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• W2605800216 abstract "Obesity and its associated complications such as insulin resistance and non-alcoholic fatty liver disease are reaching epidemic proportions. In mice, the TGF-β superfamily is implicated in the regulation of white and brown adipose tissue differentiation. The kielin/chordin-like protein (KCP) is a secreted regulator of the TGF-β superfamily pathways that can inhibit both TGF-β and activin signals while enhancing bone morphogenetic protein (BMP) signaling. However, KCP's effects on metabolism and obesity have not been studied in animal models. Therefore, we examined the effects of KCP loss or gain of function in mice that were maintained on either a regular or a high-fat diet. KCP loss sensitized the mice to obesity and associated complications such as glucose intolerance and adipose tissue inflammation and fibrosis. In contrast, transgenic mice that expressed KCP in the kidney, liver, and adipose tissues were resistant to developing high-fat diet-induced obesity and had significantly reduced white adipose tissue. Moreover, KCP overexpression shifted the pattern of SMAD signaling in vivo, increasing the levels of phospho (P)-SMAD1 and decreasing P-SMAD3. Adipocytes in culture showed a cell-autonomous effect in response to added TGF-β1 or BMP7. Metabolic profiling indicated increased energy expenditure in KCP-overexpressing mice and reduced expenditure in the KCP mutants with no effect on food intake or activity. These findings demonstrate that shifting the TGF-β superfamily signaling with a secreted protein can alter the physiology and thermogenic properties of adipose tissue to reduce obesity even when mice are fed a high-fat diet. Obesity and its associated complications such as insulin resistance and non-alcoholic fatty liver disease are reaching epidemic proportions. In mice, the TGF-β superfamily is implicated in the regulation of white and brown adipose tissue differentiation. The kielin/chordin-like protein (KCP) is a secreted regulator of the TGF-β superfamily pathways that can inhibit both TGF-β and activin signals while enhancing bone morphogenetic protein (BMP) signaling. However, KCP's effects on metabolism and obesity have not been studied in animal models. Therefore, we examined the effects of KCP loss or gain of function in mice that were maintained on either a regular or a high-fat diet. KCP loss sensitized the mice to obesity and associated complications such as glucose intolerance and adipose tissue inflammation and fibrosis. In contrast, transgenic mice that expressed KCP in the kidney, liver, and adipose tissues were resistant to developing high-fat diet-induced obesity and had significantly reduced white adipose tissue. Moreover, KCP overexpression shifted the pattern of SMAD signaling in vivo, increasing the levels of phospho (P)-SMAD1 and decreasing P-SMAD3. Adipocytes in culture showed a cell-autonomous effect in response to added TGF-β1 or BMP7. Metabolic profiling indicated increased energy expenditure in KCP-overexpressing mice and reduced expenditure in the KCP mutants with no effect on food intake or activity. These findings demonstrate that shifting the TGF-β superfamily signaling with a secreted protein can alter the physiology and thermogenic properties of adipose tissue to reduce obesity even when mice are fed a high-fat diet. Energy balance is critical for maintaining normal body weight and homeostasis. When caloric intake chronically exceeds energy expenditure, white adipose tissue (WAT) 2The abbreviations used are: WATwhite adipose tissueBATbrown adipose tissueHFDhigh-fat dietTgtransgenicBMPbone morphogenetic proteinCRcysteine-richKCPkielin/chordin-like proteinNDnormal dietPEPCKphosphoenolpyruvate carboxykinaseNEnorepinephrineeWATepididymal WATPPARγperoxisome proliferator-activated receptor γiBATinterscapular brown adipose tissueP-phospho-VO2oxygen consumptionVCO2carbon dioxide productionPFAparaformaldehydeiWATinguinal white adipose tissueANOVAanalysis of variancePGCperoxisome proliferator-activated receptor-γ coactivator. stores excess energy in the form of triglycerides, leading to obesity and complications related to insulin resistance such as type 2 diabetes and metabolic syndrome (1Rosen E.D. Spiegelman B.M. What we talk about when we talk about fat.Cell. 2014; 156: 20-44Abstract Full Text Full Text PDF PubMed Scopus (1422) Google Scholar). Metabolic disease is reaching epidemic proportions in the developed world, primarily due to the increased availability of high caloric foods and the decrease in daily physical activity (2Doria A. Patti M.E. Kahn C.R. The emerging genetic architecture of type 2 diabetes.Cell Metab. 2008; 8: 186-200Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar, 3Saltiel A.R. Insulin resistance in the defense against obesity.Cell Metab. 2012; 15: 798-804Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). WAT functions not only as a nutrient storage center but also as an endocrine organ by secreting proteins such as leptin and adiponectin that provide signals to regulate appetite and nutrient metabolism. In contrast to nutrient storage, brown adipose tissue (BAT) functions as a thermogenic organ by converting triglycerides into heat to maintain body temperature in homotherms (4Cannon B. Nedergaard J. Brown adipose tissue: function and physiological significance.Physiol. Rev. 2004; 84: 277-359Crossref PubMed Scopus (4518) Google Scholar). In humans, physiologically active BAT was identified in adults and inversely correlated with age and body mass index (5Cypess A.M. Lehman S. Williams G. Tal I. Rodman D. Goldfine A.B. Kuo F.C. Palmer E.L. Tseng Y.H. Doria A. Kolodny G.M. Kahn C.R. Identification and importance of brown adipose tissue in adult humans.N. Engl. J. Med. 2009; 360: 1509-1517Crossref PubMed Scopus (3136) Google Scholar, 6van Marken Lichtenbelt W.D. Vanhommerig J.W. Smulders N.M. Drossaerts J.M. Kemerink G.J. Bouvy N.D. Schrauwen P. Teule G.J. Cold-activated brown adipose tissue in healthy men.N. Engl. J. Med. 2009; 360: 1500-1508Crossref PubMed Scopus (2585) Google Scholar, 7Virtanen K.A. Lidell M.E. Orava J. Heglind M. Westergren R. Niemi T. Taittonen M. Laine J. Savisto N.J. Enerbäck S. Nuutila P. Functional brown adipose tissue in healthy adults.N. Engl. J. Med. 2009; 360: 1518-1525Crossref PubMed Scopus (2314) Google Scholar). Recent investigations into the origin and regulation of brown adipocytes have revealed pathways for brown adipocyte differentiation and molecular and physiological distinctions between developmentally programmed BAT, postnatally generated BAT, and inducible or beige/brite BAT (8Schulz T.J. Tseng Y.H. Brown adipose tissue: development, metabolism and beyond.Biochem. J. 2013; 453: 167-178Crossref PubMed Scopus (116) Google Scholar, 9Wu J. Cohen P. Spiegelman B.M. Adaptive thermogenesis in adipocytes: is beige the new brown?.Genes Dev. 2013; 27: 234-250Crossref PubMed Scopus (628) Google Scholar). white adipose tissue brown adipose tissue high-fat diet transgenic bone morphogenetic protein cysteine-rich kielin/chordin-like protein normal diet phosphoenolpyruvate carboxykinase norepinephrine epididymal WAT peroxisome proliferator-activated receptor γ interscapular brown adipose tissue phospho- oxygen consumption carbon dioxide production paraformaldehyde inguinal white adipose tissue analysis of variance peroxisome proliferator-activated receptor-γ coactivator. Among the most debilitating effects of obesity are inflammation and fibrosis in adipose tissue, liver, and other organs (10Donath M.Y. Shoelson S.E. Type 2 diabetes as an inflammatory disease.Nat. Rev. Immunol. 2011; 11: 98-107Crossref PubMed Scopus (2392) Google Scholar, 11Lumeng C.N. Saltiel A.R. Inflammatory links between obesity and metabolic disease.J. Clin. Investig. 2011; 121: 2111-2117Crossref PubMed Scopus (1623) Google Scholar, 12Sun K. Tordjman J. Clément K. Scherer P.E. Fibrosis and adipose tissue dysfunction.Cell Metab. 2013; 18: 470-477Abstract Full Text Full Text PDF PubMed Scopus (567) Google Scholar, 13Divoux A. Clément K. Architecture and the extracellular matrix: the still unappreciated components of the adipose tissue.Obes. Rev. 2011; 12: e494-e503Crossref PubMed Scopus (140) Google Scholar). In adipose tissue, increased numbers of macrophages are associated with the secretion of proinflammatory cytokines and increased extracellular matrix deposition (14Keophiphath M. Achard V. Henegar C. Rouault C. Clément K. Lacasa D. Macrophage-secreted factors promote a profibrotic phenotype in human preadipocytes.Mol. Endocrinol. 2009; 23: 11-24Crossref PubMed Scopus (194) Google Scholar, 15Spencer M. Yao-Borengasser A. Unal R. Rasouli N. Gurley C.M. Zhu B. Peterson C.A. Kern P.A. Adipose tissue macrophages in insulin-resistant subjects are associated with collagen VI and fibrosis and demonstrate alternative activation.Am. J. Physiol.. Endocrinol. Metab. 2010; 299: E1016-E1027Crossref PubMed Scopus (297) Google Scholar). A more recent study suggests that inflammation in adipose tissue does have an adaptive advantage for tissue remodeling in the absence of which more ectopic lipid accumulation is seen in other tissues (16Wernstedt Asterholm I. Tao C. Morley T.S. Wang Q.A. Delgado-Lopez F. Wang Z.V. Scherer P.E. Adipocyte inflammation is essential for healthy adipose tissue expansion and remodeling.Cell Metab. 2014; 20: 103-118Abstract Full Text Full Text PDF PubMed Scopus (437) Google Scholar). Obesity and insulin resistance are also closely linked to non-alcoholic fatty liver disease, which covers a broad spectrum of liver pathology that includes inflammation, fibrosis, hepatic steatosis, and cirrhosis and may ultimately progress to hepatocellular carcinoma (17Asrih M. Jornayvaz F.R. Metabolic syndrome and nonalcoholic fatty liver disease: is insulin resistance the link?.Mol. Cell. Endocrinol. 2015; 418: 55-65Crossref PubMed Scopus (218) Google Scholar). TGF-β is a proinflammatory cytokine whose role in kidney, liver, lung, and cardiac fibrosis is well documented. More recently, the importance of TGF-β superfamily signaling in determining the types of adipose tissue and the effects of a high-fat diet (HFD) on obesity and associated complications have been studied in several different experimental model systems (18Yadav H. Rane S.G. TGF-β/Smad3 signaling regulates brown adipocyte induction in white adipose tissue.Front. Endocrinol. 2012; 3: 35Crossref PubMed Scopus (32) Google Scholar, 19Zamani N. Brown C.W. Emerging roles for the transforming growth factor-β superfamily in regulating adiposity and energy expenditure.Endocr. Rev. 2011; 32: 387-403Crossref PubMed Scopus (154) Google Scholar). A deletion of SMAD3 in mice prevented HFD-induced obesity, hepatic steatosis, and insulin resistance with phenotypic changes in WAT tissue that resembled BAT (20Yadav H. Quijano C. Kamaraju A.K. Gavrilova O. Malek R. Chen W. Zerfas P. Zhigang D. Wright E.C. Stuelten C. Sun P. Lonning S. Skarulis M. Sumner A.E. Finkel T. et al.Protection from obesity and diabetes by blockade of TGF-β/Smad3 signaling.Cell Metab. 2011; 14: 67-79Abstract Full Text Full Text PDF PubMed Scopus (451) Google Scholar). The BMP proteins are also implicated in the transition of WAT to a more brown or beige phenotype. Elevated expression of BMP4 in adipose tissue of transgenic mice reduced the mass of gonadal WAT and promoted a beige phenotype (21Qian S.W. Tang Y. Li X. Liu Y. Zhang Y.Y. Huang H.Y. Xue R.D. Yu H.Y. Guo L. Gao H.D. Liu Y. Sun X. Li Y.M. Jia W.P. Tang Q.Q. BMP4-mediated brown fat-like changes in white adipose tissue alter glucose and energy homeostasis.Proc. Natl. Acad. Sci. U.S.A. 2013; 110: E798-E807Crossref PubMed Scopus (224) Google Scholar). Strikingly, a deletion of the BMP type 1A receptor in brown fat depletes these tissues in mice but results in a compensatory change in white fat to mimic a brownlike, or beige, fat phenotype (22Schulz T.J. Huang P. Huang T.L. Xue R. McDougall L.E. Townsend K.L. Cypess A.M. Mishina Y. Gussoni E. Tseng Y.H. Brown-fat paucity due to impaired BMP signalling induces compensatory browning of white fat.Nature. 2013; 495: 379-383Crossref PubMed Scopus (288) Google Scholar). Induction of BMP8 through a HFD can increase thermogenesis by enhanced signaling through the p38MAPK/cAMP-response element-binding protein pathway in BAT and regulation of neuropeptides in response to norepinephrine in the hypothalamus (23Whittle A.J. Carobbio S. Martins L. Slawik M. Hondares E. Vázquez M.J. Morgan D. Csikasz R.I. Gallego R. Rodriguez-Cuenca S. Dale M. Virtue S. Villarroya F. Cannon B. Rahmouni K. et al.BMP8B increases brown adipose tissue thermogenesis through both central and peripheral actions.Cell. 2012; 149: 871-885Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar). The regulation of TGF-β superfamily signaling is complex and encompasses mechanisms that function both within and outside of the cell. The TGF-β family ligands are disulfide-linked homo- or heterodimers that form heterotetramers with type I and type II transmembrane receptor kinases. Activated type II receptor phosphorylates the type I receptor, a serine/threonine kinase that phosphorylates the receptor-activated SMAD proteins, which transduce the signal to the nucleus. The accessibility of either TGF-β or BMPs to their corresponding receptors can be inhibited or enhanced by the actions of secreted proteins. Extracellular inhibitors of BMP signaling include vertebrate chordin, which binds directly to BMPs through the cysteine-rich (CR) domains containing CXXCXC and CCXXC motifs (24Larraín J. Bachiller D. Lu B. Agius E. Piccolo S. De Robertis E.M. BMP-binding modules in chordin: a model for signalling regulation in the extracellular space.Development. 2000; 127: 821-830Crossref PubMed Google Scholar, 25Garcia Abreu J. Coffinier C. Larraín J. Oelgeschläger M. De Robertis E.M. Chordin-like CR domains and the regulation of evolutionarily conserved extracellular signaling systems.Gene. 2002; 287: 39-47Crossref PubMed Scopus (143) Google Scholar, 26Shi Y. Massagué J. Mechanisms of TGF-β signaling from cell membrane to the nucleus.Cell. 2003; 113: 685-700Abstract Full Text Full Text PDF PubMed Scopus (4795) Google Scholar). Although chordin blocks BMP/receptor interactions, the multi-CR domain protein KCP enhances BMP/receptor interactions to increase the efficacy of signaling (27Lin J. Patel S.R. Cheng X. Cho E.A. Levitan I. Ullenbruch M. Phan S.H. Park J.M. Dressler G.R. Kielin/chordin-like protein, a novel enhancer of BMP signaling, attenuates renal fibrotic disease.Nat. Med. 2005; 11: 387-393Crossref PubMed Scopus (155) Google Scholar). However, KCP is also able to inhibit TGF-β and activin signaling (28Lin J. Patel S.R. Wang M. Dressler G.R. The cysteine-rich domain protein KCP is a suppressor of transforming growth factor β/activin signaling in renal epithelia.Mol. Cell. Biol. 2006; 26: 4577-4585Crossref PubMed Scopus (41) Google Scholar). Conversely, the CR domain protein connective tissue growth factor enhances TGF-β-mediated signaling while suppressing the BMP-dependent pathway (29Abreu J.G. Ketpura N.I. Reversade B. De Robertis E.M. Connective-tissue growth factor (CTGF) modulates cell signalling by BMP and TGF-β.Nat. Cell Biol. 2002; 4: 599-604Crossref PubMed Scopus (753) Google Scholar). Mice homozygous for a mutant kcp allele show no gross developmental abnormalities, although kcp mutations can enhance the renal developmental phenotype in mutants of Cv2 (30Ikeya M. Kawada M. Kiyonari H. Sasai N. Nakao K. Furuta Y. Sasai Y. Essential pro-Bmp roles of crossveinless 2 in mouse organogenesis.Development. 2006; 133: 4463-4473Crossref PubMed Scopus (96) Google Scholar), a gene that encodes a related multi-CR domain activator of BMPs. In animal models of renal disease, kcp−/− mice are sensitized to developing renal interstitial fibrosis (27Lin J. Patel S.R. Cheng X. Cho E.A. Levitan I. Ullenbruch M. Phan S.H. Park J.M. Dressler G.R. Kielin/chordin-like protein, a novel enhancer of BMP signaling, attenuates renal fibrotic disease.Nat. Med. 2005; 11: 387-393Crossref PubMed Scopus (155) Google Scholar), a process regulated by both BMPs and TGF-β. The kcp−/− mice exhibit lower BMP signaling and higher TGF-β signaling as measured by specific P-SMAD readouts after renal injury. Conversely, in mice that express a KCP transgene, a reciprocal shift in the balance of TGF-β and BMP signaling made animals more resistant to developing interstitial fibrosis in the kidney (31Soofi A. Zhang P. Dressler G.R. Kielin/chordin-like protein attenuates both acute and chronic renal injury.J. Am. Soc. Nephrol. 2013; 24: 897-905Crossref PubMed Scopus (27) Google Scholar). In the livers of aged mice or in mice fed a high-fat diet to induce non-alcoholic fatty liver disease, loss of KCP promoted hepatic steatosis and liver fibrosis, whereas expression of a KCP transgene was protective (32Soofi A. Wolf K.I. Ranghini E.J. Amin M.A. Dressler G.R. The kielin/chordin-like protein KCP attenuates nonalcoholic fatty liver disease in mice.Am. J. Physiol. Gastrointest. Liver Physiol. 2016; 311: G587-G598Crossref PubMed Scopus (5) Google Scholar). In this report, we describe how loss and gain of KCP function impact body mass, body composition, and the effects of diet-induced obesity on nutrient metabolism. Initial observations indicated that kcp−/− mice gained more weight over time, whereas KCP transgenic mice (kcpTg) remained lean. The KCP protein altered the amount and type of body fat to greatly impact metabolism. Thus, transgenic KCP expression protected mice from HFD-induced obesity, whereas the KCP-deficient mutant mice are more susceptible to metabolic dysfunction even on a normal diet (ND). By modulating KCP protein to alter the signaling balance between BMPs and TGF-β and associated P-SMADs, fat composition, thermoregulation, and metabolism were all impacted. These data suggest that altering TGF-β superfamily signaling by secreted regulatory proteins can attenuate the negative effects of obesity-induced metabolic syndrome. We had created a kcp−/− allele by conventional gene targeting (27Lin J. Patel S.R. Cheng X. Cho E.A. Levitan I. Ullenbruch M. Phan S.H. Park J.M. Dressler G.R. Kielin/chordin-like protein, a novel enhancer of BMP signaling, attenuates renal fibrotic disease.Nat. Med. 2005; 11: 387-393Crossref PubMed Scopus (155) Google Scholar) and a kcpTg allele using the mouse PEPCK promoter driving the KCP cDNA coding region with an amino-terminal human Igκ secretory signal and a carboxyl-terminal myc epitope tag (31Soofi A. Zhang P. Dressler G.R. Kielin/chordin-like protein attenuates both acute and chronic renal injury.J. Am. Soc. Nephrol. 2013; 24: 897-905Crossref PubMed Scopus (27) Google Scholar). Both kcp−/− and kcpTg mice were viable and fertile with no gross morphological defects. As reported previously, aged kcp−/− mice exhibit liver pathology that was accentuated when the mice were put on a high-fat diet to induce obesity (32Soofi A. Wolf K.I. Ranghini E.J. Amin M.A. Dressler G.R. The kielin/chordin-like protein KCP attenuates nonalcoholic fatty liver disease in mice.Am. J. Physiol. Gastrointest. Liver Physiol. 2016; 311: G587-G598Crossref PubMed Scopus (5) Google Scholar). To more precisely quantitate the changes in body mass, growth curves were determined for kcp−/−, kcp+/+, and kcpTg mice when fed a ND or HFD (Fig. 1, A and B). On normal diet, both kcp+/+ and kcp−/− mice were on average about 25% heavier than kcpTg. After 12 weeks on a high-fat diet, both kcp+/+ and kcp−/− were more than 80% heavier than the kcpTg mice. In fact, the kcpTg mice did not gain significantly more weight on the high-fat diet compared with the normal diet. We did not observe any gross skeletal abnormalities or evidence of dwarfism as body lengths were not significantly different between the genotypes. Body composition analyses indicated that the difference in body weights on both diets between WT or kcp−/− and the kcpTg mice was due in large part to less white adipose tissue in the kcpTg mice with no significant difference in fluid composition (Fig. 1, C and D). Obesity-induced metabolic disorder in mice can lead to glucose intolerance and insulin resistance. To assess the effects of KCP loss and overexpression on glucose metabolism, we performed glucose tolerance testing (Fig. 1, E and F). kcp+/+, kcp−/−, and kcpTg mice maintained on normal diets had similar glucose tolerance. However on the high-fat diets, the kcp+/+ and kcp−/− mice had impaired glucose tolerance compared with their lean counterparts, whereas the kcpTg mice showed glucose uptake similar to the mice on a normal diet (180 ± 25 mg/dl after 15 min). In those mice, fasting insulin levels were significantly higher in the kcp−/− animals, consistent with the development of glucose intolerance (Fig. 1G). To evaluate the mechanism of impaired weight gain in the kcpTg mice, we first measured average body temperatures as an indicator of metabolic function and thermoregulation. The kcpTg mice had significantly higher average body temperatures, almost a full degree more, than kcp+/+ or kcp−/− mice regardless of the diet conditions (Fig. 1H). Subsequently, mice of all three genotypes were fed either normal or high-fat diets for 8 weeks and then adapted to single metabolic cages for 4 weeks. Multiple metabolic parameters were then measured over a 3-day time period (Fig. 2A). Among the mice fed a normal diet, the only significant difference among the genotypes was that the kcpTg mice oxidized more glucose compared with kcp+/+ and kcp−/−. However, the mice kept on a HFD exhibited multiple differences among the three genotypes. The kcpTg mice had higher total energy expenditure compared with kcp+/+, whereas the kcp−/− mice expended less energy than kcp+/+. The differences are best illustrated in average oxygen consumption, as adjusted for lean body mass, over the light and dark cycles of a 3-day test period (Fig. 2B). The kcpTg mice used ∼14% more oxygen compared with kcp+/+, whereas the kcp−/− mice used 25% less oxygen over the 3-day period. Similar statistically significant data can be seen when energy expenditure is averaged over time for the three genotypes (Fig. 2C). The increased energy expenditure in kcpTg mice was not reflected by any more activity or any more food consumption as there were no significant differences among the three genotypes. The kcpTg mice were able to oxidize more glucose and fat compared with kcp+/+, whereas kcp−/− mice oxidized less glucose but similar amounts of fat compared with kcp+/+. The increased energy and oxygen consumption, despite no change in food intake or activity, are consistent with the significantly increased body temperature in kcpTg mice and suggest altered thermoregulation. To determine whether this was due to a neuroendocrine effect or responsiveness to norepinephrine (NE), we challenged the mice with a single dose of NE and measured the metabolic rates (Fig. 2D). Mice were kept at a thermoneutral temperature of 30 °C and anesthetized for baseline readings. Subsequently, a single injection of NE significantly increased respiration and energy expenditure in all three genotypes with no significant differences. The three genotypes were also subjected to a series of temperature challenges by first housing mice at 22 °C for 1 day, 30 °C for another day, and then 4 °C for 2 days (Fig. 2E). All three genotypes exhibited a decrease in energy expenditure and oxygen consumption at 30 °C followed by a nearly 3-fold increase in energy expenditure at 4 °C. As previous results indicated, average body temperatures at 22 °C were significantly higher in kcpTg mice, but this effect was reduced at 30 °C (Fig. 2F). Strikingly, at 4 °C, average subcutaneous body temperatures appeared lower in the kcpTg mice, which may reflect the reduced amount of insulating subcutaneous body fat in these animals. To determine the mechanisms underlying the reduced amount of white adipose tissue in kcpTg mice, we characterized both white and brown adipose tissues in mice kept on a HFD. Although kcpTg mice had less visceral fat, we were able to isolate enough epididymal WAT (eWAT) for histology and protein expression analyses. By Western blot analyses, kcpTg eWAT had significantly lower levels of P-SMAD3 but higher levels of P-SMAD1 compared with either kcp+/+ or kcp−/− eWAT (Fig. 3, A and B). These data are entirely consistent with prior results showing inhibition of TGF-β signaling and enhancement of BMP signaling by KCP protein. Furthermore, the kcpTg eWAT had higher levels of UCP1, which increases heat production and is usually associated with brown fat (Fig. 3, A and C). The kcpTg eWAT tissues also had higher levels of PPARγ, PGC1, P-AKT, and cytochrome c oxidase subunit IV, all proteins associated with a more beige fat phenotype (Fig. 3, A and C). Also of note were the increased levels of P-ERK in the kcp−/− eWAT; P-ERK is known to phosphorylate PPARγ and increase expression of diabetes-associated genes (33Banks A.S. McAllister F.E. Camporez J.P. Zushin P.J. Jurczak M.J. Laznik-Bogoslavski D. Shulman G.I. Gygi S.P. Spiegelman B.M. An ERK/Cdk5 axis controls the diabetogenic actions of PPARγ.Nature. 2015; 517: 391-395Crossref PubMed Scopus (206) Google Scholar). We also used quantitative RT-PCR to assess gene expression for adiponectin, kcp, and pgc1 (Fig. 3D), all of which were higher in the kcpTg mice and lower in the kcp−/− mice compared with the kcp+/+ animals. In mice fed a HFD, the eWAT tissue also exhibited differences in histology and in markers for inflammation among the three genotypes (Fig. 4). Trichrome staining showed extracellular collagen deposition in kcp−/− eWAT with many large adipocytes and evidence of enhanced inflammation. The average cross-sectional area of eWAT cells was approximately half in kcpTg adipocytes, whereas the kcp−/− adipocytes were even larger than kcp+/+ cells (Fig. 4, A–C and J). Whole-mount immunofluorescence examined macrophage infiltration and crownlike structures in eWAT as a measure of the inflammatory response that correlates with high-fat diet-induced obesity and glucose intolerance (Fig. 4, D–F and K). The kcpTg mice had few if any crownlike structures and MAC2-positive macrophages in eWAT. However, the obese kcp+/+ and especially the kcp−/− animals had large numbers of macrophages present in eWAT. We also looked for evidence of fibrosis by Picrosirius Red staining for collagen deposition. The kcp−/− animals showed increased collagen matrix deposition in eWAT compared with kcp+/+ mice, whereas the kcpTg mice had significantly less collagen deposition when fed a high-fat diet (Fig. 4, G–I and L). Given the increased body temperature and metabolism in kcpTg mice, we also examined interscapular brown adipose tissue (iBAT) for protein expression and histology. Protein expression levels in iBAT of mice fed a ND showed that kcpTg mice had elevated levels of P-SMAD1 and PPARγ but no significant differences in UCP1 (Fig. 5, A–C). The kcp−/− mice showed slightly more P-SMAD3 and less PPARγ but no significant differences in P-SMAD1 or UCP1 compared with kcp+/+ animals (Fig. 5, A–C). The kcpTg mice also demonstrated myc-KCP expression in iBAT, indicating that the PEPCK promoter used for the transgene is actively expressed in this tissue. When mice were kept on a HFD, differences in protein expression levels in iBAT from kcpTg and kcp−/− mice were more pronounced (Fig. 5, D–F). kcp−/− mice had less P-SMAD1 and higher levels of P-SMAD3 compared with kcpTg mice (Fig. 5F). In addition, levels of P-ERK were higher in kcp−/− mice. Levels of UCP1 were significantly higher in kcpTg mice as were PGC1 and PPARγ (Fig. 5E) compared with kcp−/− mice. Histology of iBAT indicated that the kcp−/− mice fed a normal diet exhibited increased lipid storage in iBAT, compared with WT, that became even more pronounced on the HFD (Fig. 6). Histology of BAT generally shows densely staining cytoplasm because of the density of mitochondria that fuel thermoregulation. Consistent with our metabolic data, kcpTg mice had very densely staining iBAT compared with WT on both normal diet and HFD (Fig. 6) with only minimal lipid accumulation. Finally, to test whether the differences in brown and white adipocytes were cell-autonomous and could reflect the response to TGF-β superfamily ligands, we cultured differentiated adipocytes isolated from inguinal WAT and interscapular BAT tissues of all three genotypes kept on a ND (Fig. 7). Differentiated adipocytes isolated from inguinal white adipose tissue (iWAT) showed that the kcpTg cells had a stronger response to BMP4 with higher levels of P-SMAD1 (Fig. 7A). Cells isolated from kcp−/− mice showed a higher basal level of P-SMAD3 and a stronger response to TGF-β despite having less total SMAD3. Also of note, the kcpTg cells expressed more UCP1, suggesting a more beige phenotype. The differentiated adipocytes isolated from iBAT showed a weaker BMP4 response in kcp−/− cells but no difference in the kcpTg cells. P-SMAD3 was not significantly affected in iBAT cells. However, kcp−/− cells had significantly lower UCP1 and PGC1 levels. The protein lysates were collected 2 h after addition of ligands, which did not affect expression of the non-phosphorylated proteins. Thus, the adipocytes that express myc-KCP have altered cell-autonomous responses to TGF superfamily ligands, consistent with the known role for KCP in modifying the receptor/ligand interactions. That these effects were observed on a ND indicates that they are not due to obesity or associated metabolic syndrome. The cultured cells are also removed from any infiltrating immune cells and associated cytokines, suggesting a direct effect of KCP on TGF-β superfamily signaling in adipocytes. We have utilized both gain and loss of KCP function in genetically engineered mice to demonstrate that KCP can regulate the T" @default.
• W2605800216 created "2017-04-28" @default.
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• W2605800216 date "2017-06-01" @default.
• W2605800216 modified "2023-10-15" @default.
• W2605800216 title "The kielin/chordin-like protein (KCP) attenuates high-fat diet-induced obesity and metabolic syndrome in mice" @default.
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