Plin4 is a lipid droplet protein (LDP) found predominantly in white

Plin4 is a lipid droplet protein (LDP) found predominantly in white adipose tissue (WAT). mice by gene targeting. Loss of Plin4 has meta-iodoHoechst 33258 no effect on body weight or composition or on adipose mass or meta-iodoHoechst 33258 development. However the triacylglycerol (TAG) content in heart but not other oxidative tissues such as BAT soleus muscle and liver is markedly reduced in mice. The heart of mice displays reduced Plin5 mRNA and protein levels (by ~38 and 87% respectively vs. wild-type) but unchanged mRNA levels of other perilipin family genes (Plin2 and Plin3) or genes involved in glucose and lipid metabolism. Despite reduced cardiac TAG level both young and aged mice maintain normal heart function as wild-type mice as measured by echocardiography. Interestingly Plin4 deficiency prevents the lipid accumulation in the heart that normally occurs after a prolonged (48-h) fast. It also protects the heart from cardiac steatosis induced by high-fat diet or when mice are bred into obese background. In conclusion inactivation of Plin4 downregulates Plin5 and reduces cardiac lipid accumulation in mice. also leads to a concomitant reduction of gene at both mRNA and protein levels. Our observations on the mice suggest that Plin4 in association with Plin5 may control lipid accumulation in the heart. MATERIALS AND METHODS Chemicals reagents and antibodies. We purchased tissue culture media from Invitrogen and lipid standards for thin-layer chromatography (TLC) analysis from Avanti Polar Lipids (Alabaster AL). All other chemicals were meta-iodoHoechst 33258 purchased from Sigma Chemical (St. Louis MO). Primary antibody against Plin4 (139.4 kDa) was a gift kindly provided by Dr. meta-iodoHoechst 33258 Perry E. Bickel (University of Texas Health Science Center Houston TX). Primary antibodies against Plin2 (46.6 kDa) Plin3 (47.2 kDa) and Plin5 meta-iodoHoechst 33258 (50 kDa) were polyclonal antisera generated against recombinant His-tagged Plin2 Plin3 and Plin5 as described (4). The following antibodies were also used: Plin1 (56 kDa) (Progen Heidelberg Germany) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Millipore Billerica MA). Animals. Mice with targeted deletion were generated at Regeneron Pharmaceuticals using the strategy shown in Fig. 1selection cassette (pGK-em7-Neo) was inserted right after the ATG start codon which replaced from half of exon 2 till the stop codon at exon 9 including the intronic regions of (Fig. 1mice compared with the wild-type counterparts (Fig. 1mice were back-crossed to C57BL/6J for eight generations and maintained in a temperature-controlled facility with fixed 12:12-h light-dark cycles and free access to regular chow and water. Male animals of 8-30 wk old were used throughout this study unless otherwise indicated. Some SA-2 experiments were done on animals fed a high-fat diet (HFD; 42% kcal fat; Harlan Teklad TD88137) from 6 wk old and kept for 10 wk. All animal experiments were done meta-iodoHoechst 33258 using protocols approved by the IACUC at Baylor College of Medicine. Fig. 1. Generation of (… Plasma biochemistry and whole body fat content measurement. Serum nonesterified fatty acid (NEFA; Wako) glycerol (Sigma) glucose (Therma Scientific) cholesterol and total TAG levels (Therma Scientific) were measured by enzymatic assay kits for determination of their concentrations. Whole body fat and lean masses were measured using an EchoMRI Whole Body Composition Analyzer (Echo Medical Systems Houston TX) according to the manufacturer’s instructions and normalized to body weight. Isolation of stromal vascular cells and adipocyte differentiation. Murine primary preadipocytes from the subcutaneous fat stromal vascular fraction were prepared as described (9). Briefly subcutaneous fat from 6- to 7-wk-old C57BL/6J mice were isolated minced and digested in 2 mg/ml collagenase IV (Sigma) with 20 mg/ml BSA at 37°C for 40 min. The digested mixtures were then filtered through a 100 μM cell strainer and spun at 250 for 8 min. The pellets containing the stromal vascular fraction were then resuspended in preadipocyte growth medium (Cell applications) and cultured for induction of differentiation by using the standard 3T3-L1 differentiation protocol. The intracellular triglyceride levels in D8 differentiated stromal vascular cells (SVCs) were extracted by chloroform and methanol according to Bligh and Dyer (1). The amount of triglyceride was measured with an Infinity triglyceride assay kit (Thermo.