Mutation of the RIIβ Subunit of Protein Kinase A Prevents Diet-Induced Insulin Resistance and Dyslipidemia in Mice

Sandra A. Schreyer 2 David E. Cummings 1 G. Stanley McKnight 0 Ren ee C. LeBoeuf 2 0 Department of Pharmacology, University of Washington, Seattle, Washing- ton. Department of Pathobiology , Box 353410 , University of Washington , Seattle, WA 98195 1 Department of Medicine, Division of Metabolism , Endocri- nology, and Nutrition , University of Washington , Seattle, Washington; and the 2 Department of Pathobiology, University of Washington , Seattle, Washington; the The mechanisms by which obesity contributes to diabetic phenotypes remain unclear. We evaluated the role of protein kinase A (PKA) signaling events in mediating diabetes associated with obesity. PKA comprises two regulatory subunits and two catalytic subunits and is activated by cAMP. The RII regulatory subunit is abundantly expressed in adipose tissue and brain. Knockout mice lacking this subunit are lean and display remarkable resistance to diet-induced obesity. We investigated whether these mice were also resistant to diet-induced diabetes and whether this effect was dependent on reduced adiposity. Mice were fed a high-fat, high-carbohydrate diet and weight gain and diabetes phenotypes were examined. RII / mice displayed decreased body weights, reduced insulin levels, improved insulin sensitivity, and improved total-body glucose disposal as compared with wild-type controls. Plasma levels of VLDL and LDL cholesterol were also reduced in high fat-fed RII / mice compared with wild-type mice. Taken together, these data demonstrate that loss of RII protects mice from diet-induced obesity, insulin resistance, and dyslipidemia. Diabetes 50:2555-2562, 2001 - R35% of American adults are now considered ecent reports indicate that obesity is rapidly increasing in industrialized nations. More than overweight or obese (1), and obesity is an important contributing factor to the development of type 2 diabetes (2 4). Dyslipidemia is often observed in association with obesity and diabetes and is a significant contributor to the increased mortality observed in diabetic patients (5,6). Characteristics of type 2 diabetes include impaired glucose disposal, diminished insulin production, and increased hepatic glucose output. The mechanisms by which obesity contributes to these phenotypes remain unclear (7). Signaling via protein kinase A (PKA) plays an important role in regulating metabolism and body weight (8). PKA is activated by cAMP and comprises two regulatory and two catalytic subunits (8). Four regulatory isoforms (RI, RI , RII, and RII) and two catalytic isoforms (C and C) are expressed in the mouse, and each is encoded by a separate gene. The RII subunit is expressed principally in three tissues known to regulate energy homeostasis: brown adipose tissue, white adipose tissue, and brain (8,9). Recent studies suggest that the induction of PKA in certain tissues may decrease obesity. For example, activation of the adipose-specific -adrenergic receptor (10), which signals via PKA, decreases obesity in both genetically obese (ob/ob) (11,12) and diet-induced obese mice (13), suggesting that signaling mechanisms through this pathway are important in preventing obesity. Studies in mice lacking a specific PKA subunit, RII, have revealed an unexpected role for this protein in regulating energy balance (9). RII knockout mice (RII / ) remain remarkably lean even when challenged with a high-fat diet (9). These animals have increased metabolic activity, manifested by increases in body temperature, uncoupling protein 1 concentration, and lipid hydrolysis. Biochemical studies have shown that loss of RII was compensated by increased RI regulatory subunit, which is more sensitive to cAMP activation and results in a net increase in basal PKA activity (14). These studies suggest that increasing basal PKA activity in adipose tissue and brain ameliorates obesity. In this study, we sought to determine whether loss of RII influences the development of diabetes and dyslipidemia associated with obesity. Wild-type and RII / mice, maintained on the C57BL/6 genetic background strain, were fed a high-fat, high-carbohydrate diet. This diet is known to induce obesity and diabetes in C57BL/6 mice (15,16). RII / mice were resistant to weight gain and hyperinsulinemia. In vivo insulin sensitivity and glucose disposal were dramatically improved in the RII / mice, as were plasma lipid profiles. When mice were corrected for differences in body weight, improved insulinmediated glucose disposal was still observed in the RII / mice, suggesting an obesity-independent effect of RII on promoting insulin resistance. We suggest that PKA activity in both adipose tissue and brain is important for determining body composition, food intake, and diabetogenic parameters. Thus, PKA is an attractive therapeutic target for preventing and treating obesity and the coinciding disorders of insulin resistance and dyslipidemia. RII-deficient (RII ) mice. Mice were originally generated on a 50:50 (129XC57BL/6) genetic background (9). Mice have been backcrossed to C57BL/6J (Jackson Laboratory, Bar Harbor, ME) for five generations and then interbred to produce the RII / and wild-type littermates used here. Experimental design. The two diets used in our studies were rodent chow (Wayne Rodent BLOX 8604; Teklad, Madison, WI) and a high-fat, high-sucrose diabetogenic diet (No. F1850; Bioserve, Frenchtown, NJ) containing 35% (wt/wt) fat (primarily lard) and 37% carbohydrate (primarily sucrose). For all experiments, mice were maintained in a 25C facility with a strict 12-h light/dark cycle (6:00 A.M./6:00 P.M.) and were given free access to food and water. Unless otherwise noted, food was removed from mice 4 h before the collection of blood from the retro-orbital sinus into tubes containing anticoagulant (1 mmol/l EDTA). Plasma was used immediately or stored at 70C until analysis. Mice were killed by cervical dislocation. This project was approved by the Animal Care and Use Committee of the University of Washington. Two separate experiments were performed. In the first study (Study 1), male RII / and wild-type mice were maintained on a rodent chow until they were 16 20 weeks old and then were fed the diabetogenic diet for 15 weeks. Body weights, food intake, plasma glucose, insulin, and leptin levels were quantified. Food intake was estimated from the difference in food remaining in the food trough between the afternoon, when food was given, and the next day, when food troughs were replaced. The total amount of food eaten by one to four mice per cage, in each of four cages per genotype, was averaged over 3-day periods during weeks 2, 4, 6, 8, and 11 (17). In the second experiment (Study 2), comparisons between sexes were made. Male and female RII / and wild-type mice, 9 10 weeks old, were fed the diabetogenic diet for 15 weeks. Body weights were monitored throughout the feeding study. At 12 and 14 weeks of dietary treatment, mice were subjected to an insulin sensitivity a (...truncated)