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
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ABSTRACT |
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INTRODUCTION |
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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.
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RESEARCH DESIGN AND METHODS |
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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 1620 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, 910 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 = [(glucoset=0 - glucoset=30)/glucoset=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 peroxidasecoupled goat antirabbit 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 Students t test. Pearsons correlation coefficients were used to assess relations between adiposity and glucose disposal. P < 0.05 was accepted as statistically significant.
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RESULTS |
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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.
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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.
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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.
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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 dietfed 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.
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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).
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DISCUSSION |
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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 dietfed 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 dietfed 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 insulins 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 systemmediated 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.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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REFERENCES |
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