Yolanda B. Lombardo

Lipogenic enzyme activities and glucose uptake in fat tissue of dyslipemic, insulin-resistant rats: Effects of fish oil

OBJECTIVE The purposes of the present work were twofold: (1) investigate same mechanisms involved in the development of fat cell hypertrophy in the experimental model of dyslipidemia and whole-body insulin resistance induced in rats chronically fed a sucrose-rich diet (SRD); and (2) analyze the possible beneficial effect of fish oil on these mechanisms. METHODS For 6 mo, male Wistar rats received a sucrose-rich diet (62.5% w/w sucrose, 8% corn oil) or a control diet in which sucrose was replaced by starch. After this period, the sucrose-fed animals were divided randomly into two groups: the first one continued with the same diet up to 8 mo and the second one received the same diet, but with corn oil replaced by 7% fish oil+1 % corn oil. Rats were fed with this diet for the next 2 mo. RESULTS Although an enlarged fat cell lipolysis and an impaired insulin-stimulated glucose uptake were present in the fat cells of SRD-fed rats, an increase of several key enzymes of the novo lipogenesis could be one of the possible mechanisms involved in visceral adiposity. The addition of dietary fish oil restored or improved the above abnormalities. CONCLUSION This study shows possible mechanisms conditioning the influence of nutrients on the development and management of dyslipidemia, insulin sensitivity, and fat cell accretion, all abnormalities present in the metabolic syndrome.

Dietary chia seed induced changes in hepatic transcription factors and their target lipogenic and oxidative enzyme activities in dyslipidaemic insulin-resistant rats

The present study analyses the effect of dietary chia seed rich in n-3 α-linolenic acid on the mechanisms underlying dyslipidaemia and liver steatosis developed in rats fed a sucrose-rich diet (SRD) for either 3 weeks or 5 months. The key hepatic enzyme activities such as fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC), glucose-6-phosphate dehydrogenase (G-6-PDH), carnitine palmitoyltransferase-1 (CPT-1) and fatty acid oxidase (FAO) involved in lipid metabolism and the protein mass levels of sterol regulatory element-binding protein-1 (SREBP-1) and PPARα were studied. (1) For 3 weeks, Wistar rats were fed either a SRD with 11 % of maize oil (MO) as dietary fat or a SRD in which chia seed replaced MO (SRD+Chia). (2) A second group of rats were fed a SRD for 3 months. Afterwards, half the rats continued with the SRD while for the other half, MO was replaced by chia for 2 months (SRD+Chia). In a control group, maize starch replaced sucrose. Liver TAG and the aforementioned parameters were analysed in all groups. The replacement of MO by chia in the SRD prevented (3 weeks) or improved/normalised (5 months) increases in dyslipidaemia, liver TAG, FAS, ACC and G-6-PDH activities, and increased FAO and CPT-1 activities. Protein levels of PPARα increased, and the increased mature form of SREBP-1 protein levels in the SRD was normalised by chia in both protocols (1 and 2). The present study provides new data regarding some key mechanisms related to the fate of hepatic fatty acid metabolism that seem to be involved in the effect of dietary chia seed in preventing and normalising/improving dyslipidaemia and liver steatosis in an insulin-resistant rat model.

Inverse alterations of BCKA dehydrogenase activity in cardiac and skeletal muscles of diabetic rats

Rat cardiac and skeletal muscles, which have been used as model tissues for studies of regulation of branched-chain α-keto acid (BCKA) oxidation, vary greatly in the activity state of their BCKA dehydrogenase. In the present experiment, we have investigated whether they also vary in response of their BCKA dehydrogenase to a metabolic alteration such as diabetes and, if so, to investigate the mechanism that underlies the difference. Diabetes was produced by depriving streptozotocin-treated rats of insulin administration for 96 h. The investigation of BCKA dehydrogenase in the skeletal muscle (gastrocnemius) showed that diabetes 1) increased its activity, 2) increased the protein and gene expressions of all of its subunits (E1α, E1β, E2), 3) increased its activity state, 4) decreased the rate of its inactivation, and 5) decreased the protein expression of its associated kinase (BCKAD kinase) without affecting its gene expression. In sharp contrast, the investigation of BCKA dehydrogenase in the cardiac muscle showed that diabetes 1) decreased its activity, 2) had no effect on either protein or gene expression of any of its subunits, 3) decreased its activity state, 4) increased its rate of inactivation, and 5) increased both the protein and gene expressions of its associated kinase. In conclusion, our data suggest that, in diabetes, the protein expression of BCKAD kinase is downregulated posttranscriptionally in the skeletal muscle, whereas it is upregulated pretranslationally in the cardiac muscle, causing inverse alterations of BCKA dehydrogenase activity in these muscles.Rat cardiac and skeletal muscles, which have been used as model tissues for studies of regulation of branched-chain alpha-keto acid (BCKA) oxidation, vary greatly in the activity state of their BCKA dehydrogenase. In the present experiment, we have investigated whether they also vary in response of their BCKA dehydrogenase to a metabolic alteration such as diabetes and, if so, to investigate the mechanism that underlies the difference. Diabetes was produced by depriving streptozotocin-treated rats of insulin administration for 96 h. The investigation of BCKA dehydrogenase in the skeletal muscle (gastrocnemius) showed that diabetes 1) increased its activity, 2) increased the protein and gene expressions of all of its subunits (E(1)alpha, E(1)beta, E(2)), 3) increased its activity state, 4) decreased the rate of its inactivation, and 5) decreased the protein expression of its associated kinase (BCKAD kinase) without affecting its gene expression. In sharp contrast, the investigation of BCKA dehydrogenase in the cardiac muscle showed that diabetes 1) decreased its activity, 2) had no effect on either protein or gene expression of any of its subunits, 3) decreased its activity state, 4) increased its rate of inactivation, and 5) increased both the protein and gene expressions of its associated kinase. In conclusion, our data suggest that, in diabetes, the protein expression of BCKAD kinase is downregulated posttranscriptionally in the skeletal muscle, whereas it is upregulated pretranslationally in the cardiac muscle, causing inverse alterations of BCKA dehydrogenase activity in these muscles.

Sucrose-rich feeding during rat pregnancy-lactation and/or after weaning alters glucose and lipid metabolism in adult offspring.

Adverse fetal and early life environments predispose to the development of metabolic disorders in adulthood. The present study examined whether offspring of normal Wistar dams fed a high‐sucrose diet (SRD) developed impaired lipid and glucose homeostasis when fed a control diet (CD) after weaning. In addition, we investigated whether there were more pronounced derangements in lipid and glucose homeostasis when offspring of SRD‐fed Wistar were fed an SRD after weaning compared with those in offspring of CD‐fed dams weaned on an SRD. During pregnancy and lactation, female rats were fed either an SRD or CD. After weaning, half the male offspring from both groups were fed a CD or SRD, up to 100 days of age (CD‐CD, CD‐SRD, SRD‐SRD and SRD‐CD groups). Final bodyweight was similar between all groups, although offspring of SRD‐fed dams had lighter bodyweight at birth. Plasma lipid and glucose levels were significantly higher (P < 0.05) without changes in insulin levels in the CD‐SRD, SRD‐SRD and SRD‐CD groups compared with the CD‐CD group. Dyslipidaemia in the CD‐SRD and SRD‐SRD groups resulted from increased secretion of very low‐density lipoprotein triacylglycerol, as well as decreased triacylglycerol (TAG) clearance that was associated with increased liver TAG content (P < 0.05) compared with the CD‐CD group. The hypertriglyceridaemia observed in the SRD‐CD group was mostly associated with decreased TAG clearance. Altered glucose and insulin tolerance were observed when the SRD was fed during any period of life. These data support the hypothesis that early life exposure to SRD is associated with changes in lipid and glucose metabolism, leading to an unfavourable profile in adulthood, regardless of whether offspring consumed an SRD after weaning.

Increased leptin storage with altered leptin secretion from adipocytes of rats with sucrose-induced dyslipidemia and insulin resistance: effect of dietary fish oil

This study examined the effect of long-term feeding a high-sucrose diet (SRD) on the modulation of rat adipocytes leptin secretion and storage. For this purpose, we analyzed (a) basal and insulin-stimulated leptin release and the role of isoproterenol and palmitate on insulin-stimulated leptin secretion, (b) the correlation between leptin and glycerol released, (c) the relationship between leptin contents and adiposity, and (d) the effect of fish oil (FO) administration on the above parameters. Wistar rats were fed an SRD for 6 months. Whereas half the animals continued with SRD up to month 8, the other half was fed an SRD in which FO partially replaced corn oil from months 6 to 8. Total leptin release was reduced both basally and under insulin stimulation in SRD-fed rats. However, the ratio of leptin released after hormone stimulation to basal leptin levels was similar in the 3 dietary groups. Isoproterenol inhibited insulin-stimulated leptin release in the 3 groups, but the percentage was lower in the SRD. Palmitic acid mimicked the effect of isoproterenol. Leptin release from adipocyte of SRD-fed rats negatively correlated with glycerol release. Leptin store increased in fat pads of SRD and positively correlated with adiposity. Fish oil reduced leptin content and fat pad hypertrophy, and normalized basal lipolysis, leptinemia, and glucose homeostasis. This suggests that enhanced lipolysis and altered insulin sensitivity could play a role in the decrease of leptin released in SRD-fed rats. This is consistent with the reversion of all the alterations after FO administration.

Insulin patterns prior to and after onset of diabetes

Abstract In two of three adults in whom responses to an oral glucose load were studied before and after diabetes had appeared, the increases in serum insulin in response to an oral glucose load taken during the diabetic phase were definitely greater than those recorded before the diabetes appeared. Thus, in two patients the ratio of the increment in blood sugar to the increment in insulin recorded after glucose intolerance had appeared was lower than that documented one year earlier during the pre-diabetic phase, indicating an enhanced insulin response. In other words, a true hyperinsulinemia relative to the rise in blood sugar was present in these two persons with newly-discovered diabetes. The enhanced glucose-induced hyperinsulinemia in these newly-discovered diabetic persons indicates that in them the diabetes stemmed from inadequacy of hypoglycemic action of the insulin either because the insulin molecule was defective, pro-insulin was present, insulin action was vitiated by plasma or tissue antagonism, or absorption of glucose was enhanced. The finding of diminished insulin increments in relation to increases in blood sugar following an oral glucose load in one other adult with diabetes of one years duration or less suggests that this may be another pattern in some diabetic persons. Alternatively, it is possible that the true hyperinsulinemia noted in our other two patients is a transient phase which may disappear within a year. In the one child in this series, fasting and postglucose hyperinsulinemia was present during the prediabetic phase relative to the insulin responses recorded in control studies in nondiabetic children. After diabetes had developed and prior to any therapy, an oral glucose load did not evoke a rise in serum insulin.

Fish oil reverses the altered glucose transporter, phosphorylation, insulin receptor substrate-1 protein level and lipid contents in the skeletal muscle of sucrose-rich diet fed rats

The role and underlying mechanisms by which n-3 polyunsaturated fatty acids (PUFA) prevent/reverse SRD-induced insulin resistance (IR) in the muscle are not completely understood. Therefore, we examined: triglyceride, diacylglycerol, PKCθ, Glut-4, enzymatic hexokinase activity, IRS-1 protein mass level, and fatty acid composition of muscle phospholipids. Rats were fed a SRD during 6 months. Thereafter, half the animals continued with SRD up to 8 months; the other half was fed a SRD in which CO (8% wt/wt) was replaced by FO (7%+1% CO) for 2 months. Results were compared with those obtained in rats fed a control diet (CD). In SRD-fed rats, FO oil normalized/improved lipid storage and PKCθ protein mass level. Effects of insulin were comparable with those of CD-fed rats. FO reversed impaired glucose phosphorylation, IRS-1, and, under insulin stimulation, Glut-4 protein mass level. FO normalized insulin resistance and increased n-3 PUFAs in muscle phospholipids.

Mechanisms Involved in the Improvement of Lipotoxicity and Impaired Lipid Metabolism by Dietary α-Linolenic Acid Rich Salvia hispanica L (Salba) Seed in the Heart of Dyslipemic Insulin-Resistant Rats.

This study explores the mechanisms underlying the altered lipid metabolism in the heart of dyslipemic insulin-resistant (IR) rats fed a sucrose-rich diet (SRD) and investigates if chia seeds (rich in α-linolenic acid 18:3, n-3 ALA) improve/reverse cardiac lipotoxicity. Wistar rats received an SRD-diet for three months. Half of the animals continued with the SRD up to month 6. The other half was fed an SRD in which the fat source, corn oil (CO), was replaced by chia seeds from month 3 to 6 (SRD+chia). A reference group consumed a control diet (CD) all the time. Triglyceride, long-chain acyl CoA (LC ACoA) and diacylglycerol (DAG) contents, pyruvate dehydrogenase complex (PDHc) and muscle-type carnitine palmitoyltransferase 1 (M-CPT1) activities and protein mass levels of M-CPT1, membrane fatty acid transporter (FAT/CD36), peroxisome proliferator activated receptor α (PPARα) and uncoupling protein 2 (UCP2) were analyzed. Results show that: (a) the hearts of SRD-fed rats display lipotoxicity suggesting impaired myocardial lipid utilization; (b) Compared with the SRD group, dietary chia normalizes blood pressure; reverses/improves heart lipotoxicity, glucose oxidation, the increased protein mass level of FAT/CD36, and the impaired insulin stimulated FAT/CD36 translocation to the plasma membrane. The enhanced M-CPT1 activity is markedly reduced without similar changes in protein mass. PPARα slightly decreases, while the UCP2 protein level remains unchanged in all groups. Normalization of dyslipidemia and IR by chia reduces plasma fatty acids (FAs) availability, suggesting that a different milieu prevents the robust translocation of FAT/CD36. This could reduce the influx of FAs, decreasing the elevated M-CPT1 activity and lipid storage and improving glucose oxidation in cardiac muscles of SRD-fed rats.

Changes in hepatic lipogenic and oxidative enzymes and glucose homeostasis induced by an acetyl-l-carnitine and nicotinamide treatment in dyslipidaemic insulin-resistant rats

Normal rats fed a sucrose‐rich diet (SRD) develop dyslipidaemia and insulin resistance. The present study examined whether administration of the mitochondrial nutrients nicotinamide and acetyl‐l‐carnitine reversed or improved these metabolic abnormalities. Male Wistar rats were fed an SRD for 90 days. Half the rats then received daily injections of nicotinamide (25 mg/kg, i.p.) and acetyl‐ l‐carnitine (50 mg/kg, i.p.) for a further 90 days. The remaining rats in the SRD‐fed group and those in a normal chow‐fed control group were injected with an equal volume of saline solution for the same period. The following parameters were determined in all groups: (i) liver activity of fatty acid synthase (FAS), acetyl‐CoA carboxylase (ACC) and carnitine‐palmitoyl transferase‐1 (CPT‐1); (ii) hepatic and skeletal muscle triacylglycerol content, plasma glucose, insulin, free fatty acid (FFA) and triacylglycerol levels and pancreatic insulin content; and (iii) glucose tolerance. Administration of nicotinamide and acetyl‐l‐carnitine to the SRD‐fed rats reduced dyslipidaemia, liver steatosis, muscle triacylglycerol content and hepatic FAS and ACC activities and increased CPT‐1 activity. In addition nicotinamide and acetyl‐l‐carnitine improved the glucose disappearance rate (Kg), normalized plasma glucose levels and moderately increased insulinaemia without altering pancreatic insulin content. Finally, nicotinamide and acetyl‐l‐carnitine administration reduced bodyweight gain and visceral adiposity. The results of the present study suggest that altering key hepatic lipogenic and fatty acid oxidative enzymatic activity could improve dyslipidaemia, liver steatosis and visceral adiposity. Indeed, administration of nicotinamide and acetyl‐l‐carnitine improved glucose intolerance and normalized plasma glucose levels.

β-Cell adaptation/dysfunction in an animal model of dyslipidemia and insulin resistance induced by the chronic administration of a sucrose-rich diet.

Glucose stimulated insulin secretion (GSIS) was different in rats chronically fed a sucrose-rich diet (SRD) for 3 or 30 wk. This work proposes possible mechanisms underlying insulin secretion changes from β-cell throughout these feeding periods. In isolated islets of rats fed the SRD or a control diet (CD) we examined: 1- the glucokinase and hexokinase activities and their protein mass expression; 2- pyruvate dehydrogenase activity; 3- uncoupling protein 2 (UCP2) and peroxisome proliferators-activated receptor γ (PPAR γ) protein mass expression. At 3 wk on diet the SRD-fed rats showed: a marked increase in the first peak of GSIS; increased glucokinase protein mass expression without changes in glucokinase and hexokinase activities; increased PPARγ protein mass expression without changes in the UCP2 protein mass expression. No changes in either glucose oxidation and triglyceride content within the β-cell were observed. After 30 wk of feeding, a significant decrease of both glucokinase activity and its protein mass expression was accompanied by altered glucose oxidation, a triglyceride increase within the β-cell and a significant increase of PPARγ and UCP2 protein mass expression. Moreover GSIS depicted an absence of the first peak with an increase in the second phase. Finally, the SRD chronic administration altered GSIS by different mechanisms depending on the time on diet. At an early stage, the increased protein mass expression of the glucokinase and a fatty acid cooperative effect inducing PPARγ expression seem to be the mechanisms involved. At a late stage, glucolipotoxicity appears to be the cellular mechanism contributing to progressive β-cell dysfunction.