Activation of mu-opioid receptors improves insulin sensitivity in obese Zucker rats

In the current study we investigated the effect of mu-opioid receptor activation on insulin sensitivity. In obese Zucker rats, an intravenous injection of loperamide (18 microg/kg, three times daily for 3 days) decreased plasma glucose levels and the glucose-insulin index. Both effects of loperamide were subsequently inhibited by the administration of 10 microg/kg of naloxone or 10 microg/kg of naloxonazine, doses sufficient to block mu-opioid receptors. Other metabolic defects characteristic of obese Zucker rats, such as defects in insulin signaling, the decreased expression of insulin receptor substrate (IRS)-1, the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI3 kinase), and the glucose transporter subtype 4 (GLUT 4), and the reduction of phosphorylation in IRS-1 or Akt serine, were also studied. These defects were all reversed by loperamide treatment in a dose which overcame mu-opioid receptor blockade. Moreover, loss of tolbutamide-induced plasma glucose lowering action (10 mg/kg) in wild-type mice given a fructose-rich diet was markedly delayed by repeated treatment with loperamide; however, this delay induced by loperamide did not occur in mu-opioid receptor knockout mice. These results indicate an important role of peripheral mu-opioid receptors in the loperamide-induced improvement of insulin sensitivity. Our results suggest that activation of peripheral mu-opioid receptors can ameliorate insulin resistance in animals, and provide a new target for therapy of insulin resistance.     

Response of genetically obese Zucker rats to ciprofibrate,a hypolipidemic agent,with peroxisome proliferation activity as compared to Zucker lean and Sprague-Dawley rats

Summary— Genetically obese Zucker (fa/fa) rats were used as an experimental model to study the effects of hypolipidemic agents on peroxisome proliferation; comparison was made with Zucker lean phenotype (Fa/?) and Sprague-Dawley strain/phenotype. The pharmacokinetics of a single administration of ciprofibrate (1 or 3 mg/kg), appeared to be similar in all strains/phenotypes. After a 2-week oral administration at the same dosages, there were dosage-related increases in hepatocellular peroxisomal yield and in the hepatic enzymes' cyanide-insensitive acyl-CoA oxidase and catalase. The peroxisomal yield was less increased in Zucker than in Sprague-Dawley rats, while the enzyme activities were similarly increased. Although the absolute specific activity of microsomal ω-lauryl hydroxylase (cytochrome P4504A1) was lower in Zucker rats, it was increased more in this strain than in Sprague-Dawley rats in response to drug exposure. The hypolipidemic effect (cholesterol and triglyceride reduction) was more pronounced in Zucker obese rats. Based on biochemical and morphological results, no major differences between strains/phenotypes in terms of peroxisome proliferation were observed following a 2-week administration of ciprofibrate.     

Content of essential and toxic trace elements in organs of obese Wistar and Zucker leprfa rats receiving quercetin

BackgroundThe levels of a number of essential and toxic trace elements in organs and tissues are affected by the disruptions in body homeostasis caused by obesity. Some of these elements may also be influenced by the consumption of biologically active substances of polyphenolic origin, which possess potent abilities to complex with transition metal ions.AimsThe aim of this study was to determine the content of essential and toxic trace elements in Wistar outbred and hereditary obese Zucker Lepr^fa (Z) rats consuming a standard balanced diet or hypercaloric diet with excess fat and fructose, supplemented with quercetin or not supplemented.Materials and methodsMale Wistar and Z rats were fed a control AIN-93M-based semi-synthetic diet or a high-fat-high-carbohydrate diet (HFCD, with 30% fat by weight and 20% fructose provided in the drinking water). A portion of the animals in each line and diet group was administered quercetin at 50 mg/kg body weight. Essential trace elements were included in the diets as a high-purity salt mixture. After the termination of feeding on day 63, the livers, kidneys, and brains of the rats were excised and the content of 16 elements (Fe, Mg, Cu, Mn, Co, Se, Zn, Cr, Ni, Al, Cd, As, Pb, V, Cs, and Ag) was measured by inductively coupled plasma mass spectrometry (ICP-MS).ResultsIn the livers of the Z rats, the contents of Co, Zn, Mg, Fe, Se, and V were reduced and the content of Cr was increased compared to that of the Wistar rats. Supplementation with quercetin significantly decreased liver Fe, V, and Se content, which was more noticeable in the Wistar rats than in the Z rats. In kidneys of Z rats consuming control diet, the contents of Co, Cu, and Cs were decreased whereas those of Ni, Al, and Se were increased compared with the contents in the Wistar rats. The same trend was observed with HFCD feeding except for Cs content. Quercetin reduced kidney V content in both rat lines fed both diets, whereas it reduced Se and Cs only in the Z rats fed control diet. In the brains of the Z rats, a large increase was observed in some trace elements including Pb, Cd, Al, Cr, Ni, Fe, and V compared with the levels in the Wistar rat brains. Supplementation of the control diet with quercetin decreased Al and Ni in the brains of the Z rats.ConclusionThere were significant differences in the mineral content of organs between the Wistar and Z rats, with different propensities for obesity. Moreover some of these effects had no straightforward association with decreased feed consumption or hepatic fat accumulation. When introduced into the diets, quercetin affected the content of essential and toxic elements, but with ambiguous physiological significance. Thus, indicators of essential and toxic trace elements deserve to be used in the protocols of preclinical as well as clinical trials of biologically active substances and food supplements.     

Effects of CCK on gastrointestinal function in lean and obese Zucker rats

Cholecystokinin (CCK), a hormone affecting several gastrointestinal functions, has also been shown to elicit satiety and affect daily meal patterns. Since Zucker obese rats are less sensitive to the satiety effects of CCK, two experiments were designed to determine if they are also less sensitive to the gastric emptying and intestinal transit rate effects of CCK. In the first experiment phenol red was administered to 5.5 hr fasted rats 15 minutes after intraperitoneal injection of CCK-8 or saline. Rats were sacrificed after 30 minutes, the stomach and small intestine were removed, and phenol red content was measured. More phenol red was in the stomach of obese but not lean rats treated with CCK-8. The rate of transit of the contents of the small intestine was increased by CCK-8 and the percent of phenol red in the fourth quarter of the small intestine was greater in obese than lean rats (91 vs 37%, p<0.05). In the second experiment gastrointestinal transit of ferric oxide was measured during the light and dark phases of the diurnal cycle, and when obese rats were ad lib or yoke-fed to lean pair-mates. Total gastrointestinal transit time of the ferric oxide was decreased 15% when CCK-8 was administered to yoke-fed obese rats in either the light or dark portions of the diurnal cycle but was not affected in ad lib-fed obese rats or lean rats. Thus, while Zucker obese rats are less sensitive to satiety effects of CCK, they appear to be more sensitive to the gastrointestinal effects of CCK, and therefore it is not clear what role these gastrointestinal responses have on the feeding behavior responses.     

Glucose handling by hepatocytes from obese Zucker rats

Hepatocytes isolated from obese Zucker rats showed a significantly higher rate of both [U-¹⁴C]glucose and [U-¹⁴C]lactate incorporation into [¹⁴C]lipid than those from their lean counterparts. This was associated with a marked increase in the lipogenic rate measured by the incorporation of³H₂O into the cell esterified fatty acids. Although there were no changes in the incorporation of the tracer into either [¹⁴C]glycogen or¹⁴CO₂, the [¹⁴C] total uptake was significantly higher in the obese animals. The high rate of [¹⁴C]lipid synthesis from glucose was observed both at 15 and 30 mM substrate concentrations and was linked to an enhanced uptake of the tracer into the cell as measured using the decarboxilation of [1-¹⁴C]glucose in the presence of phenazine methosulphate. The presence of insulin in the incubation medium had no effect on the uptake of glucose by the liver cells. However, the large uptake of glucose by the hepatocytes from the obese animals was not related to an enhanced rate of transport as measured using 3-O-methyl[U-¹⁴C]glucose. The activity of glucose-6-phosphate dehydrogenase together with a higher [1-¹⁴C]glucose/[U-¹⁴C]glucose descarboxylation ratio indicate a predominant very active pentose phosphate pathway which may be responsible for the enhanced glucose uptake observed in the hepatocytes from the obese animals.     

Dephosphorylation increases insulin-stimulated receptor kinase activity in skeletal muscle of obese Zucker rats

Serine/threonine phosphorylation of insulin receptor has been implicated in the development of insulin resistance. To investigate whether dephosphorylation of serine/threonine residues of the insulin receptor may restore the decreased insulin-stimulated receptor tyrosine kinase activity in skeletal muscle of obese Zucker rats, insulin receptor tyrosine kinase activity was measured before and after alkaline phosphatase treatment. Compared to lean controls, insulin-stimulated glucose transport was depressed by 61% (p < 0.05) in obese Zucker rats. The insulin receptor and insulin receptor substrate-1 contents were decreased by 14% (p < 0.05) and 16% (p < 0.05), respectively, in skeletal muscle of obese Zucker rats. In vivo insulin-induced tyrosine phosphorylation of insulin receptor and insulin receptor substrate-1 was depressed by 82% (p < 0.05) and 86% (p < 0.05), respectively. In the meantime, in vitro insulin-stimulated receptor tyrosine kinase activity in obese rats was decreased by 39% (p < 0.05). Dephosphorylation of the insulin receptor by prior alkaline phosphatase treatment increased insulin-stimulated receptor tyrosine kinase activity in both lean and obese Zucker rats, but the increase was three times greater in obese Zucker rats (p < 0.05). These findings suggest that excessive serine/threonine phosphorylation of the insulin receptor in obese Zucker rats may be a cause for insulin resistance in skeletal muscle.     

Effects of intracerebroventricular leptin administration on feeding and sexual behaviors in lean and obese female Zucker rats

Obese Zucker rats (fa/fa) are characterized by inadequate leptin signaling caused by a mutation in the leptin receptor gene. Obese Zucker females are infertile and hyporesponsive to the inductive effects of ovarian hormones on sexual behaviors. Leptin treatment reverses aspects of reproductive dysfunction due to perturbations in energy balance in other animal models. Our first experiment tested the hypothesis that intracerebroventricular (icv) leptin administration would enhance the display of sexual behaviors in obese Zucker females. A second experiment compared lean and obese Zucker females' responses to leptin, during fed and fasted conditions. Ovariectomized (OVX) Zucker rats were implanted with lateral ventricular cannulae. In Experiment 1, fasted, obese females received estradiol benzoate, progesterone, and icv injections of 3, 18, or 36 microg murine leptin or vehicle. Leptin administration reduced food intake, but did not enhance sexual behaviors. In Experiment 2, steroid-replaced, OVX lean and obese females (from a different source than those in Experiment 1) received icv injections of vehicle or 3 or 36 microg leptin under fed and fasted conditions. Leptin treatment reduced food intake and weight gain in the fed, but not the fasted, condition in both genotypes. Sexual receptivity and locomotion were not affected, but icv leptin injections reduced proceptive behaviors in ad libitum-fed rats. These data confirm previous reports that centrally administered leptin decreases food intake and weight gain in obese Zucker rats; results from Experiment 2 suggest that lean and obese females are similarly responsive to these actions of leptin. Contrary to our hypothesis, leptin treatment did not stimulate sexual behaviors; rather, the hormone appears to inhibit the display of sexual proceptivity in ad libitum-fed lean and obese Zucker female rats.     

The hepatic amino acid system A transport activity, is up-regulated in obese Zucker rats

The utilization of L-alanine by liver is dependent on amino acid uptake from blood. This uptake, mainly mediated by the A transport system, may be regulated by different nutritional and physiologic conditions. The regulation of this transport system by diets with different protein content was tested in lean and obese Zucker rats. High-protein (HP) and low-protein (LP) diets led to changes in the rats’ growth patterns, especially in lean animals. However, homeostasis was relatively well maintained, as seen in plasma values, in spite of the increased urea production in the HP groups and increased triacylglycerides in the LP groups. The obese animals took up L-alanine at a higher rate than the lean animals. Obesity led to the emergence of a high-affinity component (K_M approximately 0.1–0.2 mM) in the transport system, which was not dependent on the protein content of the diet. This component has a 10-fold increase in affinity for L-alanine, but with an approximately 3- to 5-fold reduction in maximal velocity of transport.     

The carbonyl scavenger carnosine ameliorates dyslipidaemia and renal function in Zucker obese rats

The metabolic syndrome is a risk factor that increases the risk for development of renal and vascular complications. This study addresses the effects of chronic administration of the endogenous dipeptide carnosine (β‐alanyl‐L‐histidine, L‐CAR) and of its enantiomer (β‐alanyl‐D‐histidine, D‐CAR) on hyperlipidaemia, hypertension, advanced glycation end products, advanced lipoxidation end products formation and development of nephropathy in the non‐diabetic, Zucker obese rat. The Zucker rats received a daily dose of L‐CAR or D‐CAR (30 mg/kg in drinking water) for 24 weeks. Systolic blood pressure was recorded monthly. At the end of the treatment, plasma levels of triglycerides, total cholesterol, glucose, insulin, creatinine and urinary levels of total protein, albumin and creatinine were measured. Several indices of oxidative/carbonyl stress were also measured in plasma, urine and renal tissue. We found that both L‐ and D‐CAR greatly reduced obese‐related diseases in obese Zucker rat, by significantly restraining the development of dyslipidaemia, hypertension and renal injury, as demonstrated by both urinary parameters and electron microscopy examinations of renal tissue. Because the protective effect elicited by L‐ and D‐CAR was almost superimposable, we conclude that the pharmacological action of L‐CAR is not due to a pro‐histaminic effect (D‐CAR is not a precursor of histidine, since it is stable to peptidic hydrolysis), and prompted us to propose that some of the biological effects can be mediated by a direct carbonyl quenching mechanism.     

Contrasting apoptotic responses of conjugated linoleic acid in the liver of obese Zucker rats fed palm oil or ovine fat

We hypothesized that reducing weight properties of conjugated linoleic acid (CLA) are due to adipocyte apoptosis and that CLA differentially modulates the apoptotic responses in hepatic lipotoxicity from rats fed saturated fat diets. Obese Zucker rats were fed atherogenic diets (2% w/w of cholesterol) formulated with high (15% w/w) saturated fat, from vegetable or animal origin, supplemented or not with 1% of a mixture (1:1) of cis-9, trans-11 and trans-10, cis-12 CLA isomers for 14 weeks. CLA induced no changes on retroperitoneal fat depot weight, which was in line with similar levels of apoptosis. Interestingly, CLA had a contrasting effect on cell death in the liver according to the dietary fat. CLA increased hepatocyte apoptosis, associated with upregulation of Fas protein in rats fed palm oil, compared to rats receiving palm oil alone. However, rats fed ovine fat alone displayed the highest levels of hepatic cell death, which were decreased in rats fed ovine fat plus CLA. This reducing effect of CLA was related to positively restoring endoplasmic reticulum (ER) ATF-6α, BiP and CHOP protein levels and increasing phosphorylated c-Jun NH₂-terminal kinase (JNK) and c-Jun, thus suggesting an adaptive response of cell survival. These findings reinforce the role of CLA as regulator of apoptosis in the liver. Moreover, the dietary fat composition is a key factor in activation of apoptosis.     

Primary cultures of fetal hepatocytes from the genetically obese Zucker rat: Protein synthesis

Summary Primary fetal hepatocytes derived from Zucker rats with expectedfa gene frequencies of 0.0 and 0.75 have been established and can be used to detect early effects of thefa gene on hepatocellular metabolism. Paired incubation experiments demonstrate that protein synthesis in 0.75fa gene cultures is significantly less than in 0.0fa gene cultures under basal conditions. Insulin stimulates protein synthesis in 0.0fa gene cultures but has no effect on 0.75fa gene cultures. Cycloheximide inhibits protein synthesis in both types of culture. NH₄Cl inhibits protein synthesis in 0.0 but not in 0.75fa gene cultures. These data suggest that fetal hepatocytes bearing thefa gene have in vitro a generally sluggish anabolic capacity and a blunted capacity to respond to insulin compared to fetal hepatocytes without thefa gene. These diminished capacities may be expressions of a genetic error in lysosomal function. A portion of this work was presented in preliminary form at the 1980 meeting of the Tissue Culture Association. This work was supported in part by National Institutes of Health Grants AM19382 and AM06197.     

Primary cultures of fetal hepatocytes from the genetically obese Zucker rat: Characterization and total lipogenesis

Summary Primary fetal hepatocyte cultures derived from Zucker rats and with expectedfa-gene frequencies of 0.0 and 0.75 have been established and can be used to detect early effects of thefa gene on hepatocellular metabolism. Proliferative capacity is similar in both types of culture. Changes of the growth media significantly decrease total lipogenesis in both 0.0 and 0.75fa-gene culture grown in arginine-free DME medium. Paired incubation experiments demonstrate that total lipogenesis in 0.75fa gene cultures is significantly less than in 0.0fa-gene cultures under basal conditions. Stimulation of total lipogenesis by pharmacological doses of insulin and excess substrate (glucose) is significantly less in the 0.75fa gene than in the 0.0fa-gene cultures. These data suggest that the development of obesity in the Zucker rat cannot be attributed to elevated hepatic lipogenesis in the fetus. This work constitutes part of a Ph.D. dissertation submitted to New York University by A. L. Goldstein. A portion of this work was presented in preliminary form at the 1978 meeting of the Tissue Culture Association. Supported in part by National Institutes of Health Grant AM 19382 and a Grant from the Weight Watchers Foundation, Inc.     

Calcitonin mRNA activity in young obese (fa/fa) Zucker rats

Alterations in both calcitonin (CT) secretion and plasma calcium were recently described in adult obese Zucker rats. We have investigated the CT biosynthetic activity of thyroid glands in 30-day-old obese Zucker rats (fa/fa), and their controls (Lean). Plasma calcium level was significantly increased (+0.6 mg/dl) in obese animals, but plasma phosphate was unchanged. Plasma CT levels measured by radioimmunoassay (RIA) were significantly decreased in fatty (0.50 +/- 0.03 vs 0.68 +/- 0.03 ng/ml in Leans; P less than 0.001), but thyroidal hormone content was not different between Lean and fatty rats (68.7 +/- 5.1 in Leans vs 60.5 +/- 3.6 ng/gland in fatty rats). mRNA was extracted from 10 thyroids, and translated in a rabbit reticulocyte lysate (NEN) in the presence of [35S]methionine. After polyacrylamide gel electrophoresis, specific immunoprecipitates were autoradiographed and quantified by integration. A 50% decrease in translatable CT mRNA was observed in fatty rats. In basal conditions, the biosynthetic activity of C cells in obese rats correlates with the secretion rate of the hormone in the face of unchanged thyroidal CT contents.     

Integrin expression and H2O2 production in circulating and splenic leukocytes of obese rats

Objective: Obesity is associated with oxidative stress and inflammation. We hypothesized that the pro‐inflammatory state in obesity may result in spontaneous activation and, hence, increased generation of reactive oxygen species (ROS) and integrin expression in the circulating leukocytes. Methods: Flow cytometry was used to determine integrin expression (immunostaining) as well as superoxide and hydrogen peroxide productions (fluorescent probes) in the peripheral blood and splenic leukocyte of 24‐week‐old male obese normotensive and not‐as‐yet diabetic Zucker rats (n = 6) and their lean counterparts (n = 6). Results: Obese rats had hyperlipidemia and normal arterial pressure, plasma glucose, and creatinine concentrations. Nevertheless, obese rats exhibited increased hydrogen peroxide production by circulating and splenic CD4+ and CD8+ T lymphocytes and by splenic macrophages. This was accompanied by up‐regulations of CD11a expression in the peripheral blood and splenic CD4+ T cells, CD11b in circulating macrophages, and CD11a and CD18 in circulating granulocytes. Conclusion: The study revealed direct evidence of spontaneous leukocyte activation and increased ROS generation by T lymphocytes and monocytes in the peripheral blood of obese Zucker rats before the development of diabetes or hypertension. These findings illustrate the link between obesity, oxidative stress, and inflammation.     

Insulin binding in the hypothalamus of lean and genetically obese Zucker rats

Recent reports have suggested that the obesity and hyperphagia of the genetically obese Zucker rat may be related to defective insulin action or binding in the hypothalamus. We used quantitative autoradiography to determine if insulin binding is altered in specific hypothalamic nuclei associated with food intake. Insulin binding was measured in the arcuate (ARC), dorsomedial (DMN), and ventromedial (VMN) hypothalamic nuclei of 3–4-month-old lean (Fa/Fa) and genetically obese (fa/fa) Zucker rats. A consistently reproducible 15% increase in the total specific binding of 0.1 nM [¹²⁵I]-insulin was found in the ARC of the obese genotype. A slight increase in insulin binding in the DMN was also found. No difference in specific insulin binding was found between genotypes in the VMN. Nonlinear least squares analysis of competitive binding studies showed that the K_d of the ARC insulin binding site was 33% higher in the lean rats than in the obese rats, indicating an increased affinity for insulin. No difference in site number (B_max) was found in the ARC, DMN or VMN, and no evidence was found for reduced insulin binding in the hypothalamus of the obese (fa/fa) genotype. The results suggest that hyperphagia and obesity of the obese (fa/fa) Zucker rat genotype may be associated with increased insulin binding in the arcuate nucleus.     

Copper absorption,endogenous excretion,and distribution in Sprague-Dawley and lean (Fa/Fa) Zucker rats

We previously observed a rapid reduction in plasma ceruloplasmin activity in lean Zucker (Fa/Fa) rats fed a marginal copper (Cu)-deficient diet compared to similarly fed obese Zucker (fa/fa) and lean Sprague-Dawley rats. In an effort to understand the mechanisms underlying this response, we utilized the isotope dilution method to investigate the absorption and excretion of Cu in lean Zucker rats fed control and marginal Cu diets. Sprague-Dawley (SD) and homozygous lean Zucker rats were fed either a Cu-adequate (Cont; 7.5 μg Cu/g diet) or a low Cu (Low; 1.1 μg Cu/g diet) casein-based diet for 23 d. Two weeks following initiation of the dietary treatment, each rat was injected intramuscularly (im) with 11.2 μCi of⁶⁷Cu. Urine and feces were collected daily. On the 9th d following isotope injection, rats were killed and tissues collected. Significant dietary effects were observed in the relative absorption and endogenous fecal excretion of⁶⁷Cu. The tissue distributions of nonisotopic Cu and⁶⁷Cu activity were also different between dietary treatments. Tissues from rats fed the low-Cu diet typically had high concentrations of⁶⁷Cu and low concentrations of nonisotopic Cu compared to controls. An increase in relative⁶⁷Cu absorption was evident for rats fed the low-Cu diet (57.2 and 39.3%, for SD Low, Zucker Low, respectively, and 17.9, and 28.5% SD Cont and Zucker Cont, respectively). Rats fed the low-Cu diet also had reductions in endogenous fecal excretion of⁶⁷Cu compared to their respective controls. Although strain effects were not evident for either percent Cu absorption or endogenous fecal Cu excretion, the relative adaptive changes appeared more marked for the Sprague-Dawley rats compared to the lean Zucker rats.     

Ammonium uptake and urea production in hepatocytes from lean and obese Zucker rats

The metabolic differences in vitro between genetic and dietary obese rats in the uptake of ammonium and amino acids by the liver and their use for ureogenesis have been assayed using hepatocytes isolated from Lean, Obese Zucker (Genetic obese) rats and Dietary obese rats. The hepatocytes of genetic obese animals took up more ammonium and produced higher amounts of urea from ammonium and alanine than those of lean and dietary obese groups (2 and 5 times more respectively). In the lean and dietary obese groups urea synthesis accounted for almost all the nitrogen taken up as ammonium. Thus, dietary and genetic obesity show a widely different handling of nitrogen, and the genetic obese rats need to break down protein to maintain their hepatocyte function.