Isabel R. Hsu

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

Metabolic syndrome, hyperinsulinemia, and cancer

The term metabolic syndrome describes the association between obesity, insulin resistance, and the risk of several prominent chronic diseases, including cancer. The causal link between many of these components remains unexplained, however. What is clear are the events that precede the development of the syndrome itself. In animal models, a fat-supplemented diet causes 1) lipid deposition in adipose depots, 2) insulin resistance of liver and skeletal muscle, and 3) hyperinsulinemia. One hypothesis relating fat deposition and insulin resistance involves enhanced lipolysis in the visceral depot, which leads to an increase in free fatty acid (FFA) flux. Increased mass of stored lipid and insulin resistance of visceral adipocytes favors lipolysis. Additionally, hypersensitivity of visceral adipose cells to sympathetic nervous system stimulation leads to increased lipolysis in the obese state. However, little evidence is available for enhanced plasma FFA concentrations in the fasting state. We measured FFA concentrations over a 24-h day in obese animals and found that plasma FFAs are elevated in the middle of the night, peaking at 0300. Therefore, it is possible that nocturnal lipolysis increases exposure of liver and muscle to FFAs at night, thus causing insulin resistance, which may play a role in hyperinsulinemic compensation to insulin resistance. Nocturnal lipolysis secondary to sympathetic stimulation may not only cause insulin resistance but also be responsible for hyperinsulinemia by stimulating secretion and reducing clearance of insulin by the liver. The resulting syndrome-elevated nocturnal FFAs and elevated insulin-may synergize and increase the risk of some cancers. This possible scenario needs further study.

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.

Consistency of the Disposition Index in the Face of Diet Induced Insulin Resistance: Potential Role of FFA

Objective Insulin resistance induces hyperinsulinemic compensation, which in turn maintains almost a constant disposition index. However, the signal that gives rise to the hyperinsulinemic compensation for insulin resistance remains unknown. Methods In a dog model of obesity we examined the possibility that potential early-week changes in plasma FFA, glucose, or both could be part of a cascade of signals that lead to compensatory hyperinsulinemia induced by insulin resistance. Results Hypercaloric high fat feeding in dogs resulted in modest weight gain, and an increase in adipose tissue with no change in the non-adipose tissue size. To compensate for the drop in insulin sensitivity, there was a significant rise in plasma insulin, which can be attributed in part to a decrease in the metabolic clearance rate of insulin and increased insulin secretion. In this study we observed complete compensation for high fat diet induced insulin resistance as measured by the disposition index. The compensatory hyperinsulinemia was coupled with significant changes in plasma FFAs and no change in plasma glucose. Conclusions We postulate that early in the development of diet induced insulin resistance, a change in plasma FFAs may directly, through signaling at the level of β-cell, or indirectly, by decreasing hepatic insulin clearance, result in the observed hyperinsulinemic compensation.

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.

Pulsatile changes in free fatty acids augment hepatic glucose production and preserves peripheral glucose homeostasis

Recent studies in animal and human models have revealed that free fatty acid (FFA) release from adipose tissue is oscillatory. We have shown in our laboratory that these oscillations are controlled by the sympathetic nervous system (SNS). Although FFAs have been shown to directly stimulate glucose production [endogenous glucose production (EGP)] by the liver and to reduce peripheral glucose utilization, whether the specific pattern of FFA release affects glucose metabolism is unknown. The aim of this study was to examine the effects of pulsatile vs. constant infusion of FFA on glucose homeostasis in the canine model. Euglycemic clamps with basal insulin replacement (0.1 mU.kg(-1).min(-1) insulin) were performed in dogs (n = 8) during infusion of saline (SAL) or the medium-chain fatty acid octanoate, which was given by either pulsatile infusion (PUL: 10 mmol over 2 min every 10 min) or continuous infusion (C-INF: 1 mmol/min) designed to achieve equivalent total FFA mass. Endogenous lipolytic pulses were suppressed with the beta(3)-specific adrenergic receptor antagonist bupranolol. PUL infusion elicited a pulsatile pattern of FFA in circulation with average maximum pulse height of 0.82 +/- 0.04 mM, whereas C-INF FFA levels reached 0.47 +/- 0.03 mM (fasting levels) and were maintained throughout. Glucose uptake was not affected by PUL; however, C-INF significantly reduced glucose uptake compared with both SAL and PUL. Steady-state EGP increased by >90% from basal steady state during PUL but did not change during either SAL or C-INF. Thus, pulsatile FFA infusion led to an increase in EGP while preserving glucose disposal. These data suggest that the pattern of FFA may have a role in regulation of glucose homeostasis, which may have consequences in the obese or insulin-resistant state where the SNS is known to be altered.

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.