR) and Matsuda indices. In the liver, we measured gene expression, protein content and activity of several enzymes, and metabolite concentration. RESULTS Comparable body weight changes and calorie intake were recorded in all groups after the treatments. Fructose fed rats had hyperinsulinemia, hypertriglyceridemia, higher HOMA-IR and lower Matsuda indices compared to control animals. Fructose fed rats showed increased fructokinase gene expression, protein content and activity, glucokinase and glucose-6-phosphatase gene expression and activity, glycogen storage, glucose-6-phosphate dehydrogenase mRNA and enzyme activity, NAD(P)H oxidase subunits (gp91(phox) and p22(phox)) gene expression and protein concentration and phosphofructokinase-2 protein content than control rats. All these changes were prevented by R/S-α-lipoic acid co-administration. CONCLUSIONS Fructose induces hepatic metabolic changes that presumably begin with increased fructose phosphorylation by fructokinase, followed by adaptive changes that attempt to switch the substrate flow from mitochondrial metabolism to energy storage. These changes can be effectively prevented by R/S-α-lipoic acid co-administration. GENERAL SIGNIFICANCE Control of oxidative stress could be a useful strategy to prevent the transition from impaired glucose tolerance to type 2 diabetes.

Ryanodine receptor phosphorylation by CaMKII promotes spontaneous Ca2+ release events in a rodent model of early stage diabetes: The arrhythmogenic substrate

BACKGROUND Heart failure and arrhythmias occur more frequently in patients with type 2 diabetes (T2DM) than in the general population. T2DM is preceded by a prediabetic condition marked by elevated reactive oxygen species (ROS) and subclinical cardiovascular defects. Although multifunctional Ca2+ calmodulin-dependent protein kinase II (CaMKII) is ROS-activated and CaMKII hyperactivity promotes cardiac diseases, a link between prediabetes and CaMKII in the heart is unprecedented. OBJECTIVES To prove the hypothesis that increased ROS and CaMKII activity contribute to heart failure and arrhythmogenic mechanisms in early stage diabetes. METHODS-RESULTS Echocardiography, electrocardiography, biochemical and intracellular Ca2+ (Ca2+i) determinations were performed in fructose-rich diet-induced impaired glucose tolerance, a prediabetes model, in rodents. Fructose-rich diet rats showed decreased contractility and hypertrophy associated with increased CaMKII activity, ROS production, oxidized CaMKII and enhanced CaMKII-dependent ryanodine receptor (RyR2) phosphorylation compared to rats fed with control diet. Isolated cardiomyocytes from fructose-rich diet showed increased spontaneous Ca2+i release events associated with spontaneous contractions, which were prevented by KN-93, a CaMKII inhibitor, or addition of Tempol, a ROS scavenger, to the diet. Moreover, fructose-rich diet myocytes showed increased diastolic Ca2+ during the burst of spontaneous Ca2+i release events. Mice treated with Tempol or with sarcoplasmic reticulum-targeted CaMKII-inhibition by transgenic expression of the CaMKII inhibitory peptide AIP, were protected from fructose-rich diet-induced spontaneous Ca2+i release events, spontaneous contractions and arrhythmogenesis in vivo, despite ROS increases. CONCLUSIONS RyR2 phosphorylation by ROS-activated CaMKII, contributes to impaired glucose tolerance-induced arrhythmogenic mechanisms, suggesting that CaMKII inhibition could prevent prediabetic cardiovascular complications and/or evolution.

Rat liver uncoupling protein 2: changes induced by a fructose-rich diet.

AIM To evaluate the role of uncoupling protein 2 (UCP2) and peroxisome proliferator-activated receptors (PPARs) in the response of liver to glycoxidative stress triggered by administration of a fructose-rich diet (FRD). MAIN METHODS We assessed blood glucose in the fasting state and after a glucose load (glucose-oxidase method), serum triglyceride (enzymatic measurement), insulin (radioimmunoassay), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels (colorimetric kits) in control and FRD animals. In liver, we measured UCP2, PPARα, PPARδ and PPARγ gene (real-time PCR) and protein (Western blot) expression, fatty acid synthase (FAS) and glycerol-3-phosphate acyltransferase (GPAT) gene expression, as well as triglyceride content. KEY FINDINGS Blood glucose, serum insulin and triglyceride levels, homeostasis model assessment of insulin resistance (HOMA-IR) indexes and impaired glucose tolerance were higher in FRD rats. Whereas UCP2 and PPARδ gene and protein expression increased in these animals; PPARγ levels were lower and those of PPARα remained unchanged. FRD also increased the mRNA expression of PPARδ target genes FAS and GPAT. SIGNIFICANCE Our results suggest that a) the increased UCP2 gene and protein expression measured in FRD rats could be part of a compensatory mechanism to reduce reactive oxygen species production induced by the fructose overload, and b) PPARs expression participates actively in the regulation of UCP2 expression, and under the metabolic condition tested, PPARδ played a key role. This knowledge would help to better understand the mechanisms involved in liver adaptation to fructose-induced glycoxidative stress, and to develop appropriate prevention strategies in obesity and type 2 diabetes.

Chronic Glucocorticoid-Rich Milieu and Liver Dysfunction

We investigated the impact of chronic hypercorticosteronemia (due to neonatal monosodium L-glutamate, MSG, and treatment) on liver oxidative stress (OS), inflammation, and carbohydrate/lipid metabolism in adult male rats. We evaluated the peripheral concentrations of several metabolic and OS markers and insulin resistance indexes. In liver we assessed (a) OS (GSH and protein carbonyl groups) and inflammatory (IL-1b, TNFa, and PAI-1) biomarkers and (b) carbohydrate and lipid metabolisms. MSG rats displayed degenerated optic nerves, hypophagia, low body and liver weights, and enlarged adipose tissue mass; higher peripheral levels of glucose, triglycerides, insulin, uric acid, leptin, corticosterone, transaminases and TBARS, and peripheral and liver insulin resistance; elevated liver OS, inflammation markers, and glucokinase (mRNA/activity) and fructokinase (mRNA). Additionally, MSG liver phosphofructokinase-2, glucose-6-phosphatase (mRNA and activity) and glucose-6-phosphate dehydrogenase, Chrebp, Srebp1c, fatty acid synthase, and glycerol-3-phosphate (mRNAs) were increased. In conclusion adult MSG rats developed an insulin-resistant state and increased OS and serious hepatic dysfunction characterized by inflammation and metabolic signs suggesting increased lipogenesis. These features, shared by both metabolic and Cushings syndrome human phenotypes, support that a chronic glucocorticoid-rich endogenous environment mainly impacts on hepatic glucose cycle, displacing local metabolism to lipogenesis. Whether correcting the glucocorticoid-rich environment ameliorates such dysfunctions requires further investigation.

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.

N-Acetyl-l-Cysteine treatment efficiently prevented pre-diabetes and inflamed-dysmetabolic liver development in hypothalamic obese rats

Aim: Hypothalamic obese rats are characterized by pre‐diabetes, dyslipidemia, hyperadiposity, inflammation and, liver dysmetabolism with oxidative stress (OS), among others. We studied endocrine‐metabolic dysfunctions and, liver OS and inflammation in both monosodium l‐glutamate (MSG)‐neonatally damaged and control litter‐mate (C) adult male rats, either chronically treated with N‐Acetyl‐l‐Cysteine since weaned (C‐NAC and MSG‐NAC) or not. Methodology: We evaluated circulating TBARS, glucose, insulin, triglycerides, uric acid (UA) and, aspartate and alanine amino‐transferase; insulin sensitivity markers (HOMA indexes, Liver Index of Insulin Sensitivity –LISI‐) were calculated and liver steps of the insulin‐signaling pathway were investigated. Additionally, we monitored liver OS (protein carbonyl groups, GSH and iNOS level) and inflammation‐related markers (COX‐2 and TNF&agr; protein content; gene expression level of Il1b, Tnf&agr; and Pai‐1); and carbohydrate and lipid metabolic functions (glucokinase/fructokinase activities and, mRNA levels of Srebp1c, Fas and Gpat). Key Findings: Chronic NAC treatment in MSG rats efficiently decreased the high circulating levels of triglycerides, UA, transaminases and TBARS, as well as peripheral (high insulinemia and HOMA indexes) and liver (LISI and the P‐AKT:AKT and P‐eNOS:eNOS protein ratio values) insulin‐resistance. Moreover, NAC therapy in MSG rats prevented liver dysmetabolism by decreasing local levels of OS and inflammation markers. Finally, NAC‐treated MSG rats retained normal liver glucokinase and fructokinase activities, and Srebp1c, Fas and Gpat (lipogenic genes) expression levels. Significance: Our study strongly supports that chronic oral antioxidant therapy (NAC administration) prevented the development of pre‐diabetes, dyslipidemia, and inflamed‐dysmetabolic liver in hypothalamic obese rats by efficiently decreasing high endogenous OS.