J.L. Johnson, D.P. Wan, R.J. Koletsky & Paul Ernsberger Department of Nutrition, Case Western Reserve University, Cleveland, Oh 44106

Induction of dietary obesity in the spontaneously hypertensive rat (SHR) strain would facilitate study of the interactions between obesity and hypertension. Mutant rats on the same genetic background have a nonsense mutation of the leptin receptor (SHROB) and become extremely obese on normal chow. Several other groups have fed solid diets high in fat and sugar to SHR and failed to induce weight gain, implying resistance to dietary obesity. We developed a liquid dietary supplement providing 32% sucrose + 8% heavy whipping cream +micronutrients. After 70d a 25% weight gain was achieved while controls gained less than 5%. The depot most expanded in males was the retroperitoneal (♂: 250%, ♀: 150%) and in females the gonadal (♂: 81%, ♀: 250%) while mesenteric fat increased for both genders (♂: 245%, ♀: 237%). Genetic obesity most affected the subscapular depot in both genders. Dietary obese SHR showed even higher blood pressures (227±7mmHg; N=8) than control SHR (199±8; N=8) and also higher pressures than genetically obese SHROB (190±2; N=18). Cardiac hypertrophy was present in both dietary and genetic obesities. Fasting insulin was increased in dietary obese SHR (1.35±0.07ng/ml) compared to control SHR (0.46±0.05) but were below levels in SHROB (5.0±2.2). In an oral glucose tolerance test, the glucose area under the curve showed a similar pattern of differences (dietary obese SHR: 18.5 g*min/dL control SHR: 8.07; SHROB: 24.8). Thus, comparison of dietary and genetic obesities on the SHR background will allow inferences about the role of leptin in metabolism and blood pressure regulation.

P. Ernsberger, R. A. Velliquette, J.L. Johnson and R.J. Koletsky

Fluctuating body weight may impact health. The spontaneously hypertensive obese rat (SHROB) subjected to intermittent very low calorie diet (VLCD), is a model of weight cycling. Previous findings indicated that a transient increase in blood pressure occurs during rapid weight regain, referred to as refeeding hypertension. In the present study, we tested changes in glucose tolerance and insulin resistance. We first studied metabolic events during one cycle of weight lost and regain in SHROB. After a 14d VLCD of Optifast providing one-sixth of baseline calories, SHROB were refed ad lib until their body weight equaled control ad lib fed SHROB. Triglycerides and free fatty acids fell by 55% and 48%, respectively, after VLCD and returned to baseline after refeeding. Oral glucose tolerance and insulin secretion were acutely impaired by VLCD, as shown by reduced insulin area under the curve (AUC) (2800 ± 480 ng*min/mL vs. 6600 ± 2600 ng*min/mL in controls) and elevated glucose AUC (56,700 ± 12,600 mg*min/mL vs. 33,000 ± 5200 mg*min/mL in controls). After refeeding, body weight did not differ (506 ± 21g vs. 517 ± 16g in controls) nor did total combined fat depot mass (172 ± 6 g/kg body weight vs. 170 ± 10 g/kg in controls). Weight cycled SHROB showed reduced glucose AUC (18,000 ± 890 mg*min/mL) with unchanged insulin AUC relative to controls. Subsequent studies tested glucose tolerance 30d after weight regain and the beneficial effect of weight loss was diminished but still significant. Further studies examined three cycles of weight loss and regain. Improved glucose tolerance was also seen. Following initial glucose intolerance immediately after VLCD, weight cycling may create an anabolic state characterized by increased disposal of a glucose load. Thus, improvements in glucose tolerance and insulin resistance from weight loss persist following regain of lost weight.

Zheng Sun, B.S. and Paul Ernsberger, Ph.D.. 1Cleveland, OH, United States.

Background: The obese spontaneously hypertensive rat (SHROB) is an established model for human metabolic syndrome X, expressing insulin resistance, hypertension, obesity, and hyperlipidemia. Previous studies showed reduced cellular responses to insulin in liver and skeletal muscle in SHROB relative to lean SHR littermates. Specifically, tyrosine phosphorylation of the insulin receptor and its substrate are reduced. We now tested adipocytes and examined a further downstream step insulin signaling through PKB (Akt), which has been implicated in human insulin resistance. SHROB and SHR rats were matched for gender and age, and 4 g of the gonadal depot was digested 1h at 37oC with collagenase. Floating cells were filtered through 200 m mesh, preincubated, then exposed to insulin concentrations ranging from 0 to 100nM insulin for 0 to 90 min. Cells were lysed by free-thaw in medium containing detergent. Aliquots of cell extracts were analyzed by Western blot. PKB activation was defined as the ratio of immunoreactivity for phosphorylated PKB to total PKB in each sample. Lean SHR adipocytes showed peak 25-fold activation of PKB at 3 min. SHROB adipocytes, in contrast, showed peak activation of only 4-fold, maintained for 90 min. In dose-response experiments, SHR and SHROB adipocytes showed large differences in maximum fold response (Emax) at 10 min: 18.8 +/- 2.3 vs. 3.7 +/- 0.8. Also consistent with reduced insulin sensitivity, the ED50 for insulin was lower in SHR than in SHROB (3.5 +/- 0.5 vs. 29 +/- 3.8 nM). Thus, the PKB activation step of the insulin signaling pathway in adipocytes from SHROB shows profound resistance to activation by insulin, suggesting that this step could be an important indicator or target for the treatment of insulin resistance.

Janean Johnson & Paul Ernsberger

Recent studies in human and rat adipocytes have shown that cells isolated from obese subjects are deficient in fatty acid mobilization. We tested lipolytic response not in a static incubation but in a perifusion system to mimic contact with the circulation, to reduce autocrine effects, and to allow continuous monitoring of glycerol output. We compared freshly isolated retroperitoneal adipocytes from SHROB (Koletsky) rats which have a naturally occurring knockout of the leptin receptor relative to lean SHR littermates. We also tested subscapular adipocytes from SHROB. A perifusion system was used to reduce autocrine effects and allow continuous monitoring of glycerol output in 4min fractions. Krebs' medium was perifused at 0.25mL/min and contained 1 mg/mL albumin as a lipid carrier and 2 μg/mL adenosine deaminase. The first 4 fractions defined baseline glycerol concentration, which was higher in SHROB than in SHR retroperitoneal adipocytes (27±2 vs. 21±2μM), and higher still in SHROB subscapular adipocyte perifusates (42±4μM). As shown in the figure, the response of retroperitoneal adipocytes to 4min exposure 1μM isoproterenol was robust in SHR, with a maximum increase of nearly 75% in the rate of glycerol efflux, peaking at 12 min and recovering only gradually over the next 16 min. The isoproterenol response in SHROB was attenuated by two-thirds in peak magnitude and also reduced in duration. Subscapular adipocytes showed no detectable lipolytic response to 1μM isoproterenol. Insulin (10 and 100 nM) suppressed glycerol release in both basal and isoproterenol-stimulated conditions, and 1 nM insulin almost completely suppressed responses even in insulin resistant SHROB cells. Impaired mobilization of adipocyte triglyceride in SHROB may contribute to resistance to weight loss and susceptibility to weight regain.

Magnetic resonance imaging of visceral and subcutaneous fat distribution in genetic obesity of SHROB rats and dietary obesity of overfed SHR

Wan, D.P., Johnson, D.H., Johnson, J.L., Koletsky, R.J., Flask, C. and Ernsberger, P.

Magnetic resonance imaging (MRI) is a noninvasive method that can be used to identify, map and quantify fat depots in living subjects. We sought to map the distribution of abdominal visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) in two models of obesity in the rat, one genetic and one dietary-induced. We hypothesized that the relative distribution of VAT and SAT may differ between these models, and metabolic disturbances may track with the relative proportion of VAT rather than just overall adiposity. The spontaneously hypertensive obese rat (SHROB) has a naturally occurring knockout of the leptin receptor and served as the genetic model. The spontaneously hypertensive rat (SHR), genetically identical to SHROB with the exception of the obesity-inducing mutation, was used as the lean control and was fed normal chow (4.05 kcal/g). The dietary obese model was obtained by feeding SHR a dietary supplement (1.59 kcal/mL) consisting of 24% sucrose, 8% polycose, and 8% cream supplemented with appropriate micronutrients, until a 20% increase from baseline weight was achieved. Female rats were tested at around 8 mo of age and anesthetized with enflurane. Images were acquired as T1-weighted spin echo water-cancelled scans (TE 13ms, TR 1240 ms) with 4 averaged passes on a 1.5 Tesla clinical MRI and rodent-sized coil. The acquisition was coronal with 2 mm thickness and 0.78 mm x 0.78 mm in-plane resolution. The MRI sequence data were collected by using standard software (Analyze). The peritoneum was manually traced in each slice to delineate the VAT and SAT, and organs and other non-adipose tissue were removed from the visceral area by lower-bound thresholding. In regions of poor fat-tissue contrast, adipose tissue areas were delineated manually. In lean SHR control, 22.8 mL of total adipose tissue could be divided into 13.7 mL (60.1%) of VAT and 9.1 mL (39.9%) of SAT (1.5:1 VAT:SAT ratio). In the dietary obese SHR, 85.2 mL total was measured comprising 53.3 mL (62.6%) VAT and 31.9 mL (37.4%) SAT (1.7:1 VAT:SAT). In the genetically obese SHROB model, 274.6 mL of total was calculated, with 113.4 mL (41.3%) VAT and 161.2 mL (58.7%) SAT (0.7:1 VAT:SAT). Although total VAT volume was markedly higher in the genetically obese model, the VAT:SAT ratio of the dietary obese model was markedly higher. This predicts that despite a lower total adipose tissue volume compared to genetic obesity, dietary obesity may be more closely associated with metabolic derangements, a conclusion supported by physiological data.

Experimental Biology will be combined with a meeting of the International Union of Physiology in sunny San Diego, March 31 to April 5. My student Zheng Sun and I each submitted an abstract.