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Genetic Defect in Phospholipase Cd1 Protects Mice from Obesity By ORIGINAL ARTICLE Genetic Defect in Phospholipase Cd1 Protects Mice From Obesity by Regulating Thermogenesis and Adipogenesis Masayuki Hirata,1 Mutsumi Suzuki,1 Rika Ishii,1 Reiko Satow,1 Takafumi Uchida,1 Tomoya Kitazumi,2 Tsutomu Sasaki,2 Tadahiro Kitamura,2 Hideki Yamaguchi,3,4 Yoshikazu Nakamura,1 and Kiyoko Fukami1 OBJECTIVE—Regulation of obesity development is an impor- size of adipocytes is a hallmark of obesity. The former tant issue to prevent metabolic syndromes. Gene-disrupted mice seems to be caused by proliferation and differentiation of of phospholipase Cd1(PLCd1), a key enzyme of phosphoinositide preadipocytes. On the other hand, the diet-induced in- turnover, seemed to show leanness. Here we examined whether crease in cell size is characterized by adipocyte hypertro- and how PLCd1 is involved in obesity. phy, which may be primarily caused by excessive lipid RESEARCH DESIGN AND METHODS—Weight gain, insulin overload and a decrease in metabolic rate. sensitivity, and metabolic rate in PLCd12/2 mice were compared Brown adipose tissue (BAT) is implicated in thermo- with PLCd1+/2 littermate mice on a high-fat diet. Thermogenic genesis and metabolic enhancement (5). Recent reports in- and adipogenetic potentials of PLCd12/2 immortalized brown dicated that BAT and skeletal muscle originate from adipocytes and adipogenesis of PLCd1-knockdown (KD) 3T3L1 a common precursor cell (6–9). Like skeletal muscle, BAT 2 2 cells, or PLCd1 / white adipose tissue (WAT) stromal-vascular plays a role in thermogenesis by promoting the expression of fraction (SVF) cells, were also investigated. a thermogenic gene, uncoupling protein 1 (UCP1). Upregu- RESULTS—PLCd12/2 mice showed marked decreases in weight lation of UCP1 by genetic manipulations or pharmacological gain and mass of epididymal WAT and preserved insulin sensitivity agents has been shown to reduce obesity and improve in- compared with PLCd1+/2 mice on a high-fat diet. In addition, sulin sensitivity (5). Other recent studies demonstrating that PLCd12/2 mice have a higher metabolic rate such as higher oxy- a considerable amount of metabolically active BAT exists in gen consumption and heat production. When control immortalized many adult humans have invoked an important and novel brown adipocytes were treated with thermogenic inducers, ex- role of BAT as an anti-obesity agent (10,11). Therefore, un- pression of PLCd1 was decreased and thermogenic gene uncou- derstanding the development or functions of WAT and BAT pling protein 1 (UCP1) was upregulated to a greater extent in 2 2 is indispensable for preventing obesity. PLCd1 / immortalized brown adipocytes. In contrast, ectopic expression of PLCd1inPLCd12/2 brown adipocytes induced a Phosphoinositide metabolism plays crucial roles in decrease in UCP expression, indicating that PLCd1 negatively reg- diverse cellular functions, including cell growth, cell mi- ulates thermogenesis. Importantly, accumulation of lipid droplets gration, endocytosis, and cell differentiation (12,13). Phos- was severely decreased when PLCd1-KD 3T3L1 cells, or PLCd12/2 pholipase C (PLC), a key enzyme in this system, catalyzes WAT SVF cells, were differentiated, whereas differentiation of the hydrolysis of phosphatidylinositol 4,5-bisphosphate, PLCd12/2 brown preadipocytes was promoted. leading to the generation of two second messengers, CONCLUSIONS—PLCd1 has essential roles in thermogenesis namely, diacylglycerol and inositol 1,4,5-triphosphate. Diac- ylglycerol stimulates protein kinase C (PKC) activation and and adipogenesis and thereby contributes to the development 2+ of obesity. inositol 1,4,5-triphosphate releases Ca from the intra- cellular stores. Thirteen mammalian PLC isozymes have been identified and grouped into six classes, b, g, d, ´, z,and h, on the basis of their structure and regulatory mechanisms besity is a growing concern in present society (14,15). Among these classes, the d-type PLC is evolution- because it leads to many metabolic syndromes arily conserved and therefore expected to have important that are defined by visceral obesity complicated and basic physiological functions. We have generated 2 2 by type 2 diabetes, hypertension, and increased d-type PLC knockout ( / ) mice and previously reported O – cardiovascular risk. White adipose tissue (WAT) functions that PLCd1 has an essential role in skin homeostasis (16 18). 2 2 as a lipid storage, insulin sensor, and endocrine organ that Here, we report that PLCd1 / mice were protected produce adipokines (1–4). An increase in the number and from diet-induced obesity and show a higher metabolic rate. Expression of thermogenic gene UCP1 was more enhanced in PLCd12/2-immortalized brown adipocytes 1 From the Laboratory of Genome and Biosignal, Tokyo University of Phar- when cells were treated with thermogenic inducers, sug- macy and Life Sciences, Hachioji, Tokyo, Japan; the 2Metabolic Signal Re- search Center, Institute for Molecular and Cellular Regulation, Maebashi, gesting PLCd1 has a role in thermogenesis. Furthermore, Gunma, Japan; the 3Growth Factor Division, National Cancer Center Re- knockdown (KD) of PLCd1 in 3T3L1 preadipocytes, or search Institute, Chuo-ku, Tokyo, Japan; and the 4PRESTO, Japan Science PLCd12/2 WAT stromal-vascular fraction (SVF), reduced and Technology Agency, Kawaguchi-shi, Saitama, Japan. Corresponding author: Kiyoko Fukami, [email protected]. the accumulation of lipid droplets during adipocyte dif- Received 26 October 2010 and accepted 24 April 2011. ferentiation in vitro, indicating that PLCd1 is involved in DOI: 10.2337/db10-1500 adipogenesis. This article contains Supplementary Data online at http://diabetes. diabetesjournals.org/lookup/suppl/doi:10.2337/db10-1500/-/DC1. M.H., M.S., and R.I. contributed equally to this work. Ó RESEARCH DESIGN AND METHODS 2011 by the American Diabetes Association. Readers may use this article as 2/2 long as the work is properly cited, the use is educational and not for profit, Mice. PLCd1 mice were generated previously and genotyped with tail by and the work is not altered. See http://creativecommons.org/licenses/by PCR using a mixture of the following three primers: forward (59-CAAGGA- -nc-nd/3.0/ for details. GGTGAAGGACTTCCTG-39), reverse (59- CTGGGTCAGCATCCTGTAGAAG-39), diabetes.diabetesjournals.org DIABETES 1 Diabetes Publish Ahead of Print, published online May 26, 2011 PLCd1 REGULATES THERMOGENESIS AND ADIPOGENESIS and neomycin (59- CCTGTGCTCTAGTAGCTTTACG-39) (16). Mice had ad libi- thermogenesis, differentiated brown adipocytes at the same degree were tum access to water and either regular diet (RD) (CLEA Rodent diet CE-2; 12.6% treated with 0.5 mmol/L cAMP and 0.1 mmol/L forskolin for 4 h. of calories from fat; CLEA Japan) or high-fat diet (HFD) (CLEA Rodent diet Statistical analysis. Data are expressed as the mean 6 SEM. Statistical Quick Fat; 30.6% of calories from fat; CLEA Japan). For diet-induced obesity, significance was assessed using the Student t test. A P value of , 0.05 was the mice were fed with HFD from the age of 6 weeks to 27 weeks. We per- considered statistically significant. formed experiments with male mice. Measurement of blood glucose, plasma insulin level, and plasma leptin. Blood glucose was measured directly with a blood glucose meter (Sanwa Kagaku RESULTS Kenkyusho). Plasma insulin or leptin concentration was measured by an insulin PLCd12/2 mice showed decreased weight gain and ELISA kit or leptin ELISA kit (Shibayagi). For glucose tolerance tests, mice were less accumulation of lipid droplets in metabolic tis- fasted for 16 h and injected intraperitoneally with glucose (2 g/kg body wt). For 2/2 insulin tolerance tests, mice with ad libitum access to diets were intraper- sues on a HFD. Because we noticed that PLCd1 mice +/2 itoneally injected with human regular insulin (0.25 or 0.75 units/kg for RD or seem to be leaner than PLCd1 mice, we measured the HFD, respectively; Eli Lilly). body weights of mice fed with RD or HFD from the age of 2 2 2 Energy metabolism. The 24-week-old PLCd1+/ and PLCd1 / mice fed 6 weeks to 20 weeks. PLCd12/2 mice had a decreased with RD or HFD were subjected to metabolic analysis. Indirect calorimetry body weight compared with PLCd1+/2 littermate mice on was performed with a computer-controlled open circuit calorimetry system (Oxymax; Columbus Instruments) composed of respiratory chambers. For both RD and HFD (Fig. 1A), suggesting that the absence of measurement of oxygen consumption (VO ) and carbon dioxide production the PLCd1 gene conferred protection from obesity. De- 2 2/2 (VCO2), mice were individually housed in respiratory chambers to accli- creased body fat mass was also observed in PLCd1 mate them for 1 day before measurement. Data were recorded for 2–3days. mice. The weights of epididymal WAT (eWAT) and BAT 2/2 Respiratory quotient was calculated as the VCO2 to VO2 ratio. Heat gener- were extremely lower in PLCd1 mice on both diets. The 3 2/2 ation can also be calculated with the following expression: heat = CV VO2 weight of the liver was also lower in PLCd1 mice on (CV = 3.815 + 1.232 3 VCO2/VO2). Western blot analysis. Western blot analysis was carried out as described HFD, whereas those of most other tissues were almost the previously (18). Anti-PLCd1 antibody was developed previously (18). Anti- same (Fig. 1B). Chronic exposure of mice to HFD causes UCP1 (Santa Cruz), Tim23 (BD Transduction), heat shock protein 60 (Hsp60) enlarged body mass and accumulation of lipids in eWAT, (Stressgen), cytochrome c (Cell Signaling), PKCbI (Santa Cruz), PKC´ (Cell BAT, and liver. HFD induced increased mass of eWAT Signaling,), caveolin-1 (BD Transduction), nuclear factor of activated T (NFAT)c4 in PLCd1+/2 mice but not in PLCd12/2 mice (Fig. 1C). (Santa Cruz), lamin B1 (Santa Cruz), and b-actin (Sigma) antibodies were Moreover, BAT and liver in PLCd12/2 mice on HFD were purchased, respectively. +/2 Quantitative real-time PCR. Total RNAs from tissues and cells were isolated darker than those in PLCd1 mice, indicating less accu- using an RNeasy Lipid tissue mini-kit or an RNeasy mini-kit (Qiagen).
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