Pioglitazone reverses down-regulation of cardiac PPARgamma expression in Zucker diabetic fatty rats

Peroxisome proliferator-activated receptor-gamma (PPARgamma) plays a critical role in peripheral glucose homeostasis and energy metabolism, and inhibits cardiac hypertrophy in non-diabetic animal models. The functional role of PPARgamma in the diabetic heart, however, is not fully understood. Therefore, we analyzed cardiac gene expression, metabolic control, and cardiac glucose uptake in male Zucker diabetic fatty rats (ZDF fa/fa) and lean ZDF rats (+/+) treated with the high affinity PPARgamma agonist pioglitazone or placebo from 12 to 24 weeks of age. Hyperglycemia, hyperinsulinemia, and hypertriglyceridemia as well as lower cardiac PPARgamma, glucose transporter-4 and alpha-myosin heavy chain expression levels were detected in diabetic ZDF rats compared to lean animals. Pioglitazone increased body weight and improved metabolic control, cardiac PPARgamma, glut-4, and alpha-MHC expression levels in diabetic ZDF rats. Cardiac [(18)F]fluorodeoxyglucose uptake was not detectable by micro-PET studies in untreated and pioglitazone treated ZDF fa/fa rats but was observed after administration of insulin to pioglitazone treated ZDF fa/fa rats. PPARgamma agonists favorably affect cardiac gene expression in type-2 diabetic rats via activation and up-regulation of cardiac PPARgamma expression whereas improvement of impaired cardiac glucose uptake in advanced type-2 diabetes requires co-administration of insulin.     

Impact of caloric restriction on AMPK and endoplasmic reticulum stress in peripheral tissues and circulating peripheral blood mononuclear cells from Zucker rats

The activation of endoplasmic reticulum (ER) stress and a reduction of AMP-dependent protein kinase (AMPK) phosphorylation have been described in obesity. We hypothesize that a moderate caloric restriction (CR) might contribute to reducing ER stress and increasing AMPK phosphorylation in peripheral tissues from genetically obese Zucker fa/fa rats and in peripheral blood mononuclear cells (PBMCs).Zucker Lean and Zucker fa/fa rats were fed with chow diet either ad libitum (AL) (C, as controls) or 80% of AL (CR) for 2 weeks, giving rise to four experimental groups: Lean C, Lean CR, fa/fa C and fa/fa CR.CR significantly increased AMPK phosphorylation in the liver, perirenal adipose tissue (PRAT) and PBMCs from fa/fa rats but not in the subcutaneous AT (SCAT), suggesting a reduced response of SCAT to CR. Liver samples of fa/fa rats exhibited an increased mRNA expression of PERK, EIF-2α, XBP-1(s), Chop and caspase 3, which was significantly reduced by CR. PRAT exhibited an overexpression of Edem and PDIA-4 in fa/fa rats, but only PDIA-4 expression was reduced by CR. eIF-2α phosphorylation was significantly increased in all studied tissues from fa/fa rats and reduced by CR. A negative correlation was detected between p-AMPK and p-eIF-2α in the liver, PRAT and PBMCs from fa/fa rats but not in SCAT.This study shows that a moderate CR reduces ER stress and improves AMPK phosphorylation in several peripheral tissues and in circulating PBMCs, suggesting that alterations observed in PBMCs could reflect metabolic alterations associated with obesity.     

Increased expression and activity of 11beta-HSD-1 in diabetic islets and prevention with troglitazone

To determine if increased local production of glucocorticoids by the pancreatic islets might play a role in the spontaneous noninsulin-dependent diabetes mellitus of obesity, we compared islet 11beta-HSD-1 mRNA and activity in islets of obese prediabetic and diabetic Zucker Diabetic Fatty (ZDF) (fa/fa) rats and lean wild-type (+/+) controls. In diabetic rat islets, both mRNA and enzymatic activity of the enzyme were increased in proportion to the hyperglycemia. Troglitazone (TGZ) treatment, beginning at 6 weeks of age, prevented the hyperglycemia, the hyperlipidemia, and the increase in 11beta-HSD-1. To determine if the metabolic abnormalities had caused the 11beta-HSD-1 increase, prediabetic islets were cultured in high or low glucose or in 2:1 oleate:palmitate for 3 days. Neither nutrient enhanced the expression of 11beta-HSD-1. We conclude that 11beta-HSD-1 expression and activity are increased in islets of diabetic, but not prediabetic ZDF rats, and that TGZ prevents both the increase in 11beta-HSD-1 and the diabetes.     

Correlation Between Pancreatic Islet Uncoupling Protein-2 (UCP2) mRNA Concentration And Insulin Status in Rats

Hypothesizing that UCP2 may influence insulin secretion by modifying the ATP/ADP ratio within pancreatic islets, we have investigated the expression of intraislet UCP2 gene in rats showing insulin oversecretion (non-diabetic Zucker fa/fa obese rats, glucose-infused Wistar rats) or insulin undersecretion (fasting and mildly diabetic rats). We found that in Zucker fa/fa obese rats, hyperinsulinemia (1222 ± 98 pmol/1 vs. 128 ± 22 pmol/1 in lean Zucker rats) was accompanied by a significant increase in UCP2 mRNA levels. In rat submitted to a 5 day infusion with glucose, hyperinsulinemia (1126 ± 101 pmol/l vs. 215 ± 25 pmol/1 in Wistar control rats), coincided with an enhanced intraislet UCP2 gene expression, whereas a 8h or a 2 day-infusion did not induce significant changes in UCP2 mRNA expression. In rats made hypoinsulinemic and mildly diabetic by the injection of a low dose of streptozotocin, and in 4-day-fasting rats (plasma insulin 28 ± 5 pmol/1) UCP2 gene expression was sharply decreased. A 3-day-fast was ineffective. The data show the existence of a time-dependent correlation between islet mRNA UCP2 and insulin that may be interpreted as an adaptative response to prolonged insulin excess.     

Role of sympathetic activity in controlling the expression of vascular endothelial growth factor in brown fat cells of lean and genetically obese rats

The thermogenic activity of brown adipose tissue (BAT) is heavily dependent on high perfusion, through its dense vascular system. Angiogenesis must go hand-in-hand with BAT functions, but little is known about the factors controlling it. In the present study we demonstrate that: (a) vascular endothelial growth factor (VEGF) is synthesised and released in brown adipocytes in culture; (b) VEGF mRNA isoforms and protein appear in dispersed mature brown adipocytes and whole tissue; (c) VEGF expression is increased in BAT from cold-exposed rats, and in cultured brown adipocytes exposed to noradrenaline and the β₃-adrenoceptor agonists; (e) BAT from genetically obese (fa/fa) rats exhibits reduced expression of VEGF as well as a change in the ratio of mRNA isoforms. It is concluded that sympathetic control of VEGF expression via noradrenaline acting on β₃-adrenoceptors plays a major role in developmental and adaptive angiogenesis, and defects in this contribute to the reduced thermogenic capacity of BAT in genetic obesity.     

Glucocorticoids and cyclic AMP selectively increase hepatic lipin-1 expression, and insulin acts antagonistically

Glucocorticoids (GCs) increase hepatic phosphatidate phosphatase (PAP1) activity. This is important in enhancing the liver's capacity for storing fatty acids as triacylglycerols (TAGs) that can be used subsequently for beta-oxidation or VLDL secretion. PAP1 catalyzes the conversion of phosphatidate to diacylglycerol, a key substrate for TAG and phospholipid biosynthesis. PAP1 enzymes in liver include lipin-1A and -1B (alternatively spliced isoforms) and two distinct gene products, lipin-2 and lipin-3. We determined the mechanisms by which the composite PAP1 activity is regulated using rat and mouse hepatocytes. Levels of lipin-1A and -1B mRNA were increased by dexamethasone (dex; a synthetic GC), and this resulted in increased lipin-1 synthesis, protein levels, and PAP1 activity. The stimulatory effect of dex on lipin-1 expression was enhanced by glucagon or cAMP and antagonized by insulin. Lipin-2 and lipin-3 mRNA were not increased by dex/cAMP, indicating that increased PAP1 activity is attributable specifically to enhanced lipin-1 expression. This work provides the first evidence for the differential regulation of lipin activities. Selective lipin-1 expression explains the GC and cAMP effects on increased hepatic PAP1 activity, which occurs in hepatic steatosis during starvation, diabetes, stress, and ethanol consumption.     

Brain Insulin-Like Growth Factor-II mRNA Content Is Reduced in Insulin-Dependent and Non-Insulin-Dependent Diabetes Mellitus

Abstract: Diabetic encephalopathy, characterized by structural, electrophysiological, neurochemical, and cognitive abnormalities, is observed in insulin-dependent diabetes mellitus (IDDM) and non-IDDM (NIDDM). Identification of early biochemical lesions potentially may provide clues pointing to its pathogenesis. Insulin-like growth factors (IGFs) are neurotrophic factors that recently have been implicated in the pathogenesis of diabetic neuropathy. Because IGF-II is the predominant IGF in adult brain, we tested the hypothesis that IGF-II gene expression is decreased in the CNS in both IDDM and NIDDM. Brain and spinal cord were isolated from streptozotocin-diabetic rats, a model of IDDM with weight loss and impaired insulin production. IGF-II mRNA content was measured by northern and slot blots. After 2 weeks of diabetes, IGF-II mRNA content per milligram of tissue wet weight, as well as per unit of poly(A)⁺ RNA, declined significantly (p≤ 0.05) in brain and spinal cord. Insulin replacement therapy partially restored IGF-II mRNA levels in brain, cortex, medulla, and spinal cord. The obese, hyperinsulinemic, and spontaneously diabetic (fa/fa) Zucker rat was used as a model of NIDDM. Brain weight (p < 0.025) and IGF-II mRNA contents (p < 0.01) were significantly decreased in (fa/fa) versus lean nondiabetic (+/?) rats. Therefore, the decline in IGF-II mRNA levels in diabetic brain was independent of the type of diabetes, the direction of change in body weight, and the insulinemic state. We speculate that this early biochemical lesion may contribute to the development of diabetic encephalopathy.     

Adaptations in Mitochondrial Function Parallel,but Fail to Rescue,the Transition to Severe Hyperglycemia and Hyperinsulinemia: A Study in Zucker Diabetic Fatty Rats

Cross‐sectional human studies have associated mitochondrial dysfunction to type 2 diabetes. We chose Zucker diabetic fatty (ZDF) rats as a model of progressive insulin resistance to examine whether intrinsic mitochondrial defects are required for development of type 2 diabetes. Muscle mitochondrial function was examined in 6‐, 12‐, and 19‐week‐old ZDF (fa/fa) and fa/+ control rats (n = 8–10 per group) using respirometry with pyruvate, glutamate, and palmitoyl‐CoA as substrates. Six‐week‐old normoglycemic–hyperinsulinemic fa/fa rats had reduced mitochondrial fat oxidative capacity. Adenosine diphosphate (ADP)‐driven state 3 and carbonyl cyanide p‐trifluoromethoxyphenylhydrazone (FCCP)‐stimulated state uncoupled (state u) respiration on palmitoyl‐CoA were lower compared to controls (62.3 ± 9.5 vs. 119.1 ± 13.8 and 87.8 ± 13.3 vs. 141.9 ± 14.3 nmol O₂/mg/min.). Pyruvate oxidation in 6‐week‐old fa/fa rats was similar to controls. Remarkably, reduced fat oxidative capacity in 6‐week‐old fa/fa rats was compensated for by an adaptive increase in intrinsic mitochondrial function at week 12, which could not be maintained toward week 19 (140.9 ± 11.2 and 57.7 ± 9.8 nmol O₂/mg/min, weeks 12 and 19, respectively), whereas hyperglycemia had developed (13.5 ± 0.6 and 16.1 ± 0.3 mmol/l, weeks 12 and 19, respectively). This mitochondrial adaptation failed to rescue the progressive development of insulin resistance in fa/fa rats. The transition of prediabetes state toward advanced hyperglycemia and hyperinsulinemia was accompanied by a blunted increase in uncoupling protein‐3 (UCP3). Thus, in ZDF rats insulin resistance develops progressively in the absence of mitochondrial dysfunction. In fact, improved mitochondrial capacity in hyperinsulinemic hyperglycemic rats does not rescue the progression toward advanced stages of insulin resistance.     

The effect of adrenalectomy on GDP binding to brown-adipose-tissue mitochondria of obese rats

GDP binding to brown-adipose-tissue mitochondria of young obese Zucker rats (fa/fa) was significantly lower than in lean control rats, as a result of a decrease in the number of binding sites. Adrenalectomy of fa/fa rats restored GDP binding to control values. Corticosterone replacement suppressed GDP binding in adrenalectomized obese rats.     

Glucocorticoid Receptor Changes Associate with Age in the Paraventricular Nucleus of Type II Diabetic Rat Model

Diabetes is a metabolic disorder that is associated with the dysregulation of a number of systems within the body. In the present study, we investigated glucocorticoid receptor (GR) immunoreactivity and its protein levels in the paraventricular nuclei of 4-, 12-, 20- and 30-week-old Zucker diabetic fatty (fa/fa, ZDF) and in Zucker lean control (fa/+ or +/+, ZLC) rats, because the progressive induction of diabetes is detectable in this model after 7 weeks of age and chronic diabetic conditions are maintained after 12 weeks of age. GR immunoreactivity was detected in parvocellular paraventricular nuclei and this and GR protein levels were exponentially increased according to the ages. In particular, GR immunoreactivities and protein levels were markedly more increased in 30-week-old ZDF rats than in age-matched ZLC group and in younger ZDF group. The present study suggests that GR immunoreactivity and its protein level is associated with a degenerative phenotype in the hypothalamus of from 12-weeks old in the ZDF rat type II diabetes model.     

Appropriateness of the Zucker Diabetic Fatty rat as a model for diabetic microvascular late complications

Male obese Zucker Diabetic Fatty (ZDF) rats develop type 2 diabetes around eight weeks of age, and are widely used as a model for human diabetes and its complications. The objective of the study was to test whether the complications manifested in the kidney and nerves of ZDF rats really correspond to human diabetic complications in their being related to the hyperglycaemic state. Four groups of ZDF rats were used. One lean (Fa/?) and one obese (fa/fa) untreated group served as non-diabetic and diabetic controls. In two further groups of obese (fa/fa) rats, diabetes was prevented by pioglitazone or delayed by food restriction. All rats were monitored up to 35 weeks of age with respect to their blood glucose, HbA1c and insulin levels, their kidney function (urinary glucose excretion, renal glucose filtration, glomerular filtration rate, albumin/creatinine ratio), and their nerve function (tactile and thermal sensory threshold and nerve conduction velocity). Pioglitazone prevented the development of diabetes, while food restriction delayed its onset for 8-10 weeks. Accordingly, kidney function parameters were similar to lean non-diabetic rats in pioglitazone-treated rats and significantly improved in food-restricted rats compared with obese controls. Kidney histology paralleled the functional results. By contrast, nerve functional evaluations did not mirror the differing blood glucose levels. We conclude that the ZDF rat is a good model for diabetic nephropathy, while alterations in nerve functions were not diabetes-related.     

Effects of Lactobacillus paracasei CNCM I-4034, Bifidobacterium breve CNCM I-4035 and Lactobacillus rhamnosus CNCM I-4036 on Hepatic Steatosis in Zucker Rats

We have previously described the safety and immunomodulatory effects of Lactobacillus paracasei CNCM I-4034, Bifidobacterium breve CNCM I-4035 and Lactobacillus rhamnosus CNCM I-4036 in healthy volunteers. The scope of this work was to evaluate the effects of these probiotic strains on the hepatic steatosis of obese rats. We used the Zucker rat as a genetic model of obesity. Zucker-Lepr^fa/fa rats received one of three probiotic strains, a mixture of L. paracasei CNCM I-4034 and B. breve CNCM I-4035, or a placebo for 30 days. An additional group of Zucker-lean^+/fa rats received a placebo for 30 days. No alterations in intestinal histology, in the epithelial, lamina propria, muscular layers of the ileal or colonic mucosa, or the submucosae, were observed in any of the experimental groups. Triacylglycerol content decreased in the liver of Zucker-Lepr^fa/fa rats that were fed L. rhamnosus, B. breve, or the mixture of B. breve and L. paracasei. Likewise, the area corresponding to neutral lipids was significantly smaller in the liver of all four groups of Zucker-Lepr^fa/fa rats that received probiotics than in rats fed the placebo. Zucker-Lepr^fa/fa rats exhibited significantly greater serum LPS levels than Zucker-lean^+/fa rats upon administration of placebo for 30 days. In contrast, all four groups of obese Zucker-Lepr^fa/fa rats that received LAB strains exhibited serum LPS concentrations similar to those of Zucker-lean^+/fa rats. Serum TNF-α levels decreased in the Zucker-Lepr^fa/fa rats that received B. breve, L. rhamnosus, or the mixture, whereas L. paracasei feeding decreased IL-6 levels in the serum of Zucker-Lepr^fa/fa rats. In conclusion, the probiotic strains reduced hepatic steatosis in part by lowering serum LPS, and had an anti-inflammatory effect in obese Zucker rats.     

Cyclic nucleotide PDE-3

Type 3 cyclic nucleotide phosphodiesterase (PDE-3) isoforms exhibit a high affinity (“lowK _m”) for cAMP and are specifically inhibited by cGMP and a number of pharmacological agents, which increase myocardial contractility, inhibit platelet aggregation, and increase smooth muscle relaxation. The PDE-3 family consists of at least two isozymes, PDE-3A (cardiac type) and PDE-3B (adipocyte type), with distinct tissue-specific distributions. PDE-3A mRNA is highly expressed in the cardiovascular system, whereas PDE-3B mRNA is primarily expressed in adipocytes and hepatocytes. Toward understanding potential roles of PDE-3 in diabetes mellitus, we have established a specific and sensitive RNase protection assay (RPA) for quantitating PDE-3A and PDE-3B mRNA in rat diabetic models. In fatty Zucker diabetic (ZDF) rats, PDE-3A mRNA, but not PDE-3B mRNA, was expressed in heart, whereas liver and white and brown fat tissues predominantly expressed PDE-3B mRNA. Unexpectedly, PDE-3B mRNA expression was ≈2.5 times higher than PDE-3A mRNA in aorta from both ZDF and Sprague-Dawley (SD) rats. In contrast, expression levels of PDE-3A mRNA in heart were similar in both species. With this RPA, we were thus able to compare PDE-3A and-3B mRNA levels in different tissues as well as in different rat species.     

Deposition of docosahexaenoic acid (DHA) is limited in forebrain of young obese fa/fa Zucker rats fed a diet high in α-linolenic acid but devoid of DHA

Docosahexaenoic acid (DHA) is required for neurotransmitter synthesis and learning. Conversion of α-linolenic acid (ALA) to DHA is considered adequate to support brain function in youth, but it is unknown if brain DHA can be maintained in insulin resistant states. This study investigated brain fatty acid and desaturase activities in young insulin resistant Zucker rats on diets with and without DHA. Male fa/fa and lean rats were fed diets enriched with flaxseed (FXO, ALA: 35.5% fatty acids), menhaden (MO, DHA: 9.2%) or safflower oil (SO, linoleic acid: 54.1%) for 9 weeks, n=8 per diet per genotype. Compared to lean, the 15 week old fa/fa rats were obese (56% heavier) and insulin-resistant (>18-fold in homeostasis model assessment of insulin resistance). The forebrain of fa/fa rats had higher palmitoleic (16:1n-7) and dihomo-γ-linolenic (20:3n-6) acids, and higher Δ9, Δ6 but lower Δ5 (all P≤.006) desaturase indices than lean. The Δ9 and Δ6 desaturase indices positively, while the Δ5 negatively (all P≤.01) correlated with insulin resistance. The Δ9 desaturase index positively correlated with adiposity index. The percentage of forebrain DHA of fa/fa rats was lower (P=.011) than lean rats when fed FXO diet while there was no difference (P>.05) between fa/fa and lean rats fed MO or SO diet. Thus, the alterations in the fatty acid and desaturase indices in the brain were consistent inhibited forebrain synthesis of DHA in the fa/fa rats. ALA may not have potential to effectively serve as a precursor for synthesizing DHA for youth forebrain during insulin resistance since Δ5 desaturase activity is limited.     

Lipogenesis in arterial wall and vascular smooth muscular cells: regulation and abnormalities in insulin-resistance

Background

Vascular smooth muscular cells (VSMC) express lipogenic genes. Therefore in situ lipogenesis could provide fatty acids for triglycerides synthesis and cholesterol esterification and contribute to lipid accumulation in arterial wall with aging and during atheroma.

Methods

We investigated expression of lipogenic genes in human and rat arterial walls, its regulation in cultured VSMC and determined if it is modified during insulin-resistance and diabetes, situations with increased risk for atheroma.

Results

Zucker obese (ZO) and diabetic (ZDF) rats accumulated more triglycerides in their aortas than their respective control rats, and this triglycerides content increased with age in ZDF and control rats. However the expression in aortas of lipogenic genes, or of genes involved in fatty acids uptake, was not higher in ZDF and ZO rats and did not increase with age. Expression of lipogenesis-related genes was not increased in human arterial wall (carotid endarterectomy) of diabetic compared to non-diabetic patients. In vitro, glucose and adipogenic medium (ADM) stimulated moderately the expression and activity of lipogenesis in VSMC from control rats. LXR agonists, but not PXR agonist, stimulated also lipogenesis in VSMC but not in arterial wall in vivo. Lipogenic genes expression was lower in VSMC from ZO rats and not stimulated by glucose or ADM.

Conclusion

Lipogenic genes are expressed in arterial wall and VSMC; this expression is stimulated (VSMC) by glucose, ADM and LXR agonists. During insulin-resistance and diabetes, this expression is not increased and resists to the actions of glucose and ADM. It is unlikely that this metabolic pathway contribute to lipid accumulation of arterial wall during insulin-resistance and diabetes and thus to the increased risk of atheroma observed in these situations.