Altered neuropeptide Y concentrations in specific hypothalamic regions of obese (fa/fa) Zucker rats. Possible relationship to obesity and neuroendocrine disturbances.

Neuropeptide Y (NPY) concentrations were measured by radioimmunoassay in eight microdissected hypothalamic regions of obese (fa/fa) and lean (Fa/?) Zucker rats. Freely fed obese rats showed significant (40-100%) increases in NPY concentrations in several regions, notably the paraventricular, ventromedial, and dorsomedial nuclei and the arcuate nucleus/median eminence, compared with lean rats. Hypothalamic NPY concentrations were not affected in either obese or lean rats by food restriction, which caused 25% weight loss over 3 wk. Refeeding to initial weight significantly increased NPY levels in the ventromedial and dorsomedial nuclei in lean rats but did not significantly alter NPY concentrations in any hypothalamic region in obese rats. These observations indicate fundamental differences in the regulation of hypothalamic NPY between obese and lean Zucker rats. NPY injected into the paraventricular nucleus and other regions causes hyperphagia, obesity, and increased secretion of insulin, glucagon, ACTH, and corticosterone. These behavioral and neuroendocrine abnormalities all occur in the obese Zucker syndrome and may be due to increased NPY-ergic activity in the hypothalamus.     

Characterization of bone structure in leptin receptor-deficient Zucker (fa/fa) rats.

To investigate the role of leptin in bone formation, the skeleton of the obese female leptin receptor-deficient Zucker rat was examined using pQCT, microCT, and histomorphometry. A trend toward decreasing structural and bone formation parameters in these rats as they age suggest that leptin has a small positive effect on bone. INTRODUCTION: Evidence in the literature has suggested the possible role of leptin in bone formation. Leptin deficiency or leptin receptor deficiency results in higher bone mass. In an attempt to further investigate leptin's role in bone formation, we examined the skeleton of obese leptin receptor-deficient Zucker rats. METHODS: Female leptin receptor-deficient Zucker (fa/fa) rats and their homozygous (Fa/Fa) and heterozygous (Fa/fa) lean controls were used at 9 and 15 weeks of age (n = 5). Bone mineral density of the proximal tibia was measured by peripheral quantitative computed tomography (pQCT). Microcomputed tomography (microCT) was used for the analysis of trabecular architecture in the proximal tibia metaphysis and cortical bone at the tibia-fibula junction. Static and dynamic parameters of bone resorption and formation were quantitated by histomorphometry. Statistical analysis was performed by Dunnett's one-way ANOVA. RESULTS: Analysis of the proximal tibia by pQCT show no significant differences in the bone mineral density of obese rats compared with their corresponding lean controls in either age group. Trabecular architecture measured by microCT indicate a trends toward decreasing bone volume (BV/TV) in the obese animals, evident by a decrease in trabecular number and thickness with an increase in trabecular separation. Histomorphometric evaluation further shows significant increases in osteoclast surface in the obese rats at both 9 and 15 weeks without a change in osteoclast number. Osteoid surface in the obese animals was also found to be decreased by 15 weeks of age. Fluorescent-based measurements of bone formation were not significantly different. Differences in the cortical compartment were not observed at either age. CONCLUSION: Based on the observed skeletal phenotype of the Zucker (fa/fa) rat, it is suggested that leptin exerts a positive effect on bone.     

Glucose transporter levels in tissues of spontaneously diabetic Zucker fa/fa rat (ZDF/drt) and viable yellow mouse (Avy/a).

We used antibodies to the fat/muscle glucose transporter (GLUT4) and the liver glucose transporter (GLUT2) to measure levels of these proteins in various tissues of two rodent models of non-insulin-dependent (type II) diabetes mellitus: the obese spontaneously diabetic male Zucker fa/fa rat (ZDF/drt) and the male viable yellow Avy/a obese diabetic mouse. The ZDF/drt strain generally develops overt diabetes associated with decreased plasma insulin levels. Depending on the age of the animals, the ZDF/drt rats can be arbitrarily segregated into age-matched obese, mildly diabetic (blood glucose less than 11 mM) and obese, and severely diabetic (blood glucose greater than 20 mM) groups. Avy/a mice are comparably hyperglycemic but unlike the ZDF/drt rats are severely hyperinsulinemic. In both groups of diabetic animals, GLUT4 in adipose tissue, heart, and skeletal muscle was reduced 25-55%, and GLUT2 in liver was increased 30-40%, relative to lean, age-matched controls. However, when the mildly diabetic ZDF/drt rats were compared to the lean controls, the only significant difference was a 25% reduction of GLUT4 in heart. Within all of the ZDF/drt rats (excluding the lean controls), GLUT2 in liver and GLUT4 in adipose tissue, heart, and skeletal muscle correlated significantly with glycemia. These data suggest that, in these two models of type II diabetes, glucose transporter levels in muscle, adipose tissue, and liver are regulated in a tissue-selective manner in response to changes in insulin and glucose. Furthermore, at least in the ZDF/drt rat, alterations in GLUT2 and/or GLUT4 protein levels appear not to be associated with obesity per se but appear to be secondary to the severely diabetic state.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of free fatty acids on insulin receptor binding and tyrosine kinase activity in hepatocytes isolated from lean and obese rats.

We demonstrated previously that high physiological concentrations of free fatty acids (FFA) rapidly decrease insulin binding, degradation, and action in isolated rat hepatocytes. In this study, hepatocytes from lean and obese Sprague-Dawley rats (Alab, Stockholm) were preincubated with or without 0.4 mM oleic acid, and the effect on insulin binding and tyrosine kinase activity was measured. In the absence of exogenous FFA, insulin binding was reduced in hepatocytes from obese compared with lean rats (mean +/- SE reduction 44 +/- 7%, n = 8, P less than 0.01). Furthermore, the inhibitory effect of oleic acid added to hepatocytes from lean rats (n = 8; 40 +/- 9%, P less than 0.01) was not seen in cells from obese rats. Treating obese rats with Etomoxir, a carnitine palmitoyl transferase I inhibitor, increased insulin binding to isolated hepatocytes by 41 +/- 13% (n = 5, P less than 0.05). There was no difference in total binding to partially purified insulin receptors from solubilized hepatocytes from lean and obese rats, whether cells were or were not preincubated with oleic acid. Tyrosine kinase activity of partially purified receptors from basal or insulin-stimulated cells was not affected by either obesity, treatment with Etomoxir, or preincubating the cells with oleic acid. Thus, both obesity and elevated ambient FFA levels are associated with impaired insulin cell surface binding to isolated hepatocytes, possibly through an effect of lipid oxidation on the internalization/recycling of the insulin-receptor complex without any perturbation of the receptor tyrosine kinase activity. The data suggest that the reduced insulin binding to hepatocytes from obese rats is due to elevated ambient FFA levels.     

Beneficial effect of intravenous bolus of corticotropin-releasing factor on glucose intolerance of genetically obese (fa/fa) rats.

The effect of an ovine corticotropin-releasing factor (oCRF) bolus administered intravenously at the onset of glucose ingestion during oral glucose tolerance tests (OGTTs) was evaluated in conscious lean (FA/FA) and genetically obese (fa/fa) rats. When the amount of oCRF was purposely small to not stimulate the hypothalamo-pituitary-adrenal (HPA) axis, it normalized the glucose intolerance of genetically obese rats as tested during OGTTs and decreased their insulin output, whereas it had no effect in lean rats. In obese rats, plasma xylose levels measured after the ingestion of a xylose load were unaltered by the intravenous oCRF bolus, indicating that the beneficial effect of oCRF on glucose intolerance of fa/fa rats was unlikely to be dependent on glucose absorption. When the intravenous bolus of oCRF was doubled at the onset of OGTTs, it stimulated the HPA axis and produced a worsening of glucose intolerance in obese rats together with an increase in their insulin response. Again, it had no effect in lean rats. The abnormal intravenous glucose tolerance of obese rats was unaffected by the administration of an oCRF bolus: This is in keeping with previous data showing that bypassing the oral cavity fails to elicit several sensory reflexes that markedly influence subsequent glucose clearance. It has been suggested that obese rats may have deficient oropharyngeal reflexes that could be reactivated by the oCRF bolus, thereby being responsible for the normalization of their impaired OGTT, which lies in the hepatic glucose production process.     

Isoform-specific regulation of 5' AMP-activated protein kinase in skeletal muscle from obese Zucker (fa/fa) rats in response to contraction

Glucose transport can be activated in skeletal muscle in response to insulin via activation of phosphoinositide (PI) 3-kinase and in response to contractions or hypoxia, presumably via activation of 5' AMP-activated protein kinase (AMPK). We determined the effects of insulin and muscle contraction/hypoxia on PI 3-kinase, AMPK, and glucose transport activity in epitrochlearis skeletal muscle from insulin-resistant Zucker (fa/ fa) rats. Insulin-stimulated glucose transport in isolated skeletal muscle was reduced 47% in obese versus lean rats, with a parallel 42% reduction in tyrosine-associated PI 3-kinase activity. Contraction and hypoxia elicited normal responses for glucose transport in skeletal muscle from insulin-resistant obese rats. Isoform-specific AMPK activity was measured in skeletal muscle in response to insulin, contraction, or hypoxia. Contraction increased AMPKalpha1 activity 2.3-fold in lean rats, whereas no effect was noted in obese rats. Hypoxia increased AMPKalpha1 activity to a similar extent (more than sixfold) in lean and obese rats. Regardless of genotype, contraction, and hypoxia, each increased AMPKalpha2 activity more than fivefold, whereas insulin did not alter either AMPKalpha1 or -alpha2 activity in skeletal muscle. In conclusion, obesity-related insulin resistance is associated with an isoform-specific impairment in AMPKalpha1 in response to contraction. However, this impairment does not appear to affect contraction-stimulated glucose transport. Activation of AMPKalpha2 in response to muscle contraction/ exercise is associated with a parallel and normal increase in glucose transport in insulin-resistant skeletal muscle.     

Inducible nitric oxide synthase has divergent effects on vascular and metabolic function in obesity

Previous studies have suggested an involvement of inducible nitric oxide synthase (iNOS) in obesity, but the relation, if any, between this and mechanisms underlying endothelial dysfunction in obesity is unknown. We studied mice fed an obesogenic high-fat or standard diet for up to 8 weeks. Obesity was associated with elevated blood pressure; resistance to the glucoregulatory actions of insulin; resistance to the vascular actions of insulin, assessed as the reduction in phenylephrine constrictor response of aortic rings after insulin preincubation (lean -21.7 +/- 11.5 vs. obese 18.2 +/- 15.5%; P < 0.05); and evidence of reactive oxygen species (ROS)-dependent vasodilatation in response to acetylcholine in aortic rings (change in maximal relaxation to acetylcholine after exposure to catalase: lean -2.1 +/- 6.0 vs. obese -15.0 +/- 3.8%; P = 0.04). Obese mice had increased expression of iNOS in aorta, with evidence of increased vascular NO production, assessed as the increase in maximal constriction to phenylephrine after iNOS inhibition with 1400W (lean -3.5 +/- 9.1 vs. obese 42.1 +/- 11.2%; P < 0.001). To further address the role of iNOS in obesity-induced vascular and metabolic dysfunction, we studied the effect of a high-fat diet in iNOS knockout mice (iNOS KO). Obese iNOS KO mice were protected against the development of resistance to insulin's glucoregulatory and vascular effects (insulin-dependent reduction in maximal phenylephrine response: obese wild-type 11.2 +/- 15.0 vs. obese iNOS KO -20.0 +/- 7.7%; P = 0.02). However, obese iNOS KO mice remained hypertensive (124.0 +/- 0.7 vs. 114.9 +/- 0.5 mmHg; P < 0.01) and had evidence of increased vascular ROS production. Although these data support iNOS as a target to protect against the adverse effects of obesity on glucoregulation and vascular insulin resistance, iNOS inhibition does not prevent the development of raised blood pressure or oxidative stress.     

Obesity alters rat abdominal flap survival

Obesity presents a risk factor for flap-related complications in autologous tissue breast reconstruction. In this study, an animal model was developed to examine this phenomenon. Abdominal flaps based on a superficial inferior epigastric pedicle were elevated in an experimental group of obese Zucker (fa/fa) rats (n = 8; mean weight, 413 g) and in their lean littermates (n = 9; mean weight, 276 g). Flap tissue was harvested from a subset of both groups for baseline characterization, including histology, and assays for ATP and oxidative phosphorylation uncoupler, UCP-2. Flaps were then evaluated for survival by planimetry at 4 and 7 days postprocedure. Flap survival 7 days postoperatively was reduced in obese (42.0% +/- 8.6%) versus lean (70.3% +/- 6.7%) rats (P < 0.05). At baseline, flap tissue of obese animals had decreased ATP content relative to lean counterparts (0.12 +/- 0.12 nM/microg vs 0.36 +/- 0.23 nM/microg protein, P < 0.05), whereas UCP2 mRNA was higher in obese flap tissue versus lean. Reduced viability of obese flaps may be attributable to decreased baseline energy stores due to oxidative phosphorylation uncoupling by UCP-2. This study is the first to introduce a promising animal model for examining the effect of obesity on increased flap-related complications in breast reconstruction using autologous tissue.     

Glycerol production and utilization during the early phase of human obesity.

To study the initial period of fat deposition in human obesity, we measured glycerol turnover in 12 children of 135-253% ideal body weight, who had continuously gained weight since the onset of obesity 2-9 yr previously. Hyperinsulinemia developed in these children depending on obesity duration (r = 0.74, P less than 0.01). Whole-body glycerol production was twofold greater in the obese children (311 vs. 156 mumol.min-1, P less than 0.01) and correlated with body fat (r = 0.67, P less than 0.005). Normalization of glycerol flux to fat mass revealed that the rate of triglyceride hydrolysis was in fact lower in the adipose tissue of obese children (9.4 vs. 17.7 mumol.min-1/kg body fat) and correlated with plasma insulin (r = 0.64, P less than 0.005). Euglycemic insulin clamps showed that the response of glycerol production to a unit increment in plasma insulin concentration was increased in obese children, suggesting increased insulin sensitivity of adipose tissue. As a direct consequence (r = 0.67, P less than 0.025) of their elevated plasma glycerol concentration (65 +/- 4 vs. 37 +/- 2 microM, P less than 0.05) obese children had an increased glycerol utilization by the whole body, as well as per unit of lean body mass (9.1 +/- 1 vs. 6.5 +/- 0.9 mumoles.min-1.kg lean body mass-1, P less than 0.025).     

Correlation between morphology and function in isolated islets of the Zucker rat.

Obesity in the Zucker rat is accompanied by hyperlipemia, hyperinsulinism, insulin resistance, pancreatic hyperplasia, and islet hypertrophy. This study correlates the morphologic heterogeneity of isolated pancreatic islets with secretion of insulin and glucagon in the perifusion system. Islet size was arbitrarily defined as large (greater than 0.45 mm) or small (smaller than 0.12 mm). Protein content and volume (V = 4/3pir3) were calculated for groups and individual islets, respectively. Islets from obese rats secreted more insulin in response to glucose and aminophylline than islets from lean rats (peak 7.8 +/- 2.4 vs. 1.5 +/- 0.37 microU/islet/min, P less than 0.005). Insulin release was related directly to islet size and protein content. Small islets from lean and obese animals produced less insulin per islet than large islets (P less than 0.005). In terms of islet volume, however, large islets were inefficient insulin releasers as compared to small islets (P less than 0.005). Stimulation with Br-cAMP released glucagon from islets of lean but not from large islets of obese animals (peak 11 +/- 3.3 vs. 4.1 +/- 0.3 pg/microgram protein per minute, P less than 0.05). Arginine produced the same effect on glucagon release (P less than 0.05) as stimulation with Br-cAMP. The observed increased insulin release rates and the blunted glucagon response are related to islet size in the pancreas of the Zucker rat.     

Effect of 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside infusion on in vivo glucose and lipid metabolism in lean and obese Zucker rats

Activation of AMP-activated protein kinase (AMPK) with 5-aminoimidazole-4-carboxamide-1-beta-D-ribofurano-side (AICAR) increases glucose transport in skeletal muscle via an insulin-independent pathway. To examine the effects of AMPK activation on skeletal muscle glucose transport activity and whole-body carbohydrate and lipid metabolism in an insulin-resistant rat model, awake obese Zuckerfa/fa rats (n = 26) and their lean (n = 23) littermates were infused for 90 min with AICAR, insulin, or saline. The insulin infusion rate (4 mU.kg(-1).min(-1)) was selected to match the glucose requirements during AICAR (bolus, 100 mg/kg; constant, 10 mg.kg(-1).min(-1)) isoglycemic clamps in the lean rats. The effects of these identical AICAR and insulin infusion rates were then examined in the obese Zucker rats. AICAR infusion increased muscle AMPK activity more than fivefold (P < 0.01 vs. control and insulin) in both lean and obese rats. Plasma triglycerides, fatty acid concentrations, and glycerol turnover, as assessed by [2-13C]glycerol, were all decreased in both lean and obese rats infused with AICAR (P < 0.05 vs. basal), whereas insulin had no effect on these parameters in the obese rats. Endogenous glucose production rates, measured by [U-13C]glucose, were suppressed by >50% during AICAR and insulin infusions in both lean and obese rats (P < 0.05 vs. basal). In lean rats, rates of whole-body glucose disposal increased by more than two-fold (P < 0.05 vs. basal) during both AICAR and insulin infusion; [3H]2-deoxy-D-glucose transport activity increased to a similar extent, by >2.2-fold (both P < 0.05 vs. control), in both soleus and red gastrocnemius muscles of lean rats infused with either AICAR or insulin. In the obese Zucker rats, neither AICAR nor insulin stimulated whole-body glucose disposal or soleus muscle glucose transport activity. However, AICAR increased glucose transport activity by approximately 2.4-fold (P < 0.05 vs. control) in the red gastrocnemius from obese rats, whereas insulin had no effect. In summary, acute infusion of AICAR in an insulin-resistant rat model activates skeletal muscle AMPK and increases glucose transport activity in red gastrocnemius muscle while suppressing endogenous glucose production and lipolysis. Because type 2 diabetes is characterized by diminished rates of insulin-stimulated glucose uptake as well as increased basal rates of endogenous glucose production and lipolysis, these results suggest that AICAR-related compounds may represent a new class of antidiabetic agents.     

Effects of meal ingestion on plasma amylin concentration in NIDDM and nondiabetic humans

Recent interest has focused on the potential role of amylin in the pathogenesis of non-insulin-dependent diabetes mellitus (NIDDM). This 37-amino acid peptide is found in extracellular amyloid deposits in approximately 50% of pancreatic islets of patients with NIDDM and has been shown to inhibit skeletal muscle glycogen synthesis in vitro. Immunocytochemical studies have colocalized amylin and insulin within beta-cell secretory granules in nondiabetic humans, provoking the following questions. Is amylin cosecreted with insulin? Are circulating amylin concentrations higher in patients with NIDDM either before or after food ingestion? To answer these questions, we developed a sensitive and specific immunoassay to measure plasma concentrations of amylin in humans. Use of this assay indicated that, in lean nondiabetic subjects, glucose ingestion resulted in an increase (P less than 0.001) in the plasma concentration of amylin (from 2.03 +/- 0.22 to 3.78 +/- 0.39 pM) and insulin (from 48.3 +/- 3.1 to 265 +/- 44 pM). There was a significant correlation between the concentrations of insulin and amylin (r = 0.74, P less than 0.001) and the increase in insulin and amylin concentration (r = 0.65, P less than 0.005). Fasting concentrations of amylin did not differ in diabetic and weight-matched nondiabetic subjects and showed a similar pattern of change after ingestion of a mixed meal. We conclude that amylin is secreted in response to ingestion of either glucose or a mixed meal and circulates at concentrations that do not differ in patients with NIDDM and nondiabetic subjects. It remains to be determined whether amylin at physiological concentrations influences carbohydrate metabolism and if so whether its effects differ in diabetic and nondiabetic humans.     

Increased fatty acid uptake and altered fatty acid metabolism in insulin-resistant muscle of obese Zucker rats

Altered muscle fatty acid (FA) metabolism may contribute to the presence of muscle insulin resistance in the genetically obese Zucker rat. To determine whether FA uptake and disposal are altered in insulin-resistant muscle, we measured palmitate uptake, oxidation, and incorporation into di- and triglycerides in isolated rat hindquarters, as well as muscle plasma membrane fatty acid-binding protein (FABP(PM)) content of lean (n = 16, fa/+) and obese (n = 15, fa/fa) Zucker rats (12 weeks of age). Hindquarters were perfused with 7 mmol/l glucose, 1,000 micromol/l albumin-bound palmitate, and albumin-bound [1-(14)C]palmitate at rest (no insulin). Glucose uptake was 42% lower in the obese than in the lean rats and indicated the presence of muscle insulin resistance. Fractional and total rates of palmitate uptake were 42 and 74% higher in the obese than in the lean rats and were associated with higher muscle FABP(PM) content (r(2) = 0.69, P < 0.05). The percentage of palmitate oxidized was not significantly different between groups. FA disposal to storage was altered according to fiber type. When compared with lean rats, the rate of triglyceride synthesis in red muscle was 158% higher in obese rats, and the rate of palmitate incorporation into diglycerides in white muscle was 93% higher in obese rats. Pre- and postperfusion muscle triglyceride levels were higher in both red and white muscles of the obese rats. These results show that increased FA uptake and altered FA disposal to storage may contribute to the development of muscle insulin resistance in obese Zucker rats.     

Taste-induced changes in plasma insulin and glucose turnover in lean and genetically obese rats

Cephalic-phase insulin release (CPIR) and the changes in glucose turnover induced by saccharin ingestion were studied in freely moving lean and genetically obese fa/fa rats equipped with chronic catheters for blood sampling. Six-hour-fasted lean and obese rats were trained to drink 1 ml sodium saccharin (0.15%) or 1 ml glucose (70%), and blood samples were taken before and after the stimuli. As early as 1-1.5 min poststimulus, there was a significant increase in CPIR in lean and obese rats. The amplitude of the CPIR induced either by saccharin or by glucose in the obese rats was significantly higher than it was in the lean rats. The effect of saccharin ingestion on the hepatic glucose production (HGP) and the rate of glucose disappearance (Rd) was studied in 6-h-fasted lean and obese rats, under non-steady-state conditions, according to a method previously validated. Saccharin ingestion produced a significant increase in HGP and Rd in lean and obese rats compared with basal values. The saccharin-induced increments in HGP and Rd were higher in the obese than in the lean animals. We conclude that saccharin (through taste) appears to elicit parasympathetic (insulin release) and sympathetic (HGP increase) reflexes in lean and obese rats. These taste-induced changes in plasma insulin and glucose turnover are exaggerated in the obese rats and may participate in obesity and in insulin resistance of the overall syndrome.     

Delayed insulin transport across endothelium in insulin-resistant JCR:LA-cp rats

Capillary endothelial cells are thought to limit the transport of insulin across the endothelium, resulting in attenuated insulin action at target sites. Whether endothelial insulin transport is altered in dysglycemic insulin-resistant states is not clear and was therefore investigated in the JCR:LA-cp corpulent male rat, which exhibits the metabolic syndrome of obesity, insulin resistance, hyperlipidemia, and hyperinsulinemia. Lean littermates that did not develop these alterations served as controls. Animals of both groups were normotensive (mean arterial pressure 136+/-2 mmHg). Hearts from obese and lean rats aged 7 (n = 6) or 18 (n = 8) weeks were perfused in vitro at 10 ml/min per gram wet wt over 51 min with Krebs-Henseleit buffer containing 0.1 or 0.5 U human insulin/l (equivalent to 0.6 and 3 nmol/l). Interstitial fluid was collected using a validated method, and interstitial insulin was determined with a radioimmunoassay. At 0.1 U/l, insulin transfer velocity was similar in both experimental groups (half-times of transfer: 11+/-0.2 min in obese and 18+/-4 min in lean rats; NS), but at 0.5 U/l, the respective half-times were 7+/-1 min in lean and 13+/-2 min in obese rats (P < 0.05). The steady-state level of insulin in the interstitium was 34+/-1% of the vascular level at 0.1 U/l and reached the vascular level (102+/-2%) at 0.5 U/l in both lean and obese rats. In rats aged 18 weeks, the half-times of insulin transfer were 31+/-2 and 14+/-l min in obese rats and 10+/-0.3 and 7+/-0.3 min in lean rats (P < 0.05). Again, interstitial steady-state levels were similar in both groups. Finally, postprandial insulin dynamics were simulated over a period of 120 min with a peak concentration of 0.8 U/l in rats aged 27 weeks (n = 4). The maximal interstitial level was 0.38+/-0.02 U/l in lean rats and 0.24+/-0.02 U/l in obese rats (P < 0.05), and a similar difference was noted throughout insulin infusion (areas under the transudate concentration-time curves: 17 and 11 U/min per 1, respectively). These data show, for the first time in a genetic animal model of insulin resistance, that transfer of insulin across the endothelium is substantially delayed in obese insulin-resistant rats and that it likely contributes to the postprandial alterations of glucose metabolism observed in the metabolic syndrome.     

Obesity blunts insulin-mediated microvascular recruitment in human forearm muscle

We have previously shown that skeletal muscle capillaries are rapidly recruited by physiological doses of insulin in both humans and animals. This facilitates glucose and insulin delivery to muscle, thus augmenting glucose uptake. In obese rats, both insulin-mediated microvascular recruitment and glucose uptake are diminished; however, this action of insulin has not been studied in obese humans. Here we used contrast ultrasound to measure microvascular blood volume (MBV) (an index of microvascular recruitment) in the forearm flexor muscles of lean and obese adults before and after a 120-min euglycemic-hyperinsulinemic (1 mU . min(-1) . kg(-1)) clamp. We also measured brachial artery flow, fasting lipid profile, and anthropomorphic variables. Fasting plasma glucose (5.4 +/- 0.1 vs. 5.1 +/- 0.1 mmol/l, P = 0.05), insulin (79 +/- 11 vs. 38 +/- 6 pmol/l, P = 0.003), and percent body fat (44 +/- 2 vs. 25 +/- 2%, P = 0.001) were higher in the obese than the lean adults. After 2 h of insulin infusion, whole-body glucose infusion rate was significantly lower in the obese versus lean group (19.3 +/- 3.2 and 37.4 +/- 2.6 mumol . min(-1) . kg(-1) respectively, P < 0.001). Compared with baseline, insulin increased MBV in the lean (18.7 +/- 3.3 to 25.0 +/- 4.1, P = 0.019) but not in the obese group (20.4 +/- 3.6 to 18.8 +/- 3.8, NS). Insulin increased brachial artery diameter and flow in the lean but not in the obese group. We observed a significant, negative correlation between DeltaMBV and BMI (R = -0.482, P = 0.027) in response to insulin. In conclusion, obesity eliminated the insulin-stimulated muscle microvascular recruitment and increased brachial artery blood flow seen in lean individuals.     

Decreased activation rate of insulin-stimulated glucose transport in adipocytes from obese subjects

Recent studies from our laboratory have shown that the rate at which insulin activates glucose disposal in vivo is much slower in obese subjects compared with lean controls. To determine if this was caused by an abnormality in activation of insulin-stimulated glucose transport at the cellular level, we measured the rate at which insulin stimulated glucose transport in human adipocytes from obese volunteers. Basal rates of 3-O-methylglucose transport in the absence of insulin were lower (0.20 +/- 0.04 vs. 0.40 +/- 0.11 pmol.10(-5) cells.10 s-1, P less than .25) in adipocytes from obese subjects (n = 10) than in lean control subjects (n = 5), but this did not achieve statistical significance. Maximal insulin-stimulated (4300 pM insulin) glucose transport rates were significantly decreased in obesity (2.81 +/- 0.81 vs. 1.15 +/- 0.20 pmol.10(-5) cells.10 s-1, P less than .005). It took longer for adipocytes from obese subjects to achieve half-maximal activation of insulin-stimulated glucose transport than those from lean subjects (15 +/- 2 vs. 9.4 +/- 1.2 min, P less than .05). The slower overall rates of activation of maximal insulin-stimulated glucose transport observed in adipocytes from obese subjects mirror the slower rates of stimulation of glucose disposal in vivo, which suggests that the in vivo findings are caused by a cellular abnormality in insulin action at a step beyond the binding of insulin to its receptor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Renal and metabolic effects of caffeine in obese (fa/fa(cp)), diabetic, hypertensive ZSF1 rats.

In Western society, the triad of hypertension, metabolic syndrome and obesity (which caries a high risk for renal disease) is increasing, as is the intake of caffeine. However, no information is available regarding the metabolic or renal consequences of caffeine consumption in this complex disease entity. The purpose of this study was to investigate the effects of chronic caffeine consumption on renal function and metabolic status in obese ZSF1 rats, an animal model of obesity, hypertension and the metabolic syndrome. Fifteen, 18-week-old male, obese ZSF1 rats were randomized to drink tap water (Cont, n = 8) or 0.1% solution of caffeine (Caff, n = 7) for 8 weeks. Metabolic and renal function measurements were performed at baseline and after 4 and 8 weeks of treatment. Caffeine treatment significantly (p < 0.05) reduced body weight, food, and fluid consumption and improved insulin sensitivity (fasting insulin 129.6+/-8.1 vs 97.5+/-3.6 microIU/mL; fed insulin 146.3+/-8.5 vs 110.6+/-3.4 microIU/mL; fasting glucose 138.7+/-13.4 vs 145+/-8.0 mg/dL; fed glucose 373+/-19.4 vs 283.3+/-19.6 mg/dL, Cont vs Caff, respectively). After 8 weeks of caffeine treatment, animals were less glycosuric as compared with control group. Area under glucose curves (AUC-glucose) in oral glucose tolerance test did not differ between the two groups (AUC- glucose: 592.5+/-42.7 vs 589.5+/-20.5 mg/dL x h, Cont vs Caff), whereas caffeine treatment significantly decreased AUC of insulin (AUC-insulin: 257.77+/-12.9 vs 198.0+/-5.9 microIU/mL x h, Cont vs. Caff, p<0.05). No differences were found with regard to plasma triglycerides and glycerol levels; however, caffeine significantly increased cholesterol levels after 4 and 8 weeks (2F-Anova, p<0.001). Moreover, caffeine significantly decreased creatinine clearance after 4 and 8 weeks (CrCl, Cont: 3.5+/-0.4, Caff: 2.2+/-0.2 L/kg/day, p<0.05) and increased protein/CrCl ratio (Cont: 323+/-30, Caff: 527+/-33 mg/L/day). Caffeine treatment for 8 weeks tended to increase plasma norepinephrine levels (p<0.06), but the two groups did not differ with regard to plasma renin activity, blood pressure, renal blood flow or and renal vascular resistance. The study indicates that caffeine improves insulin sensitivity but increases plasma cholesterol levels and impairs renal function in obesity with the metabolic syndrome and hypertension. Our results imply that the health consequences of chronic caffeine consumption may depend heavily on underlying pathophysiology process.     

Influence of obesity and type 2 diabetes on gluconeogenesis and glucose output in humans: a quantitative study

The contribution of gluconeogenesis (GNG) to endogenous glucose output (EGO) in type 2 diabetes is controversial. Little information is available on the separate influence of obesity on GNG. We measured percent GNG (by the 2H2O technique) and EGO (by 6,6-[2H]glucose) in 37 type 2 diabetic subjects (9 lean and 28 obese, mean fasting plasma glucose [FPG] 8.3 +/- 0.3 mmol/l) and 18 control subjects (6 lean and 12 obese) after a 15-h fast. Percent GNG averaged 47 +/- 5% in lean control subjects and was significantly increased in association with both obesity (P < 0.01) and diabetes (P = 0.004). By multivariate analysis, percent GNG was independently associated with BMI (partial r = 0.27, P < 0.05, with a predicted increase of 0.9% per BMI unit) and FPG (partial r = 0.44, P = 0.0009, with a predicted increase of 2.7% per mmol/l of FPG). In contrast, EGO was increased in both lean and obese diabetic subjects (15.6 +/- 0.5 micromol x min(-1) x kg(-1) of fat-free mass, n = 37, P = 0.002) but not in obese nondiabetic control subjects (13.1 0.7, NS) as compared with lean control subjects (12.4 +/- 1.4). Consequently, gluconeogenic flux (percent GNG x EGO) was increased in obesity (P = 0.01) and markedly elevated in diabetic subjects (P = 0.0004), whereas glycogenolytic flux was reduced only in association with obesity (P = 0.05). Fasting plasma glucagon levels were significantly increased in diabetic subjects (P < 0.05) and positively related to EGO, whereas plasma insulin was higher in obese control subjects than lean control subjects (P = 0.05) and unrelated to measured glucose fluxes. We conclude that the percent contribution of GNG to glucose release after a 15-h fast is independently and quantitatively related to the degree of overweight and the severity of fasting hyperglycemia. In obese individuals, reduced glycogenolysis ensures a normal rate of glucose output. In diabetic individuals, hyperglucagonemia contributes to inappropriately elevated rates of glucose output from both GNG and glycogenolysis.     

Beta-cell mass dynamics in Zucker diabetic fatty rats. Rosiglitazone prevents the rise in net cell death

The evolution of diabetes in the male leptin receptor-deficient (fa/fa) Zucker diabetic fatty (ZDF) rat is associated with disruption of normal islet architecture, beta-cell degranulation, and increased beta-cell death. It is unknown whether these changes precede or develop as a result of the increasing plasma glucose, or whether the increased beta-cell death can be prevented. Early intervention with thiazolidinediones prevents disruption of the islet architecture. To determine the specific effects of rosiglitazone (RSG) on beta-cell mass dynamics, male fa/fa (obese) and +/fa or +/+ (lean) rats age 6 weeks were fed either chow (control group [CN]) or chow mixed with rosiglitazone (RSG group) at a dosage of 10 micromol. kg(-1) body wt.day(-1). Rats were killed after 0, 2, 4, 6, or 10 weeks of treatment (at age 6, 8, 10, 12, or 16 weeks). Plasma glucose increased from 8.9 +/- 0.4 mmol/l at 0 weeks to 34.2 +/- 1.8 mmol/l (P = 0.0001) at 6 weeks of treatment in obese CN rats and fell from 8.0 +/- 0.3 to 6.3 +/- 0.4 mmol/l in obese RSG rats (P = 0.02). beta-cell mass fell by 51% from 2 to 6 weeks of treatment (ages 8-12 weeks) in obese CN rats (6.9 +/- 0.9 to 3.4 +/- 0.5 mg; P < 0.05), whereas beta-cell mass was unchanged in obese RSG rats. At 10 weeks of treatment (age 16 weeks), beta-cell mass in obese CN rats was only 56% of that of obese RSG rats (4.4 +/- 0.4 vs. 7.8 +/- 0.3 mg, respectively; P = 0.0001). The beta-cell replication rate fell from a baseline value of 0.95 +/- 0.12% in lean rats and 0.94 +/- 0.07% in obese rats (at 0 weeks) to approximately 0.3-0.5% in all groups by 6 weeks of treatment (age 12 weeks). After 10 weeks of treatment, beta-cell replication was higher in obese RSG rats than in CN rats (0.59 +/- 0.14 vs. 0.28 +/- 0.05%, respectively; P < 0.02). Application of our mass balance model of beta-cell turnover indicated that net beta-cell death was fivefold higher in obese CN rats as compared with RSG rats after 6 weeks of treatment (age 12 weeks). The increase in beta-cell death in obese CN rats during the 6-week observation period was well correlated with the increase in plasma glucose (r2 = 0.90, P < 0.0001). These results suggest that the development of hyperglycemia in ZDF rats is concomitant with increasing net beta-cell death. beta-cell proliferation compensates for the increased beta-cell loss at a time when plasma glucose is moderately elevated, but compensation ultimately fails and the plasma glucose levels increase beyond approximately 20 mmol/l. Treatment with rosiglitazone, previously shown to reduce insulin resistance, prevents the loss of beta-cell mass in obese ZDF rats by maintaining beta-cell proliferation and preventing increased net beta-cell death.