Resistin-like Molecule β Activates MAPKs, Suppresses Insulin Signaling in Hepatocytes, and Induces Diabetes, Hyperlipidemia, and Fatty Liver in Transgenic Mice on a High Fat Diet
2005; Elsevier BV; Volume: 280; Issue: 51 Linguagem: Inglês
10.1074/jbc.m503065200
ISSN1083-351X
AutoresAkifumi Kushiyama, Nobuhiro Shojima, Takehide Ogihara, Kouichi Inukai, Hideyuki Sakoda, Midori Fujishiro, Yasushi Fukushima, Motonobu Anai, Hiraku Ono, Nanao Horike, Amelia Y.I. Viana, Yasunobu Uchijima, Koichi Nishiyama, Tatsuo Shimosawa, Toshiro Fujita, Hideki Katagiri, Yoshitomo Oka, Hiroki Kurihara, Tomoichiro Asano,
Tópico(s)Pancreatic function and diabetes
ResumoResistin and resistin-like molecules (RELMs) are a family of proteins reportedly related to insulin resistance and inflammation. Because the serum concentration and intestinal expression level of RELMβ were elevated in insulin-resistant rodent models, in this study we investigated the effect of RELMβ on insulin signaling and metabolism using transgenic mice and primary cultured hepatocytes. First, transgenic mice with hepatic RELMβ overexpression were shown to exhibit significant hyperglycemia, hyperlipidemia, fatty liver, and pancreatic islet enlargement when fed a high fat diet. Hyperinsulinemic glucose clamp showed a decreased glucose infusion rate due to increased hepatic glucose production. In addition, the expression levels of IRS-1 and IRS-2 proteins as well as the degrees of insulin-induced phosphatidylinositol 3-kinase and Akt activations were attenuated in RELMβ transgenic mice. Similar down-regulations of IRS-1 and IRS-2 proteins were observed in primary cultured hepatocytes chronically treated (for 24 h) with RELMβ, suggesting the insulin resistance-inducing effect of RELMβ to be direct. Furthermore, it was shown that RELMβ acutely and markedly activates ERK and p38, while weakly activating JNK, in primary cultured hepatocytes. This increased basal p38 phosphorylation level was also observed in the livers of RELMβ transgenic mice. In conclusion, RELMβ, a gut-derived hormone, impairs insulin signaling probably via the activations of classic MAPKs, and increased expression of RELMβ may be involved in the pathogenesis of glucose intolerance and hyperlipidemia in some insulin-resistant models. Thus, RELMβ is a potentially useful marker for assessing insulin resistance and may also be a target for future novel anti-diabetic agents. Resistin and resistin-like molecules (RELMs) are a family of proteins reportedly related to insulin resistance and inflammation. Because the serum concentration and intestinal expression level of RELMβ were elevated in insulin-resistant rodent models, in this study we investigated the effect of RELMβ on insulin signaling and metabolism using transgenic mice and primary cultured hepatocytes. First, transgenic mice with hepatic RELMβ overexpression were shown to exhibit significant hyperglycemia, hyperlipidemia, fatty liver, and pancreatic islet enlargement when fed a high fat diet. Hyperinsulinemic glucose clamp showed a decreased glucose infusion rate due to increased hepatic glucose production. In addition, the expression levels of IRS-1 and IRS-2 proteins as well as the degrees of insulin-induced phosphatidylinositol 3-kinase and Akt activations were attenuated in RELMβ transgenic mice. Similar down-regulations of IRS-1 and IRS-2 proteins were observed in primary cultured hepatocytes chronically treated (for 24 h) with RELMβ, suggesting the insulin resistance-inducing effect of RELMβ to be direct. Furthermore, it was shown that RELMβ acutely and markedly activates ERK and p38, while weakly activating JNK, in primary cultured hepatocytes. This increased basal p38 phosphorylation level was also observed in the livers of RELMβ transgenic mice. In conclusion, RELMβ, a gut-derived hormone, impairs insulin signaling probably via the activations of classic MAPKs, and increased expression of RELMβ may be involved in the pathogenesis of glucose intolerance and hyperlipidemia in some insulin-resistant models. Thus, RELMβ is a potentially useful marker for assessing insulin resistance and may also be a target for future novel anti-diabetic agents. Insulin resistance is a major cause of type 2 diabetes, and recent studies have revealed many independent mechanisms regulating insulin sensitivity. Among them, much attention has been paid to the roles of secreted proteins in insulin resistance. Resistin (also known as FIZZ3 ADSF, mXCP4, or hXCP1) was identified as a factor that is secreted by adipocytes and causes insulin resistance (1Steppan C.M. Bailey S.T. Bhat S. Brown E.J. Banerjee R.R. Wright C.M. Patel H.R. Ahima R.S. Lazar M.A. Nature. 2001; 409: 307-312Crossref PubMed Scopus (4004) Google Scholar). This finding was supported by not only in vitro experiments using cultured cells but also animal experiments, i.e. mice with adenoviral resistin expression, infusion of recombinant resistin, the use of neutralizing antibody, or by generating resistin gene knock-out mice (2Kim K.H. Lee K. Moon Y.S. Sul H.S. J. Biol. Chem. 2001; 276: 11252-11256Abstract Full Text Full Text PDF PubMed Scopus (555) Google Scholar, 3Moon B. Kwan J.J. Duddy N. Sweeney G. Begum N. Am. J. Phys-iol. 2003; 285: E106-E115Crossref PubMed Scopus (146) Google Scholar, 4Banerjee R.R. Rangwala S.M. Shapiro J.S. Rich A.S. Rhoades B. Qi Y. Wang J. Rajala M.W. Pocai A. Scherer P.E. Steppan C.M. Ahima R.S. Obici S. Rossetti L. Lazar M.A. Science. 2004; 303: 1195-1198Crossref PubMed Scopus (624) Google Scholar, 5Rangwala S.M. Rich A.S. Rhoades B. Shapiro J.S. Obici S. Rossetti L. Lazar M.A. Diabetes. 2004; 53: 1937-1941Crossref PubMed Scopus (191) Google Scholar, 6Satoh H. Nguyen M.T. Miles P.D. Imamura T. Usui I. Olefsky J.M. J. Clin. Invest. 2004; 114: 224-231Crossref PubMed Scopus (250) Google Scholar). However, some clinical studies have failed to demonstrate a close relationship between obesity or insulin resistance and the serum resistin concentration in humans (7Fehmann H.C. Heyn J. Horm. Metab. Res. 2002; 34: 671-673Crossref PubMed Scopus (80) Google Scholar, 8Lee J.H. Chan J.L. Yiannakouris N. Kontogianni M. Estrada E. Seip R. Orlova C. Mantzoros C.S. J. Clin. Endocrinol. Metab. 2003; 88: 4848-4856Crossref PubMed Scopus (488) Google Scholar). Thus, while resistin apparently induces insulin resistance, the involvement of resistin in the pathogenesis of human diabetes and obesity remains unclear. On the other hand, there are three resistin-related proteins, termed RELMα 2The abbreviations used are: RELMresistin-like moleculeMAPKmitogen-activate protein kinaseIRSinsulin receptor substrateSAPKstress-activated protein kinaseJNKc-Jun N-terminal kinase2DG2-deoxy-d-glucosePIphosphatidylinositolSAPserum amyloid PPPARαperoxisome proliferator-activated receptor α. (resistin-like molecule, FIZZ1, or mXCP2), β (FIZZ2, mXCP3, or hXCP2), and γ (mXCP1) (9Steppan C.M. Brown E.J. Wright C.M. Bhat S. Banerjee R.R. Dai C.Y. Enders G.H. Silberg D.G. Wen X. Wu G.D. Lazar M.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 502-506Crossref PubMed Scopus (576) Google Scholar). RELMα is expressed in white adipose tissue, the lung, tongue and bone marrow, whereas the expression of RELMβ is strictly limited to the small and large intestines, especially goblet cells. Although RELMγ is expressed in mouse spleen, bone marrow, intestines, and a variety of other tissues, corresponding to each developmental stage depending on CAAT/enhancer-binding protein-ϵ (10Chumakov A.M. Kubota T. Walter S. Koeffler H.P. Oncogene. 2004; 23: 3414-3425Crossref PubMed Scopus (32) Google Scholar), its human homolog has not been identified. These proteins contain a highly conserved cysteine-rich C terminus (Cys-X11-Cys-X8-Cys-X-Cys-X2-Cys-X10-Cys-X-Cys-X-Cys-X9-Cys-Cys) and a signal peptide sequence at their N termini, as observed in resistin. Assuming that they have biological activities similar to that of resistin, these RELMs and resistin are considered to be equally important for the regulation of insulin sensitivity. Indeed, it was reported that injecting mice with either resistin or RELMβ induced insulin resistance (11Rajala M.W. Obici S. Scherer P.E. Rossetti L. J. Clin. Invest. 2003; 111: 225-230Crossref PubMed Scopus (486) Google Scholar). In addition, we found the intestinal expression and serum concentration of RELMβ to be increased in insulin resistant models such as high fat fed and db/db mice (12Shojima N. Ogihara T. Inukai K. Fujishiro M. Sakoda H. Kushiyama A. Katagiri H. Anai M. Ono H. Fukushima Y. Horike N. Viana A.Y. Uchijima Y. Kurihara H. Asano T. Diabetologia. 2005; 48: 984-992Crossref PubMed Scopus (40) Google Scholar). Thus, we speculated that the inflammatory state of the intestine, overeating, bowel movements, and/or nutrient absorption might regulate the intestinal expression and serum level of RELMβ and thereby regulate whole-body insulin sensitivity. resistin-like molecule mitogen-activate protein kinase insulin receptor substrate stress-activated protein kinase c-Jun N-terminal kinase 2-deoxy-d-glucose phosphatidylinositol serum amyloid P peroxisome proliferator-activated receptor α. Herein, to examine chronic effects of the elevated serum RELMβ observed under insulin-resistant conditions such as a high fat diet and in db/db mice, we generated transgenic mice overexpressing RELMβ and analyzed their metabolic profiles relating to insulin resistance and changes in the insulin signaling pathway. Furthermore, to investigate the physiological significance of elevated serum RELMβ, we investigated the effects of RELMβ on insulin signaling using primary cultured hepatocytes. Herein, we show that RELMβ activates three MAPKs and down-regulates IRS-1/2 proteins and suppresses insulin signaling. Our observations suggest that RELMβ may be a useful marker for assessing insulin resistance associated with obesity and may also serve as a target for future novel anti-diabetic agents. Antibodies—The affinity-purified antibodies against RELMβ, insulin receptor substrate (IRS)-1, IRS-2, tyrosine phosphorylation (4G10), and Akt/protein kinase B were prepared as previously described (13Ono H. Shimano H. Katagiri H. Yahagi N. Sakoda H. Onishi Y. Anai M. Ogihara T. Fujishiro M. Viana A.Y. Fukushima Y. Abe M. Shojima N. Kikuchi M. Yamada N. Oka Y. Asano T. Diabetes. 2003; 52: 2905-2913Crossref PubMed Scopus (141) Google Scholar). The antibodies against phospho-Ser473 of Akt, phospho-p44/p42, p44/42, phospho-p38 MAPK, p38 MAPK, phospho-SAPK/JNK, and SAPK/JNK were purchased from Cell Signaling Technology. Preparation of Recombinant RELMβ—Adenoviruses expressing RELMβ were constructed using an Ad Easy kit (Quantum Biotechnology). HEK293 T cells transfected with RELMβ adenovirus produced 50–100 mg/liter RELMβ protein in the medium. Serum-free medium CD293 (Invitrogen) was used to collect the secreted protein from confluent HEK293 T cells for 2 days. The medium was harvested and spun down to remove cells. The medium was then purified and concentrated using a high S column and Biologic LP system (Bio-Rad) as described previously (4Banerjee R.R. Rangwala S.M. Shapiro J.S. Rich A.S. Rhoades B. Qi Y. Wang J. Rajala M.W. Pocai A. Scherer P.E. Steppan C.M. Ahima R.S. Obici S. Rossetti L. Lazar M.A. Science. 2004; 303: 1195-1198Crossref PubMed Scopus (624) Google Scholar). Greater than 95% purity was confirmed by silver staining with a Silver Staining Kit (BEXEL Biotechnology), and quantities were determined by Western blotting, using commercially available recombinant RELMβ (Peprotech) as the standard. The medium of HEK293T cells transfected with β-galactosidase expressing adenovirus was used to prepare a control solution. The pH values of RELMβ and control solution were adjusted to 7.4. Construction of RELMβ and Generation of Transgenic Mice—The open reading frame of RELMβ was obtained employing PCR based on the previously reported sequence (9Steppan C.M. Brown E.J. Wright C.M. Bhat S. Banerjee R.R. Dai C.Y. Enders G.H. Silberg D.G. Wen X. Wu G.D. Lazar M.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 502-506Crossref PubMed Scopus (576) Google Scholar). This RELMβ cDNA was cloned into a pAT15-3 vector containing the SAP promoter and rat β-globin intron (Fig. 1a), which was used for the generation of RELMβ transgenic mice. Animals and High Fat Diet—The C57/Bl6 line was used to generate RELMβ transgenic mice. All animal studies were conducted according to the Japanese guidelines for the care and use of experimental animals. All animal experiments were performed after 4-week high fat diet loading, unless otherwise indicated. The high fat diet had, basically, the previously described composition (14Anai M. Funaki M. Ogihara T. Kanda A. Onishi Y. Sakoda H. Inukai K. Nawano M. Fukushima Y. Yazaki Y. Kikuchi M. Oka Y. Asano T. Diabetes. 1999; 48: 158-169Crossref PubMed Scopus (89) Google Scholar), except that the skim was added to the formulation. Food intakes were determined daily for 5 consecutive days. Food was withdrawn 12 h before each experiment. Immunoblotting of RELMβ—Serum RELMβ concentrations in transgenic mice and their littermates were determined by immunoblotting. Two microliters of serum was boiled in Laemmli sample buffer containing 100 mm dithiothreitol. Samples were subjected to SDS-PAGE, transferred to 0.1-μm pore nitrocellulose, and immunoblotted using anti-RELMβ antibody (1:1000). Proteins were visualized with enhanced chemiluminescence (ECL or ECL plus) and exposed to ECL film (Amersham Biosciences) Serum Glucose, Lipids, and Hepatic Triglyceride and Glycogen— Blood glucose was measured with a portable blood glucose monitor, Freestyle Kissei (Kissei Pharmaceutical, Japan). The plasma insulin level was determined with an enzymatic immunoassay kit (Amersham Biosciences). Serum triglyceride, cholesterol, and free fatty acids were assayed with Triglyceride E test Wako, Cholesterol E test Wako, and NEFA C test Wako (Wako Chemicals, Japan), respectively. Serum adiponectin was assayed with an adiponectin measurement kit (Otsuka Pharmaceuticals, Japan). Hepatic total lipid was extracted and assayed using the Folch method, as described previously (15Folch J. Lees M. Sloane Stanley G.H. J. Biol. Chem. 1957; 226: 497-509Abstract Full Text PDF PubMed Google Scholar). The triglyceride content was assayed as described above. Hepatic glycogen content was measured as previously described (16Bergmeyer H. Bergmeyer J. Grassl M. Methods of Enzymatic Analys-is. 3rd ed. Wiley, Hoboken, NJ1984: 11-18Google Scholar). Tolerance Test—Glucose (2 g/kg glucose load), insulin (0.75 unit/kg insulin), and pyruvate (2 g/kg pyruvate) tolerance tests were performed as previously described (17Miyake K. Ogawa W. Matsumoto M. Nakamura T. Sakaue H. Kasuga M. J. Clin. Invest. 2002; 110: 1483-1491Crossref PubMed Scopus (134) Google Scholar). Glucose Clamp Study—The glucose clamp study was performed, as previously described (18Onishi Y. Honda M. Ogihara T. Sakoda H. Anai M. Fujishiro M. Ono H. Shojima N. Fukushima Y. Inukai K. Katagiri H. Kikuchi M. Oka Y. Asano T. Biochem. Biophys-. Res. Commun. 2003; 303: 788-794Crossref PubMed Scopus (83) Google Scholar, 19Miles P.D. Barak Y. Evans R.M. Olefsky J.M. Am. J. Phys-iol. 2003; 284: E618-E626Crossref PubMed Scopus (47) Google Scholar, 20Fueger P.T. Bracy D.P. Malabanan C.M. Pencek R.R. Granner D.K. Wasserman D.H. Diabetes. 2004; 53: 306-314Crossref PubMed Scopus (63) Google Scholar, 21Fisher S.J. Kahn C.R. J. Clin. Invest. 2003; 111: 463-468Crossref PubMed Scopus (171) Google Scholar), with some modifications. In brief, mice were implanted with catheters that were exteriorized at the back of the neck and encased in silastic tubing. Four days after surgery, the animals were fasted for 3 h and used for the experiments. [d-3H]Glucose (Amersham Biosciences) was injected (bolus 10 μCi, 0.1 μCi/min, for 240 min) intravenously. After a 90-min basal period, a blood sample was collected for determination of glucose-specific activity and the blood glucose level. At time 0, hyperinsulinemic-euglycemic clamps were started, and 10 milliunits/kg/min human insulin (Novolin R, Novo Nordisk) was continuously infused for 150 min. The blood glucose concentration was clamped at 90–120 mg/dl, for at least 60 min, by estimating the blood glucose concentration at 5-min intervals and adjusting the rate of glucose solution infusion. Blood samples were taken to determine blood glucose, insulin, and plasma [d-3H]glucose every 30 min for 120 min. Then, 12 μCi of [14C]2-deoxy-d-glucose ([14C]2DG, Amersham Biosciences) was injected, and blood samples were taken at 122, 125, 130, and 150 min to determine blood glucose and plasma [14C]2DG. At 150 min, the gastrocnemius (type IIB fibers) muscle, soleus (type I and type IIA fibers) muscles, and epididymal fat were immediately excised and frozen in liquid nitrogen, then stored at –80 °C until future tissue analysis. Plasma and Tissue Assays in Glucose Clamp Study—After deproteinization with barium hydroxide (Ba(OH)2, 0.3 n) and zinc sulfate (ZnSO4, 0.3 n), [d-3H]glucose and [14C]2DG radioactivities of plasma were determined by dual channeled liquid scintillation counting. Hepatic glucose production and the glucose disposal rate were calculated for the basal period and the steady-state portion of the glucose clamp as previously described (21Fisher S.J. Kahn C.R. J. Clin. Invest. 2003; 111: 463-468Crossref PubMed Scopus (171) Google Scholar). Muscle and fat samples were weighed and homogenized in 0.5% perchloric acid. Homogenates were centrifuged and neutralized with NaHCO3. The sample was then separated into two aliquots. One was counted directly to determine [14C]2DG and [14C]2DG-6-phosphate ([14C]2DGP) radioactivities. The other was treated with Ba(OH)2 and ZnSO4 to remove [14C]2DGP and any tracer incorporated into glycogen and then counted to determine [14C]2DG radioactivity. [14C]2DGP is the difference between the two aliquots. Tissue glucose uptake was calculated as described (20Fueger P.T. Bracy D.P. Malabanan C.M. Pencek R.R. Granner D.K. Wasserman D.H. Diabetes. 2004; 53: 306-314Crossref PubMed Scopus (63) Google Scholar). In Vivo Insulin Stimulation—In vivo insulin stimulation was performed, as previously described (22Ogihara T. Shin B.C. Anai M. Katagiri H. Inukai K. Funaki M. Fukushima Y. Ishihara H. Takata K. Kikuchi M. Yazaki Y. Oka Y. Asano T. J. Biol. Chem. 1997; 272: 12868-12873Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar), with some modifications. In brief, mice were anesthetized with pentobarbital sodium. The portal vein was exposed, and 0.4 ml of normal saline (0.9% NaCl) with or without insulin (25 milliunits/g body wt) was injected. The livers were removed 30 s later, and hind limb skeletal muscles were removed 90 s thereafter and immediately homogenized with a Polytron homogenizer in 6 volumes of solubilization buffer. Both extracts were centrifuged at 15,000 × g for 30 min at 4 °C, and the supernatants were used as samples for immunoprecipitation, immunoblotting, or kinase assay of PI 3-kinase and Akt/protein kinase B. Immunoprecipitation and Immunoblotting—Supernatants containing equal amounts of protein (8 mg) were incubated with anti-IRS-1 and anti-IRS-2 antibodies (5 mg/ml each) and then incubated with 100 μlof protein A- and G-Sepharose. The samples were washed and then boiled in Laemmli sample buffer containing 100 mm dithiothreitol. Total lysates were also boiled to allow detection of Akt, AktSer473 phosphorylation, the three MAPKs, and their phosphorylations. Total lysates or immunoprecipitants were subjected to Western blotting, blotted with one of the antibodies or 4G10 antibody. Band intensities were quantified with Image J (National Institutes of Health). Measurement of PI 3-Kinase and Akt/Protein Kinase B Activity—For PI 3-kinase assay, the supernatants containing equal amounts of protein were immunoprecipitated for 2 h at 4°C with anti-IRS-1, anti-IRS-2 or 4G10 antibody and protein A- or G-Sepharose. PI 3-kinase activities in the immunoprecipitants were assayed as previously described (23Ogihara T. Asano T. Katagiri H. Sakoda H. Anai M. Shojima N. Ono H. Fujishiro M. Kushiyama A. Fukushima Y. Kikuchi M. Noguchi N. Aburatani H. Gotoh Y. Komuro I. Fujita T. Diabetologia. 2004; 47: 794-805Crossref PubMed Scopus (121) Google Scholar). For the Akt kinase assay, an Akt kinase assay kit (Cell signaling) was used according to the manufacturer's instructions. Glucose Uptake by Isolated Soleus Muscle in Vitro—Insulin-stimulated glucose uptake by the soleus muscle was measured as described previously (24Sakoda H. Ogihara T. Anai M. Fujishiro M. Ono H. Onishi Y. Katagiri H. Abe M. Fukushima Y. Shojima N. Inukai K. Kikuchi M. Oka Y. Asano T. Am. J. Phys-iol. 2002; 282: E1239-E1244Crossref PubMed Scopus (81) Google Scholar). Mouse soleus muscles were isolated and incubated for 30 min in KHB buffer, with or without human insulin (2 milliunits/ml). The muscles were then rinsed for 10 min at 29 °C and incubated for 20 min at 29 °C in KHB buffer containing 8 mm 2-deoxy-d-[1,2-3H]glucose (2-DG) (2.25 μCi/ml) and 32 mm [14C]mannitol (0.3 μCi/ml). After incubation, the muscles were rapidly solubilized. Radioactivity in the resultant samples was counted, and 2-DG uptake rates were corrected for extracellular trapping with mannitol counts. Effects of RELMβ on Insulin Action in Primary Hepatocytes—Hepatocytes were isolated from fasted C57/bl6 mice by collagenase perfusion, as described previously (25Sakoda H. Gotoh Y. Katagiri H. Kurokawa M. Ono H. Onishi Y. Anai M. Ogihara T. Fujishiro M. Fukushima Y. Abe M. Shojima N. Kikuchi M. Oka Y. Hirai H. Asano T. J. Biol. Chem. 2003; 278: 25802-25807Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). To determine the effects of RELMβ on insulin signaling, the dishes were split into two groups corresponding the presence and absence of RELMβ stimulation for 24 h. The RELMβ concentration was 1 μg/ml. The cells were then serum-starved for 3 h and stimulated with 10–6 m insulin for 5 min at 37 °C. IRS-1 and IRS-2 protein contents were evaluated by immunoblotting as described above. Insulin-induced IRS-1 and IRS-2 phosphorylations were evaluated by 4G10 immunoprecipitation and immunoblotting as described above. Effect of RELMβ on Phosphorylations of MAPKs—To determine the effects of RELMβ on MAPK phosphorylations with their time course and dose dependence, primary hepatocytes were stimulated with 1 μg/ml RELMβ for 10 or 30 min, and with 0.01, 0.1, or 1 μg/ml RELMβ for 15 min. Phosphorylations of p44/p42 (ERK1/2), p38 MAPK, and p54/p46 (SAPK/JNK) were evaluated by immunoblotting as described previously (26Fujishiro M. Gotoh Y. Katagiri H. Sakoda H. Ogihara T. Anai M. Onishi Y. Ono H. Abe M. Shojima N. Fukushima Y. Kikuchi M. Oka Y. Asano T. Mol. Endocrinol. 2003; 17: 487-497Crossref PubMed Scopus (165) Google Scholar). Tissue Hematoxylin-Eosin Staining—The liver and pancreas were removed from transgenic mice and their littermates and formalin-fixed. Samples were routinely embedded in paraffin. Approximately 5-μm-thick slices obtained from these samples were stained with hematoxylin and eosin. Mean pancreatic islet area was histomorphometrically analyzed as previously described (27Tomimoto S. Hashimoto H. Shintani N. Yamamoto K. Kawabata Y. Hamagami K. Yamagata K. Miyagawa J. Baba A. J. Pharmacol. Exp. Ther. 2004; 309: 796-803Crossref PubMed Scopus (23) Google Scholar), using ImageJ (National Institutes of Health). Ribonuclease Protection Assay—Riboprobes of enzymes were amplified from mouse embryonic cDNA using PCR primers and subcloned as already described (13Ono H. Shimano H. Katagiri H. Yahagi N. Sakoda H. Onishi Y. Anai M. Ogihara T. Fujishiro M. Viana A.Y. Fukushima Y. Abe M. Shojima N. Kikuchi M. Yamada N. Oka Y. Asano T. Diabetes. 2003; 52: 2905-2913Crossref PubMed Scopus (141) Google Scholar). Total RNA from the liver and primary cultured hepatocytes was isolated using TRIzol reagent (Isogen, Nippon Gene, Japan). A 10-μg RNA sample was used for each assay. RNase protection assays were carried out according to the manufacturer's instructions (RPA III kit, Ambion, Austin, TX). Intensities of the resultant bands were determined using BAS2000 (Fuji film, Japan). Statistical Analysis—Results are expressed as means ± S.E., and significance was assessed using unpaired Student's t tests, unless otherwise indicated. Generating RELMβ Transgenic Mice—We generated transgenic mice, the livers of which express RELMβ, by inserting RELMβ cDNA downstream from the serum amyloid P (SAP) promoter. As expected, liver-specific expression in these transgenic mice was confirmed (data not shown), and two lines were established. In the livers of line 1 mice, RELMβ was highly overexpressed, and the serum RELMβ concentration exceeded that of the non-transgenic mice by 10-fold (Fig. 1c). In line 2 mice, RELMβ was moderately overexpressed, and the resulting elevation of serum RELMβ was approximately double that in non-transgenic mice (Fig. 1c). Hepatic RELMβ expression was reportedly increased in response to high fat feeding. Thus, increases in serum RELMβ with a high fat diet were observed in wild-type and line 2 mice, whereas there was no marked increase in the line 1 mice in which hepatic RELMβ overexpression was high. These observations indicate that a high fat diet increases endogenous RELMβ expression but not transgene-derived RELMβ expression. No significant difference was observed in terms of growth or adolescence between RELMβ transgenic mice and their littermates. At the time of sacrifice (age 16 weeks, after 4 weeks of being fed a high fat diet) and within the observation period, there was no significant difference in body weight or food intake. Glucose and Lipid Metabolic Profiles of RELMβ Transgenic Mice— The body weight, fasting serum glucose, serum insulin, and lipid concentrations of RELMβ transgenic mice did not differ from those of control mice when both were fed normal chow, at the age of 16 weeks. Furthermore, glucose, insulin, and pyruvate tolerance tests showed no significant differences between 16-week-old RELMβ-overexpressing mice and their littermates (Fig. 2, a–d, and TABLE ONE). However, the serum glucose concentration was significantly more elevated in both lines of transgenic mice (n = 25, p < 0.01) when fed a high fat diet (TABLE TWO), than those in the control mice. Under these conditions, hyperinsulinemia (n = 6, p < 0.05), hyperlipidemia (n = 6, p < 0.05), and increased hepatic triglyceride content (n = 8, p < 0.05) were observed in the transgenic mice.TABLE ONEGlucose and lipid metabolic profiles of 16-week-old RELMβ transgenic mice on normal chowControl (line 1)Transgenic (line 1)Control (line 2)Transgenic (line 2)BW (g)17.2 ± 0.817.9 ± 0.515.4 ± 1.116.4 ± 0.6FBS (mg/dl)88.9 ± 4.192.0 ± 2.481.2 ± 2.786.8 ± 2.6Serum insulin (ng/ml)0.08 ± 0.020.11 ± 0.02Serum TC (mg/dl)38.2 ± 2.236.7 ± 3.3Serum TG (mg/dl)49.6 ± 8.149.0 ± 5.8Serum free fatty acid (mEq/liter)0.80 ± 0.080.63 ± 0.07 Open table in a new tab TABLE TWOGlucose and lipid metabolic profiles of RELMβ transgenic mice, at the age of 16 weeks, which had been fed a high fat diet for 4 weeksControl (line 1)Transgenic (line 1)Control (line 2)Transgenic (line 2)Body weight (g)37.1 ± 2.636.5 ± 1.431.5 ± 1.533.9 ± 2.6Food intake (g/day)4.6 ± 0.44.5 ± 0.54.1 ± 0.24.3 ± 0.8FBS (mg/dl)109.5 ± 8.3156.1 ± 10.6aStatistical significance: p < 0.05 for control mice versus transgenic mice.117.3 ± 3.9132.0 ± 2.8aStatistical significance: p < 0.05 for control mice versus transgenic mice.Serum insulin (ng/ml)3.8 ± 0.712.3 ± 2.7aStatistical significance: p < 0.05 for control mice versus transgenic mice.5.4 ± 1.37.6 ± 1.4Serum TC (mg/dl)48.0 ± 3.661.2 ± 7.9aStatistical significance: p < 0.05 for control mice versus transgenic mice.50.3 ± 4.576.4 ± 13.6Serum TG (mg/dl)51.7 ± 6.484.6 ± 6.4aStatistical significance: p < 0.05 for control mice versus transgenic mice.75.9 ± 8.0103.3 ± 14.0aStatistical significance: p < 0.05 for control mice versus transgenic mice.Serum free fatty acid (mEq/l)0.59 ± 0.020.56 ± 0.04NTbNT, not tested.NTSerum adiponectin (μg/ml)14.1 ± 1.411.1 ± 0.9NTNTLiver TG (mg/g liver)14.3 ± 1.921.0 ± 2.9aStatistical significance: p < 0.05 for control mice versus transgenic mice.NTNTLiver glycogen (mg/g liver)1.96 ± 0.081.97 ± 0.10NTNTIslet area (μm2)36310 ± 635792760 ± 9571aStatistical significance: p < 0.05 for control mice versus transgenic mice.NTNTa Statistical significance: p < 0.05 for control mice versus transgenic mice.b NT, not tested. Open table in a new tab Starting at the age of 16 weeks (line 1), or 20 weeks (line 2), mice were given a high fat diet for 4–6 weeks, and glucose, insulin, and pyruvate tolerance tests were then performed (Fig. 3). The glucose tolerance test confirmed an elevated fasting serum glucose concentration (Fig. 3, a and d) and revealed glucose intolerance in the transgenic mice. The insulin tolerance test revealed insulin resistance in the transgenic mice (Fig. 3, b and e). Finally, the pyruvate tolerance test showed greater elevation of the serum glucose concentration in the transgenic mice, as compared with the control mice (Fig. 3, c and f), indicating insufficient suppression of hepatic gluconeogenesis in the transgenic mice. Taking these results together, RELMβ transgenic mice are insulin-resistant and glucose-intolerant, particularly in the liver. These tendencies were more evident in the highly RELMβ-overexpressing line. In terms of adipocytokines, the levels of serum free fatty acid and adiponectin were not altered (n = 8–10). Histological Analysis of Liver and Pancreas—Histology showed fatty liver and islet hyperplasia in transgenic mice as compared with control mice (n = 3, age 16 weeks, after 4 weeks on a high fat diet, Fig. 4). Quantitatively, the mean islet area in RELMβ-overexpressing mice was significantly increased, as compared with that in control mice, by approximately 2.5-fold (TABLE TWO). In contrast, no significant difference in adipocyte size or mass was seen in epididymal or subcutaneous fat at the time of sacrifice (data not shown). Glucose Clamp Study and Glucose Uptake in Vivo and in Vitro—Six-month-old mice fed a high fat diet for 4 weeks were used for the glucose clamp study. In the basal state, glucose levels were 113.3 ± 2.0 versus 158 ± 9.5 mg/dl (control versus transgenic), and insulin levels were 5.0 ± 1.2 versus 12.1 ± 5.4 ng/ml.
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