Karyn J. Catalano

Why visceral fat is bad : Mechanisms of the metabolic syndrome

A consensus has emerged that fat stored in the central segment of the body is particularly damaging in that it portends greater risk for diabetes, cardiovascular disease, hypertension, and certain cancers (1–3). It is also accepted that insulin resistance is a related characteristic that may be an essential link between central fat and disease risk. Additionally, it is possible that the hyperinsulinemia that accompanies insulin resistance in non-diabetic but at-risk individuals may magnify, or even mediate, some of the detrimental effects of visceral adiposity (4–6). However, there is less information regarding the mechanisms that may link visceral fat with risk for disease. For example, there is controversy regarding the specific mechanisms by which fat in the visceral compartment confers greater risk than subcutaneous fat. Many investigators have suggested that one or more moieties secreted by the visceral adipocyte might mediate insulin resistance. Among the socalled “bad actors” are free fatty acids (FFAs) themselves (“portal theory”) (7–9) or the adipose tissue–released cytokines (adipokines) such as interleukin-1, interleukin-6, tumor necrosis factor, resistin, or a reduction in adiponectin, which has been repeatedly shown to be associated with reduced insulin resistance (10–13). Of course, insulin itself could be involved, as other adipose-secreted protein compounds not yet identified. But why visceral fat? Is it because of the unique anatomical position of the visceral fat depot, with effluent entering the liver, or is it because of molecular characteristics of visceral fat itself, which may favor release of damaging molecules into the systemic circulation? These questions remain unanswered. However, in our laboratory, we have developed the obese dog model, which has led to some understanding of the pathogenesis of the metabolic syndrome. The dog model has not been widely used for the study of the metabolic syndrome, but we have found it to have several important characteristics that we have been able to exploit: the ability to make longitudinal measurements and the ability to access the portal vein. In that sense the dog is a unique model, in that these latter measurements are daunting in rodents, and carrying out repetitive, invasive clinical measurements in non-human primates is challenging. Also, the dog with visceral obesity has turned out to be a reasonable model for a similar syndrome in humans (Figure 1). In fact, the dog is genetically more similar to humans than is the rodent. Here we summarize a significant amount of evidence in which we examined what we considered to be the simplest hypothesis composed of two postulates: 1) that FFAs per se are among the most important products of the visceral adipocyte to cause insulin resistance (and hence the metabolic syndrome) and 2) that the anatomical position of the visceral adipose depot (i.e., portal drainage into the liver) plays an important role in the pathogenesis of the metabolic syndrome. While we cannot say that these postulates are proven, there are data that support them, and Occam’s razor instructs us to accept them until proven untrue. Whether true or not, it appears that examining them has led us to a deeper understanding of the physiological basis for the metabolic syndrome itself. One similarity between dogs and humans is the wide variance in fat deposition in a “wild” or “natural” population. We measure distribution of fat about the truncal region using magnetic resonance imaging [Figure 2; 11 axial slices: 1-cm landmark slice at the umbilicus (left renal artery) 5 cm]. Similar to human subjects (14,15), there is surprising variability in distribution. Some animals are strikingly lean, with total fat varying over a factor of 5, from 10 to 50 cm/cm non-fat tissue. Interestingly, there is a tendency for visceral adiposity to increase rapidly as one examines animals with increasing body fat; the visceral fat depot tends to plateau, and subcutaneous fat increases more rapidly with overall obesity. This tendency for visceral fat to Department of Physiology and Biophysics, Keck School of Medicine, University of Southern California, Los Angeles, California. Address correspondence to Richard N. Bergman, Department of Physiology and Biophysics, MMR 630, 1333 San Pablo Street, Los Angeles CA 90033. E-mail: rbergman@usc.edu Copyright

Critical role of the mesenteric depot versus other intra-abdominal adipose depots in the development of insulin resistance in young rats

OBJECTIVE Age-associated insulin resistance may be caused by increased visceral adiposity and older animals appear to be more susceptible to obesity-related resistance than young animals. However, it is unclear to what extent the portally drained mesenteric fat depot influences this susceptibility. RESEARCH DESIGN AND METHODS Young high-fat–fed and old obese rats were subjected to 0, 2, 4, or 6 weeks of caloric restriction. Insulin sensitivity (SI) was assessed by hyperinsulinemic clamp and lean body mass (LBM) and total body fat were assessed by 18O-water administration. RESULTS Six weeks of caloric restriction caused a similar reduction in body weight in young and old animals (P = 0.748) that was not due to reduced subcutaneous fat or LBM, but rather preferential loss of abdominal fat (P < 0.05). Most notably, mesenteric fat was reduced equivalently in young and old rats after 6 weeks of caloric restriction (∼↓53%; P = 0.537). Despite similar visceral fat loss, SI improved less in old (↑32.76 ± 9.80%) than in young (↑82.91 ± 12.66%) rats versus week 0. In addition, there was significantly more reversal of fat accumulation in the liver in young (% reduction: 89 ± 2) versus old (64 ± 5) rats (P < 0.0001). Furthermore, in young rats, SI changed much more rapidly for a given change in mesenteric fat versus other abdominal depots (slope = 0.53 vs. ≤0.27 kg/min/mg per % fat). CONCLUSIONS Improved SI during caloric restriction correlated with a preferential abdominal fat loss. This improvement was refractory in older animals, likely because of slower liberation of hepatic lipid. Furthermore, mesenteric fat was a better predictor of SI than other abdominal depots in young but not old rats. These results suggest a singular role for mesenteric fat to determine insulin resistance. This role may be related to delivery of lipid to liver, and associated accumulation of liver fat.

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.

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.

CB1 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.

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 R2 = 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 &bgr;-cell glucose responsiveness. The isolation variables associated with the outcomes in our canine model confirm previous reports in other species, including humans.

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.

Variable Hepatic Insulin Clearance with Attendant Insulinemia is the Primary Determinant of Insulin Sensitivity in the Normal Dog

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.

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: Factors Determining Fasting Insulin Sensitivity

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.