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

¹ Department of Pathobiology, University of Washington, Seattle, Washington
² Department of Medicine, Division of Metabolism, Endocrinology, and Nutrition, University of Washington, Seattle, Washington
³ Department of Pharmacology, University of Washington, Seattle, Washington

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESEARCH DESIGN AND METHODS RESULTS DISCUSSION REFERENCES

 
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 RIIß 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. RIIß^-/- 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 RIIß^-/- mice compared with wild-type mice. Taken together, these data demonstrate that loss of RIIß protects mice from diet-induced obesity, insulin resistance, and dyslipidemia.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESEARCH DESIGN AND METHODS RESULTS DISCUSSION REFERENCES

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, RIß, RII, and RIIß) and two catalytic isoforms (C and Cß) are expressed in the mouse, and each is encoded by a separate gene. The RIIß 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 ß-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, RIIß, have revealed an unexpected role for this protein in regulating energy balance (9). RIIß knockout mice (RIIß^-/-) 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 RIIß 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 RIIß influences the development of diabetes and dyslipidemia associated with obesity. Wild-type and RIIß^-/- 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). RIIß^-/- mice were resistant to weight gain and hyperinsulinemia. In vivo insulin sensitivity and glucose disposal were dramatically improved in the RIIß^-/- mice, as were plasma lipid profiles. When mice were corrected for differences in body weight, improved insulin-mediated glucose disposal was still observed in the RIIß^-/- mice, suggesting an obesity-independent effect of RIIß 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.

	RESEARCH DESIGN AND METHODS

TOP ABSTRACT INTRODUCTION RESEARCH DESIGN AND METHODS RESULTS DISCUSSION REFERENCES

 
RIIß-deficient (RIIß^-/-) 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 RIIß^-/- 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 25°C 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 -70°C 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 RIIß^-/- 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 RIIß^-/- 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 assay and an intraperitoneal glucose tolerance test (IPGTT), respectively. At 15 weeks, mice were bled and killed, and the individual fat pads were weighed (reproductive and pairs of inguinal, retroperitoneal/renal, and brown adipose tissue).

Analytical procedures.
Plasma insulin and leptin were measured using radioimmunoassay kits (No. RI-13K and No. ML-82K; Linco, St. Louis, MO) with rat insulin and mouse leptin as the respective standards. Plasma glucose levels were determined colorimetrically (Cat. #315-100; Sigma, St. Louis, MO). Plasma triglyceride concentrations were assessed after the removal of free glycerol (Diagnostic Kit #450032; Boehringer Mannheim, Indianapolis, IN) (18). Plasma cholesterol levels were determined using a colorimetric kit (Diagnostic Chemicals Ltd., Oxford, CT). Plasma lipoproteins were separated by fast-performance liquid chromatography gel filtration using a Superose 6 column (Pharmacia LKB Biotechnology, Uppsala, Sweden). A 200-µl aliquot of plasma from each of three mice per diet group was analyzed at a flow rate of 0.2 ml/min using phosphate-buffered saline (PBS). Next, 100-µl aliquots from each 0.5-ml fraction were used for total cholesterol determinations.

IPGTT.
IPGTTs were performed essentially as described (18). Mice were fasted overnight (18 h) and injected intraperitoneally with 10% glucose in PBS at a dose of 2 g glucose/kg body wt. Plasma glucose was monitored before glucose injection and at 30, 60, 120, and 240 min after injection.

Insulin sensitivity assay.
Mice were fasted overnight and injected intraperitoneally with pork insulin (Eli Lilly, Indianapolis, IN) at a dose of 1.0 units insulin/kg body wt (18). A pilot experiment was performed in which six male mice of each genotype were fed the diabetogenic diet for 12 weeks. Mice were injected with insulin and glucose levels were determined after 0, 15, 30, 60, and 90 min. Because glucose disposal was not delayed due to the knockout genotype (data not shown) and for ease of study, 30 min was chosen as a time point to represent glucose response to injected insulin. Thus, for the current studies, plasma glucose was monitored before and 30 min after insulin injection. The percentage decrease in glucose between these time points was then calculated as follows: % glucose disposed = [(glucose_t=0 - glucose_t=30)/glucose_t=0] x 100.

Immunoblot procedures.
Apolipoproteins were quantified using immunoblotting procedures essentially as described (19,20). Plasma samples (2 µl) from male mice fed the diabetogenic diet for 15 weeks were applied to SDS-PAGE, electrophoresed, and transferred to nitrocellulose membranes. Specific protein bands were detected after incubation of filters with monospecific antibodies (diluted 1:1,000) for apolipoprotein (apo)-B, apoA-1, or apoE (Cat. #K23300R, #K23500R, and #K23100R; Biodesign International, Kennebunk, ME), followed by incubation with iodinated protein A (apoA-1 and apoE) (Cat. #NE146 l; NEN Life Sciences, Boston, MA) or enhanced chemiluminescence (apoB) (Cat. #RPN2209; Amersham Pharmacia Biotech, Piscataway, NJ). Band intensities were measured using a scanning densitometer (Molecular Analyst GS 700; BioRad, Hercules, CA) or the Cyclone Phosphorimaging analysis system (Packard Instruments, Downers Grove, IL).

Levels of the PKA subunits, RIIß, RII, and RI, were evaluated using procedures as described (9). Briefly, brown adipose tissue, testis, or islet protein homogenates (40 µg per lane) were resolved by 10% SDS-PAGE and transferred to nitrocellulose membranes. Uniformity of protein loading was confirmed by Coomassie Blue staining (not shown). Polyclonal antibodies raised to recombinant murine PKA subunits were provided by J. Scott and diluted as follows: RIIß 1:10,000; RII 1:1,000; and RI 1:200. Primary antibodies were visualized using horseradish peroxidase–coupled goat anti–rabbit Ig (1:40,000) and enhanced chemiluminescence (Amersham).

Hepatic lipid measurements.
Lipids were extracted from mouse livers as described (19). Triglyceride and cholesterol content were then measured on extracted samples using the colorimetric kits.

Statistics.
Data are presented as means ± SE. Differences between genotypes were determined using the Student’s t test. Pearson’s correlation coefficients were used to assess relations between adiposity and glucose disposal. P < 0.05 was accepted as statistically significant.

	RESULTS

TOP ABSTRACT INTRODUCTION RESEARCH DESIGN AND METHODS RESULTS DISCUSSION REFERENCES

 
Longitudinal analysis of RIIß^-/- mice.
Body weights increased steadily for male mice of both genotypes fed the diabetogenic diet (Study 1) (Fig. 1A). RIIß^-/- mice were 20% lighter than wild-type mice at initial (0 weeks) and final (15 weeks) time points (Table 1). Although mice of both genotypes showed comparable changes in percent body weight by the end of the study (e.g., 60% at 15 weeks), RIIß^-/- mice gained weight more slowly than did wild-type mice.

View larger version (39K): [in this window] [in a new window]   FIG. 1. Body weights and plasma levels of leptin, glucose, and insulin for male wild-type and RIIß-/- mice fed a high-fat, high-sucrose (diabetogenic) diet. Mice were introduced to the diet at 16–20 weeks of age. A: Body weights of wild-type mice () were significantly higher (P < 0.001) than those of mutant mice (•) at all time points. B: Leptin levels were significantly higher for wild-type mice than for RIIß-/- mice between 0 and 10 weeks of diet feeding (P < 0.05). C: Glucose levels were significantly different between genotypes at 4 weeks (P < 0.05). (Inset: Ratios of insulin to glucose were significantly higher for wild-type mice than mutant mice for weeks 3–15 [P < 0.05]). D: Plasma insulin was significantly higher in wild-type mice than in mutant mice at 4 and 8 weeks (P < 0.05). Values are presented as means ± SE for n = 10–12 mice per set.

Body weights correlated significantly with leptin levels for both wild-type (r = 0.88, P < 0.01) and RIIß^-/- (r = 0.95, P < 0.008) mice over the 15 weeks of study. Food intake was measured to determine whether differences in weight and leptin levels were reflected as differences in caloric consumption. Average food intake over the 15-week period was higher for wild-type mice than for RIIß^-/- mice (15.4 ± 0.6 vs. 13.8 ± 0.6 cal/mouse/day, P = 0.06). However, when food consumption was corrected to differences in body weight, the RIIß^-/- mice actually consumed more calories. Wild-type mice consumed 0.37 ± 0.02 cal/g mouse/day, whereas RIIß^-/- mice consumed 0.44 ± 0.02 cal/g mouse/day (P = 0.005). Since the leptin levels were significantly different between the genotypes (Fig. 1B), leptin levels did not reflect food consumption in these mice.

Mice fed rodent chow (0 weeks) had plasma glucose levels that were comparable between genotypes (Table 1 and Fig. 1C). When the mice were fed the high-fat diet, glucose levels increased in both strains. Glucose levels for wild-type mice increased from 131 to 215 mg/dl, with final values after 15 weeks of 184 ± 8 mg/dl (P < 0.05 vs. 0 weeks). RIIß^-/- mice exhibited a steady but gradual increase in glucose with time on the diet to a final value of 205 ± 18 mg/dl (P < 0.001 vs. week 0). Except for the time point at 3 weeks (P < 0.005), plasma glucose levels did not differ significantly between genotypes.

In contrast to glucose, plasma insulin levels were consistently higher in wild-type than in RIIß^-/- mice (Fig. 1D). Initial insulin values were 26% higher for wild-type mice (Table 1) and were approximately two- to fivefold higher at all other time points. Final insulin levels were significantly higher than initial levels for both wild-type (P < 0.001) and RIIß^-/- (P < 0.001) mice. Overall, both genotypes were able to adjust insulin levels to compensate for increases in glucose, but insulin requirements were higher for wild-type mice than for RIIß^-/- mice.

The observation that RIIß^-/- mice were able to achieve glucose compensation with less insulin suggests improved insulin sensitivity in this strain. This is better reflected by examining the ratio of insulin to glucose, which would be expected to be higher for insulin-resistant mice. Indeed, over the course of the study, the average ratio of insulin to glucose for wild-type mice was 40% higher than that for RIIß^-/- mice (Fig. 1C inset). However, another interpretation of the reduced insulin levels in RIIß^-/- mice may be that loss of RIIß causes defects in insulin secretion. Immunoblotting of islet tissue taken from wild-type mice showed that RIIß protein is absent from this tissue (Fig. 2). Thus, it is unlikely that RIIß contributes directly to signaling pathways in ß-cells involved with insulin secretion. Taken together, these data are consistent with increased insulin sensitivity in the RIIß^-/- strain.

View larger version (36K): [in this window] [in a new window]   FIG. 2. Protein levels of PKA regulatory subunits in tissues taken from wild-type mice and assessed by Western blot. Tissue homogenates (40 µg per lane) were resolved by 10% SDS-PAGE and immunoblotting was performed using antibodies specific for RII and RI subunts, as described in RESEARCH DESIGN AND METHODS. RIIß protein is present in brown adipose tissue (BAT) but is absent in islets (Is). RI and RII proteins are observed in testis (T) and Is.

Body weight and adiposity.
At week 0 of the study (8 weeks of age), body weights for RIIß^-/- mice were 15% lower than those for wild-type mice within each sex (Fig. 3), consistent with data obtained for the older males (Fig. 1A). Absolute body weights were somewhat lower than those seen in Study 1 because the mice in Study 2 were younger. For instance, males weighed 20.3 ± 0.4 vs. 24.2 ± 0.8 g (P < 0.001) and females weighed 15.6 ± 0.7 vs. 19.7 ± 0.5 g (P < 0.0006) for RIIß^-/- and wild-type mice, respectively. When they were fed the diabetogenic diet, mice of both sexes experienced steady increases in body weight. Final body weights were 27 ± 1 vs. 43 ± 2 g for male RIIß^-/- versus wild-type mice, respectively (P < 0.0002), and 23 ± 1 vs. 31 ± 4 g for female RIIß^-/- versus wild-type mice, respectively (P < 0.03). RIIß^-/- mice of both sexes maintained lighter phenotypes than wild-type mice when fed either the rodent chow diet or the diabetogenic diet.

View larger version (18K): [in this window] [in a new window]   FIG. 3. Body weights for female (A) and male (B) mice fed the diabetogenic diet for 14 weeks. Wild-type () and RIIß-/- (•) mice were 8–10 weeks old at initiation of the diet. Values are presented as means ± SE for n = 4–7 mice per set. *P < 0.05 vs. wild-type mice.

Based on initial characterizations of RIIß^-/- mice (9), differences in actual body weights between genotypes were probably due to differences in adiposity. To test this, the investigators killed male and female mice (Study 2) after 15 weeks of consuming the diabetogenic diet, and four fat pad pairs (reproductive, inguinal, retroperitoneal/renal, and intrascapular brown adipose tissue) were collected and weighed (Fig. 4). Loss of RIIß resulted in reduced adiposities involving all fat pads with the exception of brown adipose tissue in females.

View larger version (18K): [in this window] [in a new window]   FIG. 4. Fat depot weights as a percent of body weight for female (A) and male (B) mice fed the diabetogenic diet for 15 weeks. Wild-type () and RIIß-/- () mice were killed after a 4-h fast and individual fat depots were removed and weighed. White adipose tissue fat pads measured were as follows: reproductive (epididymal or periuterine), inguinal, retroperitoneal plus perirenal, and the sum of these tissue weights (WAT). Intrascapular brown adipose tissue (BAT) weights were also measured. Values are presented as means ± SE for n = 4–7 mice per set. Significant differences between genotypes were seen at *P < 0.05 and **P < 0.001.

View larger version (15K): [in this window] [in a new window]   FIG. 5. IPGTT for wild-type () and RIIß-/- (•) mice fed the chow (A) or diabetogenic diet (B and C). Chow-fed male mice, 16 weeks old, were subjected to IPGTT after an overnight fast (A). Female (B) or male (C) mice were fed the diabetogenic diet for 14 weeks and fasted overnight before the IPGTT was performed. Values are presented as means ± SE for n = 5–6 mice per set. Significant differences between genotypes were seen at *P < 0.05 and **P < 0.001.

View larger version (23K): [in this window] [in a new window]   FIG. 6. Insulin-mediated glucose disposal measurements in chow diet– and diabetogenic diet–fed mice. A: Insulin-mediated glucose disposal for wild-type (WT) or RIIß-/- mice fed the chow or diabetogenic diet. Chow-fed male mice were subjected to the assay at 16 weeks of age (left panel). Male and female mice were fed the diabetogenic diet for 12 weeks before performing the insulin sensitivity assay (right panel). Mice from both Study 1 and Study 2 were used in this experiment. B: Relation between adipose weight and insulin-mediated glucose disposal for male or female mice fed the diabetogenic diet. Total weights of all white adipose depots (WAT) were used to estimate adiposity. Wild-type mice are presented by the open symbols and RIIß-/- mice are presented by the filled symbols. C: Insulin-mediated glucose disposal per gram body weight for mice fed the chow or diabetogenic diet. Values are presented as means ± SE for n = 4–13 mice per set. Significant differences between genotypes were seen at *P < 0.05 and **P < 0.001.

Plasma lipids.
Dyslipidemia is often observed in conjunction with obesity and diabetes. Plasma total cholesterol levels were significantly higher in wild-type mice than in RIIß^-/- male mice fed the diabetogenic diet for 15 weeks (250 ± 37 and 159 ± 12 mg/dl for wild-type and RIIß^-/- mice, respectively; P = 0.02). Female mice showed significantly lower levels of plasma total cholesterol than males (P < 0.05), and there was a trend toward lower levels in RIIß^-/- mice than in wild-type mice (132 ± 17 and 117 ± 10 mg/dl). No significant differences in plasma triglyceride or free fatty acid levels were observed between genotypes.

Plasma lipoprotein profiles were determined for diabetogenic diet–fed wild-type and RIIß^-/- mice (Fig. 7), and the results were consistent with total cholesterol levels. Although all mice showed an HDL fraction of comparable size and abundance, male RIIß^-/- mice had a dramatic decrease in the VLDL and LDL fraction as compared with wild-type mice. Female RIIß^-/- mice also showed reduced VLDL/LDL cholesterol compared with wild-type mice, although the difference was not as striking.

View larger version (20K): [in this window] [in a new window]   FIG. 7. Representative lipoprotein profiles for female (A) and male (B) mice fed the diabetogenic diet for 15 weeks. Lipoprotein profiles were assessed for n = 2–3 mice per group by fast-performance liquid chromatography as described in RESEARCH DESIGN AND METHODS. Typical results are presented for wild-type () and RIIß-/- (•) mice. VLDL/LDL and HDL elution profiles are illustrated.

Hepatic lipid content.
Hepatic cholesterol and triglyceride levels were quantified for male mice fed the diabetogenic diet for 15 weeks. Liver cholesterol levels were similar between genotypes (3.1 mg cholesterol/g liver), whereas the RIIß^-/- mice showed a 2.5-fold reduction in triglyceride levels (wild-type: 22.3 ± 2.2 mg/g; RIIß^-/-: 8.5 ± 1.2 mg/g; P < 0.0003). Thus, RIIß^-/- mice were relatively resistant to diet-induced fatty livers, as reported earlier on a different diet (9).

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESEARCH DESIGN AND METHODS RESULTS DISCUSSION REFERENCES

 
We show that a major consequence of PKA RIIß deficiency is to protect mice from obesity, insulin resistance, and dyslipidemia induced by a high-fat, high-carbohydrate diet. RIIß^-/- mice displayed reduced weight gain, lower insulin levels, and improved total-body glucose disposal as compared with wild-type mice fed a high-fat, high-sucrose diet. The magnitude of effect was dependent on both the sex and age of the mice studied. These findings demonstrate that targeting RIIß activity would provide an excellent therapy to reduce both obesity and metabolic disorders associated with obesity.

RIIß expression is limited to a few tissues, which include white and brown adipose tissue and brain (8,25). In murine brain, targeted disruption of the RIIß gene results in marked reduction of total PKA activity in the striatum, cortex, and hypothalamus (26,27). In adipose tissue, RI substitutes for RIIß and causes a four- to fivefold increase in basal PKA activity, resulting in chronic stimulation of thermogenesis and basal lipolysis. Since RIIß is absent from pancreatic islets, effects on insulin secretion in response to circulating glucose would be indirect. Taken together, these findings imply that PKA activity in both brain and adipose tissue is involved in regulating both susceptibility to adiposity and insulin-mediated glucose clearance.

There are several mechanisms that are likely contributing to the lean phenotype observed in the RIIß^-/- mice. Possible contributing factors include increased thermogenesis, increased lipolysis, and decreased food intake. RIIß^-/- mice fed normal chow diets have elevations in thermogenesis (9) and basal lipolysis (28), both of which may promote leanness. These attributes may contribute significantly to the leanness seen in diabetogenic diet–fed mice, since food intake was similar based on total body weight and significantly higher based on gram mouse weight for the RIIß^-/- mice. Therefore, differences in caloric consumption cannot fully account for the unusual leanness in the RIIß^-/- mice. Leptin was markedly reduced in RIIß mutants compared with wild types at the onset of this study and during most of the time on the diabetogenic diet. Leptin is a long-term satiety hormone secreted by adipocytes in proportion to adiposity (29,30,31). Since the RIIß^-/- mice ate comparably to the wild-type mice despite having lower leptin levels, this suggests that RIIß deficiency dysregulates leptin-induced satiety responses.

It is likely that RIIß^-/- mice are protected against developing insulin resistance when fed the diabetogenic diet at least in part because of their relative resistance to diet-induced obesity. We were unable to obtain a sufficient number of RIIß^-/- mice that had white adipose tissue weights similar to those of wild-type mice. The availability of this population would allow us to directly test whether RIIß has direct effects on influencing insulin resistance. However, when we corrected insulin-mediated glucose disposal to differences in body weights, we observed that diabetogenic diet–fed RIIß^-/- mice retained a similar amount of insulin-mediated glucose disposed per gram mouse weight as compared with chow-fed RIIß^-/- mice, whereas wild-type mice showed decreased glucose disposal per gram mouse weight. This finding suggests that elimination of RIIß improves insulin-mediated glucose disposal through a mechanism independent of alterations in body composition. PKA is known to antagonize insulin’s activation of the mitogen-activated protein kinase cascade, probably by blocking events at the levels of ras or raf (32). It is possible that this action requires an RIIß- containing PKA holoenzyme and cannot be subserved by the compensating RI isoform present in RIIß knockout mice. Insulin action in adipose may thus be enhanced in RIIß^-/- mice due to the absence of PKA-mediated counter-regulation of insulin signaling.

Elevations in LDL and VLDL occur commonly among mice fed high-fat diets (33,34,35,36). In fact, the major lipoprotein increase is seen in ß-VLDL (36), cholesterol-rich particles containing both apoB and apoE (24). Unique to this report was the marked loss of lipoproteins from the VLDL/LDL fraction in the RIIß^-/- mice. Concomitant with this loss was a twofold reduction in plasma levels of apoE, supporting the concept that ß-VLDL is nearly absent in RIIß^-/- animals fed a high-fat diet. Remaining apoE is presumed to reside in the HDL fraction (35). It remains to be determined whether the difference in ß-VLDL between RIIß^-/- and wild-type mice results from direct effects of RIIß on lipoprotein production or clearance, as opposed to secondary effects arising from differences in diet-induced insulin resistance.

A consistent feature of obesity and insulin resistance in rodents is sex dimorphism. Male mice are more susceptible to body weight gain and diabetes due to eating high-fat diets (22) or to genetic mutations (21) as compared with female mice. Molecular mechanisms for this difference are not clear but may involve the expression of hepatic sex steroid sulfotransferases (37). Here, female RIIß^-/- mice gained proportionally more body weight than male mice, although they retained insulin sensitivity. This is a somewhat unusual finding among diabetic rodent models and may reflect a role for PKA in modulating hormonal control of body weight.

In early studies of RIIß^-/- mice from the mixed 129 and C57BL/6 genetic background, we found greater disparities in diet-induced weight gain between genotypes than were seen for the >97% C57BL/6 background mice presented in this report. This may be due to allelic contributions from 129, known to present a lean phenotype in other studies. Thus, the influence of the RIIß mutation is magnified in mice containing 129 genes and demonstrates that the influence of RIIß function is dependent on unknown loci present within the 129 genome. Importantly, however, the effects of the RIIß mutation on body weight are significant regardless of genetic background, which suggests that targeting this gene in human therapies will benefit a range of human populations.

Overall, our studies support independent roles for PKA RIIß in the modulation of weight gain and insulin resistance associated with obesity. It remains to be determined which of these effects are modulated by PKA activity within adipose versus central nervous system–mediated responses. Our results suggest that alleles at loci coding for PKA regulatory subunits should be included in the list of candidate genes determining susceptibility to obesity and diabetes associated with obesity.

	ACKNOWLEDGMENTS

 
This work was supported by National Institutes of Health grants HL-52848 (to R.C.L.) and GM-32875 (to G.S.M.), and the University of Washington Clinical Nutrition Research Unit Laboratory core NIH/NIDDK DK-35816.

	FOOTNOTES

 
Address correspondence and reprint requests to Dr. Renée C. LeBoeuf, Department of Pathobiology, Box 353410, University of Washington, Seattle, WA 98195. E-mail: leboeuf{at}u.washington.edu.

Received for publication 22 January 2001 and accepted in revised form 2 August 2001.

apo, apolipoprotein; IPGTT, intraperitoneal glucose tolerance test; PBS, phosphate-buffered saline; PKA, protein kinase A.

	REFERENCES

TOP ABSTRACT INTRODUCTION RESEARCH DESIGN AND METHODS RESULTS DISCUSSION REFERENCES

 

1. Flegal KM, Carrol MD, Kuczmarski RJ, Johnson CL: Overweight and obesity in the United States: prevalence and trends, 1960–1994. Int J Obes22:39–47, 1998
2. Bosello O, Armellini F, Zamboni M, Fitchet M: The benefits of modest weight loss in type II diabetes. Int J Obes Relat Metab Disord 1:S10–S13, 1997
3. Jensen MD: Health consequences of fat distribution. Horm Res 48 (Suppl. 5):88–92, 1997
4. Edelman SV: Type II diabetes mellitus. Adv Intern Med 43:449–500, 1998[Medline]
5. Garg A: Treatment of diabetic dyslipidemia. Am J Cardiol 81:47B–51B, 1998[Medline]
6. Garber AJ: Vascular disease and lipids in diabetes. Med Clin North Am 82:931–948, 1998[Medline]
7. Montague CT, O’Rahilly S: The perils of portliness: causes and consequences of visceral adiposity. Diabetes 49:883–888, 2000[Abstract]
8. McKnight GS, Cummings DE, Amieux PS, Sikorski MA, Brandon EP, Planas JV, Motamed K, Idzerda RL: Cyclic AMP, PKA, and the physiological regulation of adiposity. Recent Prog Hormone Res 53:139–161, 1998
9. Cummings DE, Brandon EP, Planas JV, Motamed K, Idzerda RL, McKnight GS: Genetically lean mice result from targeted disruption of the RIIß subunit of protein kinase A. Nature 382:622–626, 1996[Medline]
10. Emorine L, Blin N, Strosberg AD: The human beta 3-adrenoceptor: the search for a physiological function. Trends Pharmacol Sci 15:3–7, 1994[Medline]
11. Meier MK, Alig L, Burgi-Saville ME, Muller M: Phenethanolamine derivatives with calorigenic and antidiabetic qualities. Int J Obes 8:215–225, 1984
12. Largis EE, Burns MG, Meunkel HA, Dolan JA, Claus TH: Antidiabetic and antiobesity effects of a highly selective ß3-adrenoceptor agonist. Drug Dev Res 32:69–76, 1994
13. Himms-Hagen J, Cui J, Danforth E Jr, Tatjes DJ, Lang SS, Waters BL, Claus TH: Effect of CL-316,243, a thermogenic beta 3-agonist, on energy balance and brown and white adipose tissues in rats. Am J Physiol 266:R1371–R1382, 1994[Abstract/Free Full Text]
14. Amieux PS, Cummings DE, Motamed K, Brandon EP, Wailes LA, Le K, Idzerda RL, McKnight GS: Compensatory regulation of RI protein levels in protein kinase A mutant mice. J Biol Chem 272:3993–3998, 1997[Abstract/Free Full Text]
15. Surwit RS, Kuhn CM, Cochrane C, McCubbin JA, Feinglos MN: Diet induced type II diabetes in C57BL/6J mice. Diabetes 37:1163–1170, 1988[Abstract]
16. Surwit RS, Seldin MF, Kuhn CM, Cochrane C, Feinglos MN: Control of expression of insulin resistance and hyperglycemia by different genetic factors in diabetes C57BL/6J mice. Diabetes 40:82–87, 1991[Abstract]
17. Harte RA, Kirk EA, Rosenfeld ME, LeBoeuf RC: The initiation of hyperinsulinemia and hyperleptinemia is diet dependent in C57BL/6 mice. Horm Metab Res 31:570–575, 1999[Medline]
18. Schreyer SA, Wilson DL, LeBoeuf RC: C57BL/6 mice fed high fat diets as models for diabetes-accelerated atherosclerosis. Atherosclerosis 136:17–24, 1998[Medline]
19. LeBouef RC, Caldwell M, Kirk E: Regulation by nutritional status of lipids and apolipoproteins A-1, A-II, and A-IV in inbred mice. J Lipid Res 35:121–133, 1994[Abstract]
20. Lusis AJ, Taylor BA, Quon D, Zollman S, LeBoeuf R: Genetic factors controlling structure and expression of apolipoproteins B and E in mice. J Biol Chem 262:7594–7604, 1987[Abstract/Free Full Text]
21. Leiter EH, Le PH, Coleman DL: Susceptibility to db gene and streptozotocin-induced diabetes in C57BL mice: control by gender-associated, MHC-unlinked traits. Immunogenetics 26:6–13, 1987[Medline]
22. Schreyer SA, Chua SC Jr, LeBoeuf RC: Obesity and diabetes in TNF-alpha receptor-deficient mice. J Clin Invest 102:402–411, 1998[Medline]
23. Camus MC, Chapman MJ, Forgez P, Laplaud PM: Distribution and characterization of the serum lipoproteins and apoproteins in the mouse, Mus musculus. J Lipid Res 24:1210–1228, 1983[Abstract]
24. Weisgraber KH, Mahley RW: Characterization of apolipoprotein E–containing lipoproteins. Methods Enzymol 129:145–166, 1986[Medline]
25. Brandon EP, Idzerda RL, McKnight GS: PKA isoforms, neural pathways, and behaviour: making the connection. Curr Opin Neurobiol 7:397–403, 1997[Medline]
26. Brandon EP, Logue SF, Adams MR, Qi M, Sullivan SP, Matsumoto AM, Dorsa DM, Wehner JM, McKnight GS: Defective motor behaviour and neural gene expression in RIIß-protein kinase A mutant mice. J Neurosci 18:3639–3649, 1998[Abstract/Free Full Text]
27. Adams MR, Brandon EP, Chartoff JH, Idzerda RL, Dorsa DM, McKnight GS: Loss of haloperidol induced gene expression and catalepsy in protein kinase A–deficient mice. Proc Natl Acad Sci U S A 94:12157–12161, 1998[Abstract/Free Full Text]
28. Planas JV, Cummings DE, Idzerda RL, McKnight GS: Mutation of the RIIß subunit of protein kinase A differentially affects lipolysis but not gene induction in white adipose tissue. J Biol Chem 274:36281–36287, 1999[Abstract/Free Full Text]
29. Pelleymounter MA, Cullen MJ, Baker MB, Hecht R, Winters D, Boone T, Collins F: Effects of the obese gene product on body weight reduction in ob/ob mice. Science 269:540–543, 1995[Abstract/Free Full Text]
30. Halaas JL, Gajiwala KS, Maffei M, Cohen SL, Chait BT, Rabinowitz D, Lallone RL, Burley SK, Friedman JM: Weight-reducing effects of the plasma protein encoded by the obese gene. Science 269:543–546, 1995[Abstract/Free Full Text]
31. Inui A: Feeding and body-weight regulation by hypothalamic neuropeptide-mediation of the actions of leptin. Trends Neurosci 22:62–67, 1999[Medline]
32. Sevetson BR, Kong X, Lawrence JC Jr: Increasing cAMP attenuates activation of mitogen activated protein kinase. Proc Natl Acad Sci U S A 90:10305–10309, 1993[Abstract/Free Full Text]
33. Kirk EA, Moe GL, Caldwell MT, Lernmark JA, Wilson DL, LeBoeuf RC: Hyper- and hypo-responsiveness to dietary fat and cholesterol among inbred mice: searching for level and variability genes. J Lipid Res 36:1522–1532, 1995[Abstract]
34. Srivastava RA, Jiao S, Tang JJ, Pfleger BA, Kitchens RT, Schonfeld G: In vivo regulation of low-density lipoprotein receptor and apolipoprotein B gene expressions by dietary fat and cholesterol in inbred strains of mice. Biochim Biophys Acta 1086:29–43, 1991[Medline]
35. Ishida BY, Blanche PJ, Nichols AV, Yashar M, Paigen B: Effects of atherogenic diet consumption on lipoproteins in mouse strains C57BL/6 and C3H. J Lipid Res 32:559–568, 1991[Abstract]
36. LeBoeuf RC, Tsao W, Kirk E, Childs MT: Cholesterol feeding induced cholesterol-rich VLDL in atherosclerosis-susceptible mice regardless of dietary fat content. Nutrition Res 13:549–561, 1993
37. Leiter EH, Chapman HD, Coleman DL: The influence of genetic background on the expression of mutations at the diabetes locus in the mouse. V. Interaction between the db gene and hepatic sex steroid sulfotransferases correlates with gender-dependent susceptibility to hyperglycemia. J Lipid Res 36:1522–1532, 1989

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