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• W2096594075 abstract "To explore the role of leptin in PKCβ action and to determine the protective potential of PKCβ deficiency on profound obesity, double knockout (DBKO) mice lacking PKCβ and ob genes were created, and key parameters of metabolism and body composition were studied. DBKO mice had similar caloric intake as ob/ob mice but showed significantly reduced body fat content, improved glucose metabolism, and elevated body temperature. DBKO mice were resistant to high-fat diet-induced obesity. Moreover, PKCβ deficiency increased β-adrenergic signaling by inducing expression of β1- and β3-adrenergic receptors (β-ARs) in white adipose tissue (WAT) of ob/ob mice. Accordingly, p38MAPK activation and expression of PGC-1α and UCP-1 were increased in WAT of DBKO mice. Consistent with results of in vivo studies, inhibition of PKCβ in WAT explants from ob/ob mice also increased expression of above β-ARs. In contrast, induction of PGC-1α and UCP-1 expression in brown adipose tissue of DBKO mice was not accompanied by changes in the expression of these β-ARs. Collectively, these findings suggest that PKCβ deficiency may prevent genetic obesity, in part, by remodeling the catabolic function of adipose tissues through β-ARs dependent and independent mechanisms. To explore the role of leptin in PKCβ action and to determine the protective potential of PKCβ deficiency on profound obesity, double knockout (DBKO) mice lacking PKCβ and ob genes were created, and key parameters of metabolism and body composition were studied. DBKO mice had similar caloric intake as ob/ob mice but showed significantly reduced body fat content, improved glucose metabolism, and elevated body temperature. DBKO mice were resistant to high-fat diet-induced obesity. Moreover, PKCβ deficiency increased β-adrenergic signaling by inducing expression of β1- and β3-adrenergic receptors (β-ARs) in white adipose tissue (WAT) of ob/ob mice. Accordingly, p38MAPK activation and expression of PGC-1α and UCP-1 were increased in WAT of DBKO mice. Consistent with results of in vivo studies, inhibition of PKCβ in WAT explants from ob/ob mice also increased expression of above β-ARs. In contrast, induction of PGC-1α and UCP-1 expression in brown adipose tissue of DBKO mice was not accompanied by changes in the expression of these β-ARs. Collectively, these findings suggest that PKCβ deficiency may prevent genetic obesity, in part, by remodeling the catabolic function of adipose tissues through β-ARs dependent and independent mechanisms. Leptin is an adipocyte-derived hormone that is required for normal energy homeostasis (1Zhang Y. Proenca R. Maffei M. Barone M. Leopold L. Friedman J.M. Positional cloning of the mouse obese gene and its human homologue.Nature. 1994; 372: 425-432Crossref PubMed Scopus (11741) Google Scholar, 2Friedman J.M. Obesity in the new millennium.Nature. 2000; 404: 632-634Crossref PubMed Scopus (629) Google Scholar–3Ahima R.S. Flier J.S. Leptin.Annu. Rev. Physiol. 2000; 62: 413-437Crossref PubMed Scopus (1487) Google Scholar). It plays a key role in the control of body weight by suppressing food intake through actions on hypothalamic receptors and by increasing energy expenditure via activation of sympathetic activity and brown adipose tissue (BAT) thermogenesis. This is best illustrated by loss of function mutations in genes encoding leptin or the leptin receptor, which result in severe obesity in rodents and humans. Leptin is also known to play a dual role in glucose metabolism and insulin signaling, acting as an insulin sensitizer and as an antagonizer. In vivo, leptin has been reported to enhance insulin action in inhibiting hepatic glucose output while antagonizing insulin action on the expression of metabolic genes (4Cohen B. Novick D. Rubinstein M. Modulation of insulin activities by leptin.Science. 1996; 274: 1185-1188Crossref PubMed Scopus (651) Google Scholar). The insulin and leptin signaling pathways are known to share downstream targets such as Janus kinase-2, insulin receptor substrates, phosphatidyl-inositol 3-kinase, protein kinase B, mitogen-activated protein kinase, and protein kinase C (PKC). Recent data provide evidence that PKC is activated by leptin via increasing calcium concentration and stimulating inositol triphosphate (IP-3) production (5Takekoshi K. Ishii K. Kawakami Y. Isobe K. Nanmoku T. Nakai T. Ca(2+) mobilization, tyrosine hydroxylase activity, and signaling mechanisms in cultured porcine adrenal medullary chromaffin cells: effects of leptin.Endocrinology. 2001; 142: 290-298Crossref PubMed Scopus (0) Google Scholar). PKC-dependent phosphorylation of Ser-318 in insulin receptor substrate-1 has been implicated in mediating the inhibitory signal of leptin on the insulin-signaling cascade (6Hennige A.M. Stefan N. Kapp K. Lehmann R. Weigert C. Beck A. Moeschel K. Mushack J. Schleicher E. Haring H.U. Leptin down-regulates insulin action through phosphorylation of serine-318 in insulin receptor substrate 1.FASEB J. 2006; 20: 1206-1208Crossref PubMed Scopus (83) Google Scholar). Several other interactions in different physiological systems have been described between PKC and leptin (7Kellerer M. Mushack J. Seffer E. Mischak H. Ullrich A. Haring H.U. Protein kinase C isoforms alpha, delta and theta require insulin receptor substrate-1 to inhibit the tyrosine kinase activity of the insulin receptor in human kidney embryonic cells (HEK 293 cells).Diabetologia. 1998; 41: 833-838Crossref PubMed Scopus (100) Google Scholar, 8Lee J.W. Swick A.G. Romsos D.R. Leptin constrains phospholipase C-protein kinase C-induced insulin secretion via a phosphatidylinositol 3-kinase-dependent pathway.Exp. Biol. Med.(Maywood). 2003; 228: 175-182Crossref PubMed Scopus (16) Google Scholar, 9Barrenetxe J. Sainz N. Barber A. Lostao M.P. Involvement of PKC and PKA in the inhibitory effect of leptin on intestinal galactose absorption.Biochem. Biophys. Res. Commun. 2004; 317: 717-721Crossref PubMed Scopus (23) Google Scholar–10Payne G.A. Borbouse L. Kumar S. Neeb Z. Alloosh M. Sturek M. Tune J.D. Epicardial perivascular adipose-derived leptin exacerbates coronary endothelial dysfunction in metabolic syndrome via a protein kinase C-beta pathway.Arterioscler. Thromb. Vasc. Biol. 2010; 30: 1711-1717Crossref PubMed Scopus (147) Google Scholar). PKCs comprise a large family of serine/threonine protein kinases that plays a key role in signal transduction and regulation of gene expression (11Newton A.C. Protein kinase C: poised to signal.Am. J. Physiol. Endocrinol. Metab. 2010; 298: E395-E402Crossref PubMed Scopus (397) Google Scholar, 12Rosse C. Linch M. Kermorgant S. Cameron A.J. Boeckeler K. Parker P.J. PKC and the control of localized signal dynamics.Nat. Rev. Mol. Cell Biol. 2010; 11: 103-112Crossref PubMed Scopus (344) Google Scholar, 13Freeley M. Kelleher D. Long A. Regulation of protein kinase C function by phosphorylation on conserved and non-conserved sites.Cell. Signal. 2011; 23: 753-762Crossref PubMed Scopus (83) Google Scholar–14Griner E.M. Kazanietz M.G. Protein kinase C and other diacylglycerol effectors in cancer.Nat. Rev. Cancer. 2007; 7: 281-294Crossref PubMed Scopus (778) Google Scholar). Twelve distinct members have been discovered in mammalian cells, and these have been subdivided into three distinct subfamilies as follows: conventional PKCs (α, βI, βII, and γ), novel PKCs (δ, ∊, ν, and θ), and atypical PKCs (∊ and ι/λ). These PKC isoforms are unique not only with respect to their primary structures but also in their expression patterns, subcellular localization, in vitro activation, and responsiveness to extracellular signals. Most importantly, these isoforms show differences in cofactor dependence and responsiveness to calcium and phospholipid metabolites. Conventional PKCs bind to and are activated by sn-1,2-diacylglycerol, which increases the specificity of the enzyme for phosphatidylserine and its affinity for calcium. Novel PKCs are also activated by DAG and require phosphatidylserine as a cofactor but have lost the requirement for calcium. Atypical PKCs do not respond to DAG or calcium but apparently still require phosphatidylserine as a cofactor. Recent studies have shown that DAG-PKC signaling is activated in diabetic conditions, and the induction appears to be restricted to a few “diabetic-related” isoforms (15Rask-Madsen C. King G.L. Proatherosclerotic mechanisms involving protein kinase C in diabetes and insulin resistance.Arterioscler. Thromb. Vasc. Biol. 2005; 25: 487-496Crossref PubMed Scopus (153) Google Scholar, 16Geraldes P. King G.L. Activation of protein kinase C isoforms and its impact on diabetic complications.Circ. Res. 2010; 106: 1319-1331Crossref PubMed Scopus (608) Google Scholar). PKCβ is one isoform that has been most directly linked to important aspects of hyperglycemia in in vivo and in vitro. PKCβ was also one of the earliest isoforms recognized in insulin signaling and appears to play dual roles in insulin signaling pathways (17Patel N.A. Apostolatos H.S. Mebert K. Chalfant C.E. Watson J.E. Pillay T.S. Sparks J. Cooper D.R. Insulin regulates protein kinase CbetaII alternative splicing in multiple target tissues: development of a hormonally responsive heterologous minigene.Mol. Endocrinol. 2004; 18: 899-911Crossref PubMed Scopus (31) Google Scholar, 18Yamamoto T. Watanabe K. Inoue N. Nakagawa Y. Ishigaki N. Matsuzaka T. Takeuchi Y. Kobayashi K. Yatoh S. Takahashi A. et al.Protein kinase Cbeta mediates hepatic induction of sterol-regulatory element binding protein-1c by insulin.J. Lipid Res. 2010; 51: 1859-1870Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 19Formisano P. Oriente F. Fiory F. Caruso M. Miele C. Maitan M.A. Andreozzi F. Vigliotta G. Condorelli G. Beguinot F. Insulin-activated protein kinase Cbeta bypasses Ras and stimulates mitogen-activated protein kinase activity and cell proliferation in muscle cells.Mol. Cell. Biol. 2000; 20: 6323-6333Crossref PubMed Scopus (67) Google Scholar, 20Aguirre V. Werner E.D. Giraud J. Lee Y.H. Shoelson S.E. White M.F. Phosphorylation of Ser307 in insulin receptor substrate-1 blocks interactions with the insulin receptor and inhibits insulin action.J. Biol. Chem. 2002; 277: 1531-1537Abstract Full Text Full Text PDF PubMed Scopus (767) Google Scholar, 21Ishizuka T. Kajita K. Natsume Y. Kawai Y. Kanoh Y. Miura A. Ishizawa M. Uno Y. Morita H. Yasuda K. Protein kinase C (PKC) beta modulates serine phosphorylation of insulin receptor substrate-1 (IRS-1)–effect of overexpression of PKCbeta on insulin signal transduction.Endocr. Res. 2004; 30: 287-299Crossref PubMed Scopus (20) Google Scholar–22Osterhoff M.A. Heuer S. Pfeiffer M. Tasic J. Kaiser S. Isken F. Spranger J. Weickert M.O. Mohlig M. Pfeiffer A.F. Identification of a functional protein kinase Cbeta promoter polymorphism in humans related to insulin resistance.Mol. Genet. Metab. 2008; 93: 210-215Crossref PubMed Scopus (14) Google Scholar). PKCβ does not appear to regulate glucose-induced insulin secretion in vivo (23Biden T.J. Schmitz-Peiffer C. Burchfield J.G. Gurisik E. Cantley J. Mitchell C.J. Carpenter L. The diverse roles of protein kinase C in pancreatic β-cell function.Biochem. Soc. Trans. 2008; 36: 916-919Crossref PubMed Scopus (22) Google Scholar), even though it has been reported to undergo translocation to the plasma membrane subsequent to stimulation by glucose in primary islet cells (24Pinton P. Tsuboi T. Ainscow E.K. Pozzan T. Rizzuto R. Rutter G.A. Dynamics of glucose-induced membrane recruitment of protein kinase C βII in living pancreatic islets β-cells.J. Biol. Chem. 2002; 277: 37702-37710Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). We recently showed that PKCβ is markedly elevated in white adipose tissue (WAT) of leptin-deficient (ob/ob) mice and is significantly induced by intake of high-fat diet (HFD) (25Huang W. Bansode R. Mehta M. Mehta K.D. Loss of protein kinase Cbeta function protects mice against diet-induced obesity and development of hepatic steatosis and insulin resistance.Hepatology. 2009; 49: 1525-1536Crossref PubMed Scopus (53) Google Scholar, 26Bansode R.R. Huang W. Roy S.K. Mehta M. Mehta K.D. Protein kinase C deficiency increases fatty acid oxidation and reduces fat storage.J. Biol. Chem. 2008; 283: 231-236Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). We also assessed the impact of PKCβ deficiency on glucose and lipid homeostasis in vivo and found that deficiency of PKCβ signaling resulted in adipose atrophy, hypoleptinemia, hyperphagia, and altered expression of genes involved in energy homeostasis in the adipose tissue. The lean phenotype of PKCβ−/− mice was associated with reduced serum leptin and compensatory increased food intake (26Bansode R.R. Huang W. Roy S.K. Mehta M. Mehta K.D. Protein kinase C deficiency increases fatty acid oxidation and reduces fat storage.J. Biol. Chem. 2008; 283: 231-236Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Furthermore, adiposity is not increased when PKCβ-deficient (PKCβ−/−) mice are challenged with a HFD. These studies identified the PKCβ signaling pathway as a novel modulator of adipose tissue homeostasis. Unlike PKCβ−/− mice, ob/ob mice exhibit marked obesity, hyperphagia, insulin resistance, hypothermia, and increased food efficiency. To explore the protective potential of PKCβ deficiency on profound obesity and to better understand the regulatory pathways that govern energy metabolism, we examined the effects of PKCβ gene disruption in genetically obese ob/ob mice on diverse elements of energy balance, focusing particularly on the β-AR signaling. We report that deletion of PKCβ in ob/ob mice (DBKO) decreases food efficiency through increasing energy expenditure and thermogenesis and through enhanced insulin sensitivity, thus improving the energy balance of ob/ob mice. A significant component of the effect of PKCβ deficiency on energy expenditure is independent of leptin and involves signaling through β-ARs in WAT. In fact, enhanced β-adrenergic signaling may account for hypoleptinemia in PKCβ−/− mice. A double knockout mouse simultaneously lacking the leptin and PKCβ genes was generated by intercrossing male ob/ob+/− mice with female PKCβ−/− mice on a C57BL/6J background (Jackson Laboratories, Bar Harbor, ME) to generate ob+/− x PKCβ−/−. These leptin heterozygous and PKCβ homozygous mice were used to generate double knockout ob/ob x PKCβ−/− mice. Genotyping for ob/ob and PKCβ were performed as previously described (25Huang W. Bansode R. Mehta M. Mehta K.D. Loss of protein kinase Cbeta function protects mice against diet-induced obesity and development of hepatic steatosis and insulin resistance.Hepatology. 2009; 49: 1525-1536Crossref PubMed Scopus (53) Google Scholar). Unless indicated, all experiments were performed on male animals. Male mice were weaned at 21 days of age, genotyped, and maintained at a room temperature of 22 ± 2°C on a 12:12 light-dark cycle with a relative humidity of 50%. Animals had free access to water and were fed ad libitum. Body weight and food intake were registered weekly. Body temperature was assessed by measuring rectal temperature using a rectal thermometer. Seven- to eight-week-old ob/ob and DBKO mice were fed ad libitum for the indicated period continuously either on a HFD (D12492; Research Diets, New Brunswick, NJ) in which 60% of the total calories were derived from fat (soybean oil and lard) or a standard diet containing 17% kcal from fat (7912 rodent chow; Harlan Laboratories, Inc., Indianapolis, IN) (25Huang W. Bansode R. Mehta M. Mehta K.D. Loss of protein kinase Cbeta function protects mice against diet-induced obesity and development of hepatic steatosis and insulin resistance.Hepatology. 2009; 49: 1525-1536Crossref PubMed Scopus (53) Google Scholar). All procedures on mice followed guidelines established by the Ohio State University College of Medicine Animal Care Committee. Unless indicated, all experiments were performed on mice starved for approximately 16 h. Eighteen-week-old mice were fasted for 6 h and euthanized by CO2 inhalation. Blood samples were obtained by submandibular bleeding, and plasma or sera were collected after centrifugation (4°C) at 12,000 rpm for 15 min and stored at −20°C. Epididymal, inguinal, and retroperitoneal white adipose tissues, together with brown fat from the interscapular depot, and livers were carefully excised. Tissue samples were weighed and then immediately frozen in liquid nitrogen. For morphological assessment, parts of adipose tissue was fixed in 4% buffered formaldehyde overnight and then dehydrated in graded ethanols and embedded in paraffin. Sections (10 μm) were cut and mounted on slides and stained with hematoxylin and eosin or UCP-1 antibody (1:500)-HRP according to standard protocols. Plasma concentrations of triglycerides, total cholesterol, and serum-free fatty acids were measured by enzymatic methods using commercially available kits. Serum insulin and adiponectin were determined by ELISA. A glucose tolerance test and insulin tolerance test were performed on fasted (16 h) mice. Mice were weighed and injected intraperitoneally with glucose (1.5 mg/kg body weight) or insulin (0.8 U/kg body weight). Blood samples were collected via tail bleeds, and glucose concentrations were measured before and 15, 30, 60, 90, and 120 min after the challenge. Glucose was determined by glucometer. Oxygen consumption, CO2 production, and spontaneous physical movement were measured simultaneously over 24 h for each mouse using a computer-controlled, open-circuit Oxymax/ CLAMS System (Columbus Instruments, Columbus, OH). Each mouse was measured individually in a resting state at 22°C in the presence of food and water. Tissues were homogenized in buffer containing 20 mM Tris, 50 mM NaCl, 250 mM sucrose, 1% Triton X-100, and phosphatase and protease inhibitors cocktail, and protein content was measured as described earlier (25Huang W. Bansode R. Mehta M. Mehta K.D. Loss of protein kinase Cbeta function protects mice against diet-induced obesity and development of hepatic steatosis and insulin resistance.Hepatology. 2009; 49: 1525-1536Crossref PubMed Scopus (53) Google Scholar). Equal amounts of protein were run in 12% SDS-PAGE, transferred to nitrocellulose membranes, and blocked in Tris-buffered saline with Tween 20 containing 5% nonfat dry milk or BSA for 1 h at room temperature. Blots were incubated overnight at 4°C with primary antibodies against UCP-1 (Abcam, Cambridge, MA) at 1:2,000; or P-p38MAPK (Cell Signaling Technology, Inc., Boston, MA) at 1:1,000, or p38MAPK (Santa Cruz Biotechnology, Santa Cruz, CA) at 1:2,000. The antigen-antibody complexes were visualized using peroxidase-conjugated anti-rabbit antibodies (1:5,000) and the enhanced chemiluminescence ECL detection system (Life Technologies, Grand Island, NY). All assays were performed in duplicate. Inguinal, epididymal WAT, or interscapular BAT were surgically removed from ob/ob and DBKO mice (n = 3 per group). Freshly dissected fat pads were minced to a size of 2–3 mm3 for iWAT and 1–2 mm3 for BAT on an ice-cold Petri dish containing Krebs-Ringer HEPES buffer (5 mM D-glucose, 2% BSA, 135 mM NaCl, 2.2 mM CaCl2, 1.25 mM MgSO4, 0.45 mM KH2PO4, 2.17 mM Na2HPO4, and 10 mM HEPES) and then rinsed twice with Kreb-Ringer HEPES buffer and once with DMEM containing 10% BSA. Samples were passed through a 200-μm mesh. The tissue explants were transferred to 6-well plate with an equal amount of the tissue in each well. The explants were allowed to stabilize in DMEM medium for 1 h before the treatment. All procedures were performed under sterile conditions. LY333,531 from Alexus Biochemicals was added to each well as indicated for 16 h. Explants were transferred to a 2 ml tube and rinsed with PBS twice before being snap-frozen in liquid nitrogen for later analysis of gene expression. Total RNA was extracted from iWAT and BAT samples by homogenization using TRIzol reagent. Samples were treated with a DNA-free kit (Life Technologies, Grand Island, NY). For first-strand cDNA synthesis, constant amounts of 2 μg of total RNA were reverse transcribed in a 20 μl final volume using random hexamers as primers and 50 units of MultiScribe™ Reverse Transcriptase (High-capacity cDNA Reverse Transcription Kit, Life Technologies, Grand Island, NY) (25Huang W. Bansode R. Mehta M. Mehta K.D. Loss of protein kinase Cbeta function protects mice against diet-induced obesity and development of hepatic steatosis and insulin resistance.Hepatology. 2009; 49: 1525-1536Crossref PubMed Scopus (53) Google Scholar, 26Bansode R.R. Huang W. Roy S.K. Mehta M. Mehta K.D. Protein kinase C deficiency increases fatty acid oxidation and reduces fat storage.J. Biol. Chem. 2008; 283: 231-236Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). The transcript levels for indicated genes were quantified as described earlier. Relative mRNA expression was expressed as fold expression over the control mice. All samples were run in triplicate, and the average values were calculated. The results are shown as means ± SEM. All statistical analysis was performed by Student t-test or ANOVA in Excel; P < 0.05 was considered significant. To explore the effects of PKCβ deficiency on ob/ob phenotype, we intercrossed ob−/+ with PKCβ−/− mice to generate DBKO mice. Body weight and fat content were compared between ob/ob and DBKO mice. Compared with the ob/ob mice of the same age, DBKO mice have lower body weight (Fig. 1A–C). Noteworthy differences in body weights were apparent by as early as 7 weeks of age and became even more pronounced with aging. At 18 weeks of age, weight was reduced by 34% in female mice and by 29% in male mice. It was accompanied by significantly reduced inguinal and retroperitoneal white fat depots per body weight as compared with ob/ob mice in male (Fig. 1D, E) and female mice (results not shown). The wet weight of inguinal white adipose tissue (iWAT) was reduced by ∼41% in DBKO in comparison to ob/ob mice. The weights of other tissues, including liver and brown adipose tissue (BAT), were similar between genotypes (Fig. 1F, G). Histology of inguinal WAT (iWAT) of DBKO mice revealed unilocular white adipocytes that were smaller than those in ob/ob mice and abundant, more densely stained and much smaller cells, many with a multilocular appearance (Fig. 2A).Fig. 2Histological and biochemical changes in liver, adipose, and muscle TG content of male ob/ob and DBKO mice. A: Hematoxylin and eosin (H+E) staining of iWAT, interscapular BAT, and liver from male ob/ob and DBKO mice. Results are representative of n = 6 in each group. Bar, 5 μM. B: Thin-layer chromatography of total lipid extracts from liver and muscle of both genotypes is also shown. Each lane represents lipids from the liver and muscle of three mice of each genotype. C: Relative amounts of TG contents in the liver and muscle of male mice of both genotypes (fasted 16 h). Each value represents the means ± SE (n = 6 per genotype). **P < 0.01.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Brown fat is critical for adaptive thermogenesis in mice. DBKO mice also tend to show reduced absolute weight of BAT compared with ob/ob mice, but the difference did not reach statistical significance when body weight was considered (Fig. 1F). Compared with the control ob/ob mice, the size of brown adipocytes was much smaller in the DBKO mice (Fig. 2A). Furthermore, brown adipocytes showed small and multilocular lipid droplets in DBKO mice, whereas ob/ob mice exhibited large and unilocular lipid droplets, suggesting higher thermogenic activity in DBKO mice leading to reduction in fat accumulation. ob/ob mice have massively enlarged livers that are engorged with lipid (Fig. 1G). Histological sections of ob/ob liver showed large lipid-filled vacuoles, whereas those of DBKO mice showed significantly smaller lipid-filled vacuoles (Fig. 2A). Consistent with histological examination, hepatic TG content was greatly reduced in DBKO mice (Fig. 2B, C). In addition to a reduced amount of TG in the liver, the amount of TG in the skeletal muscle of DBKO mice was also significantly reduced (Fig. 2B, C). Therefore, the reduced TG levels in adipose tissue were not due to alternative lipid storage in other tissues, such as liver and muscle. It remains to be seen whether reduction in TG biosynthesis contributes to the overall reduction in the body's TG contents. Adiposity is influenced by the rate of food consumption and the rate at which energy is expanded metabolically. To determine whether change in food intake and/or energy expenditure was responsible for decreased obesity of DBKO mice relative to ob/ob mice, we compared their rates of food intake and oxygen consumption. With or without normalization for body weight, Fig. 3A and 3B show that male DBKO mice consumed a similar level of food as male ob/ob mice; this rate of food intake was also seen with female mice (results not shown), suggesting that the decrease in food intake cannot account for the failure of DBKO mice to gain weight. Interestingly, basal oxygen consumption at night was significantly higher in male DBKO mice than in male ob/ob mice (Fig. 3C, D). Thus, increased metabolic rate may help to normalize energy balance in DBKO mice. We cannot rule out whether the differences in body weight contribute to increased oxygen consumption of DBKO mice compared with ob/ob mice (27Butler A.A. Kozak L.P. A recurring problem with the analysis of energy expenditure in genetic models expressing lean and obese phenotypes.Diabetes. 2010; 59: 323-329Crossref PubMed Scopus (216) Google Scholar, 28Kaiyala K.J. Morton G.J. Leroux B.G. Ogimoto K. Wisse B. Schwartz M.W. Identification of body fat mass as a major determinant of metabolic rate in mice.Diabetes. 2010; 59: 1657-1666Crossref PubMed Scopus (124) Google Scholar). The increased oxygen consumption could also be due in part to the maintenance of a higher body temperature of DBKO mice (Fig. 3E). These data strongly suggest that PKCβ deficiency may increase energy expenditure in ob/ob mice. Changes in adiposity are often associated with alterations in glucose and insulin homeostasis. The ob/ob mice develop a form of diabetes similar to human type-2 diabetes, a condition commonly associated with obesity. To evaluate the effect of PKCβ deficiency on glucose metabolism in chow-fed mice, we first evaluated plasma glucose and insulin levels between genotypes. Both were significantly lower in fasted DBKO mice compared with those in ob/ob mice (Fig. 4A). Moreover, glucose to insulin ratios in DBKO mice was also significantly elevated (Fig. 4A), indicating improved insulin sensitivity. Meanwhile, DBKO mice showed a significant decrease in plasma glucose compared with ob/ob mice after intraperitoneal injection of glucose or insulin, indicating improved glucose metabolism by PKCβ deficiency in ob/ob mice (Fig. 4B). In agreement with the above findings, DBKO mice showed a significant increase in the whole body glucose uptake, further supporting improved glucose metabolism (Huang et al., unpublished results). We also compared responses of ob/ob and DBKO mice under conditions of severe dietary stress. Both genotypes were fed HFD beginning at 8 weeks of age. Body weights and food intake were monitored weekly, and at the end we examined weights of adipose tissues and various metabolic parameters. A significant increase in ob/ob mice body weights were evident even after 2 weeks on HFD, and this trend continued throughout the dietary protocol (Fig. 5A, B). DBKO mice fed HFD gained less weight than ob/ob mice and exhibited an obese-resistant phenotype (Fig. 5B). The difference in body weights between ob/ob and DBKO mice was further reflected by marked reduction in iWAT and rWAT mass in DBKO mice, indicating that these mice are strongly protected from HFD-induced obesity (Fig. 5C, D). Histological analysis of iWAT revealed smaller adipocytes than those from ob/ob mice fed HFD (Fig. 5C). The weights of DBKO BAT and livers were almost comparable between these genotypes (Fig. 5E, F). Liver tissue appearance in ob/ob male mice was more whitish in color than that in DBKO mice. Similarly, ob/ob mice had higher hepatic TG content than DBKO mice (Fig. 5G) and exhibit increased glucose/insulin ratio (Fig. 5H). It is again clear that the decreased body weight was not a result of reduced food intake of DBKO mice compared with ob/ob mice (Fig. 6). Indirect calorimetry measurements revealed a significant increase in oxygen consumption (Fig. 7A, B) in HFD-fed DBKO mice compared with ob/ob mice. In the DBKO mice, the increase in energy expenditure was more prominent at night when mice actively took food (Fig. 7A). The respiratory quotient was comparable in the ob/ob and DBKO mice (Fig. 7C, D), indicating similar utilization of carbohydrates and fat as energy sources.Fig. 6Average daily food consumption. Food intake was determined by measuring 1 day or 7 day intake of HFD by 12-week-old male mice (n = 12 for each genotype). Values represent the means ± SE of ob/ob and DBKO mice.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 7Energy expenditure and body temperature of male ob/ob and DBKO mice fed HFD. A–C: Metabolic rate and oxygen consumption of 12- to 14-week-old mice on a chow diet (n = 5 each genotype) as measured by indirect calorimetry. Mice were monitored for 24 h continuously, from 10:00 AM to 10:00 AM the next day. To allow for acclimation, data from the initial 3 h are omitted. All values are given a" @default.
• W2096594075 created "2016-06-24" @default.
• W2096594075 creator A5003583851 @default.
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• W2096594075 date "2012-03-01" @default.
• W2096594075 modified "2023-10-16" @default.
• W2096594075 title "Protein kinase Cβ deficiency attenuates obesity syndrome of ob/ob mice by promoting white adipose tissue remodeling" @default.
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