CB1R antagonist increases hepatic insulin clearance in fat-fed dogs likely via upregulation of liver adiponectin receptors.

The improvement of hepatic insulin sensitivity by the cannabinoid receptor 1 (CB1R) antagonist rimonabant (RIM) has been recently been reported to be due to upregulation of adiponectin. Several studies demonstrated that improvement in insulin clearance accompanies the enhancement of hepatic insulin sensitivity. However, the effects of RIM on hepatic insulin clearance (HIC) have not been fully explored. The aim of this study was to explore the molecular mechanism(s) by which RIM affects HIC, specifically to determine whether upregulation of liver adiponectin receptors (ADRs) and other key genes regulated by adiponectin mediate the effects. To induce insulin resistance in skeletal muscle and liver, dogs were fed a hypercaloric high-fat diet (HFD) for 6 wk. Thereafter, while still maintained on a HFD, animals received RIM (HFD+RIM; n = 11) or placebo (HFD+PL; n = 9) for an additional 16 wk. HIC, calculated as the metabolic clearance rate (MCR), was estimated from the euglycemic-hyperinsulinemic clamp. The HFD+PL group showed a decrease in MCR; in contrast, the HFD+RIM group increased MCR. Consistently, the expression of genes involved in HIC, CEACAM-1 and IDE, as well as gene expression of liver ADRs, were increased in the HFD+RIM group, but not in the HFD+PL group. We also found a positive correlation between CEACAM-1 and the insulin-degrading enzyme IDE with ADRs. Interestingly, expression of liver genes regulated by adiponectin and involved in lipid oxidation were increased in the HFD+RIM group. We conclude that in fat-fed dogs RIM enhances HIC, which appears to be linked to an upregulation of the adiponectin pathway.

Gastric electrical stimulation for obesity.

Obesity is a growing health problem worldwide with a major impact on health and healthcare expenditures. Medical therapy in the form of diet and pharmacotherapy has limited effect on weight. Standard bariatric surgery is effective but is associated with morbidity and mortality, creating an unmet need for alternative therapies. One such therapy, the application of electrical stimulation to the stomach, has been studied extensively for the last two decades. Though pulse parameters differ between the various techniques used, the rationale behind this assumes that application of electrical current can interfere with gastric motor function or modulate afferent signaling to the brain or both. Initial studies led by industry failed to show an effect on body weight. However, more recently, there has been a renewed interest in this therapeutic modality with a number of concepts being evaluated in large human trials. If successful, this minimally invasive and low-risk intervention would be an important addition to the existing menu of therapies for obesity.

Hepatic vascular endothelial growth factor regulates recruitment of rat liver sinusoidal endothelial cell progenitor cells.

BACKGROUND & AIMS: After liver injury, bone marrow-derived liver sinusoidal endothelial cell progenitor cells (BM SPCs) repopulate the sinusoid as liver sinusoidal endothelial cells (LSECs). After partial hepatectomy, BM SPCs provide hepatocyte growth factor, promote hepatocyte proliferation, and are necessary for normal liver regeneration. We examined how hepatic vascular endothelial growth factor (VEGF) regulates recruitment of BM SPCs and their effects on liver injury. METHODS: Rats were given injections of dimethylnitrosamine to induce liver injury, which was assessed by histology and transaminase assays. Recruitment of SPCs was analyzed by examining BM SPC proliferation, mobilization to the circulation, engraftment in liver, and development of fenestration (differentiation). RESULTS: Dimethylnitrosamine caused extensive denudation of LSECs at 24 hours, followed by centrilobular hemorrhagic necrosis at 48 hours. Proliferation of BM SPCs, the number of SPCs in the bone marrow, and mobilization of BM SPCs to the circulation increased 2- to 4-fold by 24 hours after injection of dimethylnitrosamine; within 5 days, 40% of all LSECs came from engrafted BM SPCs. Allogeneic resident SPCs, infused 24 hours after injection of dimethylnitrosamine, repopulated the sinusoid as LSECs and reduced liver injury. Expression of hepatic VEGF messenger RNA and protein increased 5-fold by 24 hours after dimethylnitrosamine injection. Knockdown of hepatic VEGF with antisense oligonucleotides completely prevented dimethylnitrosamine-induced proliferation of BM SPCs and their mobilization to the circulation, reduced their engraftment by 46%, completely prevented formation of fenestration after engraftment as LSECs, and exacerbated dimethylnitrosamine injury. CONCLUSIONS: BM SPC recruitment is a repair response to dimethylnitrosamine liver injury in rats. Hepatic VEGF regulates recruitment of BM SPCs to liver and reduces this form of liver injury.

CB(1) antagonism restores hepatic insulin sensitivity without normalization of adiposity in diet-induced obese dogs.

The endocannabinoid system is highly implicated in the development of insulin resistance associated with obesity. It has been shown that antagonism of the CB(1) receptor improves insulin sensitivity (S(I)). However, it is unknown whether this improvement is due to the direct effect of CB(1) blockade on peripheral tissues or secondary to decreased fat mass. Here, we examine in the canine dog model the longitudinal changes in S(I) and fat deposition when obesity was induced with a high-fat diet (HFD) and animals were treated with the CB(1) antagonist rimonabant. S(I) was assessed (n = 20) in animals fed a HFD for 6 wk to establish obesity. Thereafter, while HFD was continued for 16 additional weeks, animals were divided into two groups: rimonabant (1.25 mg·kg(-1)·day(-1) RIM; n = 11) and placebo (n = 9). Euglycemic hyperinsulinemic clamps were performed to evaluate changes in insulin resistance and glucose turnover before HFD (week -6) after HFD but before treatment (week 0) and at weeks 2, 6, 12, and 16 of treatment (or placebo) + HFD. Magnetic resonance imaging was performed to determine adiposity- related changes in S(I). Animals developed significant insulin resistance and increased visceral and subcutaneous adiposity after 6 wk of HFD. Treatment with RIM resulted in a modest decrease in total trunk fat with relatively little change in peripheral glucose uptake. However, there was significant improvement in hepatic insulin resistance after only 2 wk of RIM treatment with a concomitant increase in plasma adiponectin levels; both were maintained for the duration of the RIM treatment. CB(1) receptor antagonism appears to have a direct effect on hepatic insulin sensitivity that may be mediated by adiponectin and independent of pronounced reductions in body fat. However, the relatively modest effect on peripheral insulin sensitivity suggests that significant improvements may be secondary to reduced fat mass.

High-fat diet-induced insulin resistance does not increase plasma anandamide levels or potentiate anandamide insulinotropic effect in isolated canine islets.

BACKGROUND: Obesity has been associated with elevated plasma anandamide levels. In addition, anandamide has been shown to stimulate insulin secretion in vitro, suggesting that anandamide might be linked to hyperinsulinemia. OBJECTIVE: To determine whether high-fat diet-induced insulin resistance increases anandamide levels and potentiates the insulinotropic effect of anandamide in isolated pancreatic islets. DESIGN AND METHODS: Dogs were fed a high-fat diet (n = 9) for 22 weeks. Abdominal fat depot was quantified by MRI. Insulin sensitivity was assessed by the euglycemic-hyperinsulinemic clamp. Fasting plasma endocannabinoid levels were analyzed by liquid chromatography-mass spectrometry. All metabolic assessments were performed before and after fat diet regimen. At the end of the study, pancreatic islets were isolated prior to euthanasia to test the in vitro effect of anandamide on islet hormones. mRNA expression of cannabinoid receptors was determined in intact islets. The findings in vitro were compared with those from animals fed a control diet (n = 7). RESULTS: Prolonged fat feeding increased abdominal fat content by 81.3±21.6% (mean±S.E.M, P<0.01). In vivo insulin sensitivity decreased by 31.3±12.1% (P<0.05), concomitant with a decrease in plasma 2-arachidonoyl glycerol (from 39.1±5.2 to 15.7±2.0 nmol/L) but not anandamide, oleoyl ethanolamide, linoleoyl ethanolamide, or palmitoyl ethanolamide. In control-diet animals (body weight: 28.8±1.0 kg), islets incubated with anandamide had a higher basal and glucose-stimulated insulin secretion as compared with no treatment. Islets from fat-fed animals (34.5±1.3 kg; P<0.05 versus control) did not exhibit further potentiation of anandamide-induced insulin secretion as compared with control-diet animals. Glucagon but not somatostatin secretion in vitro was also increased in response to anandamide, but there was no difference between groups (P = 0.705). No differences in gene expression of CB1R or CB2R between groups were found. CONCLUSIONS: In canines, high-fat diet-induced insulin resistance does not alter plasma anandamide levels or further potentiate the insulinotropic effect of anandamide in vitro.

Hepatic insulin clearance is the primary determinant of insulin sensitivity in the normal dog.

OBJECTIVE: Insulin resistance is a powerful risk factor for Type 2 diabetes and a constellation of chronic diseases, and is most commonly associated with obesity. We examined if factors other than obesity are more substantial predictors of insulin sensitivity under baseline, nonstimulated conditions. METHODS: Metabolic assessment was performed in healthy dogs (n = 90). Whole-body sensitivity from euglycemic clamps (SICLAMP ) was the primary outcome variable, and was measured independently by IVGTT (n = 36). Adiposity was measured by MRI (n = 90), and glucose-stimulated insulin response was measured from hyperglycemic clamp or IVGTT (n = 86 and 36, respectively). RESULTS: SICLAMP was highly variable (5.9-75.9 dl/min per kg per µU/ml). Despite narrow range of body weight (mean, 28.7 ± 0.3 kg), adiposity varied approximately eight-fold and was inversely correlated with SICLAMP (P < 0.025). SICLAMP was negatively associated with fasting insulin, but most strongly associated with insulin clearance. Clearance was the dominant factor associated with sensitivity (r = 0.53, P < 0.00001), whether calculated from clamp or IVGTT. CONCLUSIONS: These data suggest that insulin clearance contributes substantially to insulin sensitivity, and may be pivotal in understanding the pathogenesis of insulin resistance. We propose the hyperinsulinemia due to reduction in insulin clearance is responsible for insulin resistance secondary to changes in body weight.

Simplified method to isolate highly pure canine pancreatic islets.

OBJECTIVES: The canine model has been used extensively to improve the human pancreatic islet isolation technique. At the functional level, dog islets show high similarity to human islets and thus can be a helpful tool for islet research. We describe and compare 2 manual isolation methods, M1 (initial) and M2 (modified), and analyze the variables associated with the outcomes, including islet yield, purity, and glucose-stimulated insulin secretion (GSIS). METHODS: Male mongrel dogs were used in the study. M2 (n = 7) included higher collagenase concentration, shorter digestion time, faster shaking speed, colder purification temperature, and higher differential density gradient than M1 (n = 7). RESULTS: Islet yield was similar between methods (3111.0 ± 309.1 and 3155.8 ± 644.5 islets/g, M1 and M2, respectively; P = 0.951). Pancreas weight and purity together were directly associated with the yield (adjusted R(2) = 0.61; P = 0.002). Purity was considerably improved with M2 (96.7% ± 1.2% vs 75.0% ± 6.3%; P = 0.006). M2 improved GSIS (P = 0.021). Independently, digestion time was inversely associated with GSIS. CONCLUSIONS: We describe an isolation method (M2) to obtain a highly pure yield of dog islets with adequate ß-cell glucose responsiveness. The isolation variables associated with the outcomes in our canine model confirm previous reports in other species, including humans.

Large size cells in the visceral adipose depot predict insulin resistance in the canine model.

Adipocyte size plays a key role in the development of insulin resistance. We examined longitudinal changes in adipocyte size and distribution in visceral (VIS) and subcutaneous (SQ) fat during obesity-induced insulin resistance and after treatment with CB-1 receptor antagonist, rimonabant (RIM) in canines. We also examined whether adipocyte size and/or distribution is predictive of insulin resistance. Adipocyte morphology was assessed by direct microscopy and analysis of digital images in previously studied animals 6 weeks after high-fat diet (HFD) and 16 weeks of HFD + placebo (PL; n = 8) or HFD + RIM (1.25 mg/kg/day; n = 11). At 6 weeks, mean adipocyte diameter increased in both depots with a bimodal pattern only in VIS. Sixteen weeks of HFD+PL resulted in four normally distributed cell populations in VIS and a bimodal pattern in SQ. Multilevel mixed-effects linear regression with random-effects model of repeated measures showed that size combined with share of adipocytes >75 µm in VIS only was related to hepatic insulin resistance. VIS adipocytes >75 µm were predictive of whole body and hepatic insulin resistance. In contrast, there was no predictive power of SQ adipocytes >75 µm regarding insulin resistance. RIM prevented the formation of large cells, normalizing to pre-fat status in both depots. The appearance of hypertrophic adipocytes in VIS is a critical predictor of insulin resistance, supporting the deleterious effects of increased VIS adiposity in the pathogenesis of insulin resistance.

Diet-induced obesity prevents interstitial dispersion of insulin in skeletal muscle.

OBJECTIVE: Obesity causes insulin resistance, which has been interpreted as reduced downstream insulin signaling. However, changes in access of insulin to sensitive tissues such as skeletal muscle may also play a role. Insulin injected directly into skeletal muscle diffuses rapidly through the interstitial space to cause glucose uptake. When insulin resistance is induced by exogenous lipid infusion, this interstitial diffusion process is curtailed. Thus, the possibility exists that hyperlipidemia, such as that seen during obesity, may inhibit insulin action to muscle cells and exacerbate insulin resistance. Here we asked whether interstitial insulin diffusion is reduced in physiological obesity induced by a high-fat diet (HFD). RESEARCH DESIGN AND METHODS: Dogs were fed a regular diet (lean) or one supplemented with bacon grease for 9-12 weeks (HFD). Basal insulin (0.2 mU x min(-1) x kg(-1)) euglycemic clamps were performed on fat-fed animals (n = 6). During clamps performed under anesthesia, five sequential doses of insulin were injected into the vastus medialis of one hind limb (INJ); the contralateral limb (NINJ) served as a control. RESULTS: INJ lymph insulin showed an increase above NINJ in lean animals, but no change in HFD-fed animals. Muscle glucose uptake observed in lean animals did not occur in HFD-fed animals. CONCLUSIONS: Insulin resistance induced by HFD caused a failure of intramuscularly injected insulin to diffuse through the interstitial space and failure to cause glucose uptake, compared with normal animals. High-fat feeding prevents the appearance of injected insulin in the interstitial space, thus reducing binding to skeletal muscle cells and glucose uptake.

Experimental hyperlipidemia dramatically reduces access of insulin to canine skeletal muscle.

A complex sequence of steps is required for insulin to cause glucose uptake. Impairment of any one of these steps can contribute to insulin resistance. We observed the effect of insulin resistance induced by hyperlipidemia on the dynamics of insulin injected into skeletal muscle. Basal insulin euglycemic clamps (0.2 mU/min/kg) with or without lipid infusions (20% at 1.5 ml/min) were done on anesthetized dogs. Sequential insulin doses were administered by intramuscular injection directly into the vastus medialis of one hindlimb, using the contralateral leg for comparison. Intramuscular insulin injection in normal animals caused a clear dose-dependent increment in interstitial insulin levels, as well as dose-dependent increase in leg glucose uptake. In a second group of animals, lipid was infused before and during intramuscular insulin injection to cause systemic increase in free fatty acids (FFAs). In sharp contrast, systemic lipid infusion caused insulin resistance, indicated by reduced glucose infusion required to maintain euglycemia, and prevented injection-induced increase in lymphatic insulin and leg glucose uptake observed without lipid. The injected insulin was instead detected in the venous outflow from the leg. Lipid infusion caused intramuscular insulin to be diverted from interstitium into the capillary circulation, preventing a rise in interstitial insulin and any increase in local leg glucose uptake. The diversion of insulin from the interstitium under hyperlipidemic conditions may play a role in the insulin resistance observed coincident with elevated nocturnal FFAs as is observed in obesity.

Rimonabant prevents additional accumulation of visceral and subcutaneous fat during high-fat feeding in dogs.

We investigated whether rimonabant, a type 1 cannabinoid receptor antagonist, reduces visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) in dogs maintained on a hypercaloric high-fat diet (HHFD). To determine whether energy expenditure contributed to body weight changes, we also calculated resting metabolic rate. Twenty male dogs received either rimonabant (1.25 mg.kg(-1).day(-1), orally; n = 11) or placebo (n = 9) for 16 wk, concomitant with a HHFD. VAT, SAT, and nonfat tissue were measured by magnetic resonance imaging. Resting metabolic rate was assessed by indirect calorimetry. By week 16 of treatment, rimonabant dogs lost 2.5% of their body weight (P = 0.029), whereas in placebo dogs body weight increased by 6.2% (P < 0.001). Rimonabant reduced food intake (P = 0.027), concomitant with a reduction of SAT by 19.5% (P < 0.001). In contrast with the VAT increase with placebo (P < 0.01), VAT did not change with rimonabant. Nonfat tissue remained unchanged in both groups. Body weight loss was not associated with either resting metabolic rate (r(2) = 0.24; P = 0.154) or food intake (r(2) = 0.24; P = 0.166). In conclusion, rimonabant reduced body weight together with a reduction in abdominal fat, mainly because of SAT loss. Body weight changes were not associated with either resting metabolic rate or food intake. The findings provide evidence of a peripheral effect of rimonabant to reduce adiposity and body weight, possibly through a direct effect on adipose tissue.

Direct administration of insulin into skeletal muscle reveals that the transport of insulin across the capillary endothelium limits the time course of insulin to activate glucose disposal.

OBJECTIVE: Intravenous insulin infusion rapidly increases plasma insulin, yet glucose disposal occurs at a much slower rate. This delay in insulin's action may be related to the protracted time for insulin to traverse the capillary endothelium. An increased delay may be associated with the development of insulin resistance. The purpose of the present study was to investigate whether bypassing the transendothelial insulin transport step and injecting insulin directly into the interstitial space would moderate the delay in glucose uptake observed with intravenous administration of the hormone. RESEARCH DESIGN AND METHODS: Intramuscular injections of saline (n = 3) or insulin (n = 10) were administered directly into the vastus medialis of anesthetized dogs. Injections of 0.3, 0.5, 0.7, 1.0, and 3.0 units insulin were administered hourly during a basal insulin euglycemic glucose clamp (0.2mU x min(-1) x kg(-1)). RESULTS: Unlike the saline group, each incremental insulin injection caused interstitial (lymph) insulin to rise within 10 min, indicating rapid diffusion of the hormone within the interstitial matrix. Delay in insulin action was virtually eliminated, indicated by immediate dose-dependent increments in hindlimb glucose uptake. Additionally, bypassing insulin transport by direct injection into muscle revealed a fourfold greater sensitivity to insulin of in vivo muscle tissue than previously reported from intravenous insulin administration. CONCLUSIONS: Our results indicate that the transport of insulin to skeletal muscle is a rate-limiting step for insulin to activate glucose disposal. Based on these results, we speculate that defects in insulin transport across the endothelial layer of skeletal muscle will contribute to insulin resistance.

Abdominal obesity: role in the pathophysiology of metabolic disease and cardiovascular risk.

Visceral adiposity has been identified as an independent risk factor for cardiovascular disease and the so-called metabolic syndrome. The canine obesity model closely recapitulates the correlation between human visceral adiposity and insulin resistance. A recent canine study indicates that insulin expands the volume of distribution associated with skeletal muscle, and that its ability to enhance macromolecular distribution within this space is blunted in the fat-fed obese canine model. Our canine study supports the portal theory of insulin resistance, in which free fatty acids (FFAs) from visceral fat directly enter the liver and have a detrimental effect on insulin action. The role of adipokines in this condition remains less clear. Sympathetic nervous system hyperactivity in obesity may also contribute to excessive FFA release, hypertension, and insulin resistance. Pathologies interrelated with insulin resistance include beta-cell hypersecretion, reduced insulin clearance, and resultant hyperinsulinemia. An observed nocturnal increase in plasma FFA levels may account for both insulin resistance and compensatory hyperinsulinemia and warrants further investigation. The elucidation of these interrelated pathologies may help reveal points where medical intervention can reduce metabolic disease.

Nocturnal free fatty acids are uniquely elevated in the longitudinal development of diet-induced insulin resistance and hyperinsulinemia.

Obesity is strongly associated with hyperinsulinemia and insulin resistance, both primary risk factors for type 2 diabetes. It has been thought that increased fasting free fatty acids (FFA) may be responsible for the development of insulin resistance during obesity, causing an increase in plasma glucose levels, which would then signal for compensatory hyperinsulinemia. But when obesity is induced by fat feeding in the dog model, there is development of insulin resistance and a marked increase in fasting insulin despite constant fasting FFA and glucose. We examined the 24-h plasma profiles of FFA, glucose, and other hormones to observe any potential longitudinal postprandial or nocturnal alterations that could lead to both insulin resistance and compensatory hyperinsulinemia induced by a high-fat diet in eight normal dogs. We found that after 6 wk of a high-fat, hypercaloric diet, there was development of significant insulin resistance and hyperinsulinemia as well as accumulation of both subcutaneous and visceral fat without a change in either fasting glucose or postprandial glucose. Moreover, although there was no change in fasting FFA, there was a highly significant increase in the nocturnal levels of FFA that occurred as a result of fat feeding. Thus enhanced nocturnal FFA, but not glucose, may be responsible for development of insulin resistance and fasting hyperinsulinemia in the fat-fed dog model.

Thermodynamic stability of a kappaI immunoglobulin light chain: relevance to multiple myeloma.

Immunoglobulin light chains have two similar domains, each with a hydrophobic core surrounded by beta-sheet layers, and a highly conserved disulfide bond. Differential scanning calorimetry and circular dichroism were used to study the folding and stability of MM-kappaI, an Ig LC of kappaI subtype purified from the urine of a multiple myeloma patient. The complete primary structure of MM-kappaI was determined by Edman sequence analysis and mass spectrometry. The protein was found to contain a cysteinyl post-translational modification at Cys(214). Protein stability and conformation of MM-kappaI as a function of temperature or denaturant conditions at pH 7.4 and 4.8 were investigated. At pH 4.8, calorimetry demonstrated that MM-kappaI undergoes an incomplete, cooperative, partially reversible thermal unfolding with increased unfolding temperature and calorimetric enthalpy as compared to pH 7.4. Secondary and tertiary structural analyses provided evidence to support the presence of unfolding intermediates. Chemical denaturation resulted in more extensive protein unfolding. The stability of MM-kappaI was reduced and protein unfolding was irreversible at pH 4.8, thus suggesting that different pathways are utilized in thermal and chemical unfolding.