Perilipin and adipophilin, two significant lipid droplet (LD)-specific proteins, participate in storing fat or ectopic lipid deposition and fat mobilization in many types of mammalian cells. Acylation stimulating protein (ASP) is a novel adipocyte-derived hormone known for a major determinant for triglyceride synthesis (TGS) and lipid metabolism. The present study was aimed to investigate: (1) whether ASP, rather than insulin, is a powerful potentiator which could physiologically and directly influence TGS during 3T3-L1 preadipocyte differentiation; (2) whether ASP exposure at indicated time points during 3T3-L1 preadipocyte differentiation could influence the gene/protein expression of adipophilin and perilipin. 3T3-L1 preadipocytes were differentiated by traditional hormone cocktail and divided into control, ASP and insulin groups according to the treatment of ASP (1 mmol/L) or insulin (100 nmol/L). ASP-stimulated and insulin-stimulated TGS rate at indicated time points (0 d, 3 d, 6 d, 9 d) were assayed by measuring the incorporation of [(3)H]-oleic acid into TG, and the corresponding glucose transport was assayed by [(3)H]-2-DG uptake. The effects of ASP or insulin on gene/protein expression of adipophilin and perilipin at indicated time points were evaluated by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. The results obtained were as follows: (1) on the 3rd and 6th day of differentiation, ASP dramatically enhanced TGS rate compared with control group (P<0.05, P<0.01); There was no significant difference in TGS rate between insulin group and control group; (2) on the 6th and 9th day of differentiation, both ASP and insulin promoted glucose uptake (P<0.05, P<0.01), and the promoting effect in ASP group was greater than that in insulin group; (3) ASP elevated adipophilin gene and protein expression at the very early stage of differentiation (P<0.05, P<0.001) and had no significant effect from the 4th day of differentiation. Perilipin gene and protein expression increased throughout preadipocyte differentiation and its expression was up-regulated following ASP stimulation from the 3rd day of differentiation (P<0.05, P<0.001) to the end of differentiation (P<0.05); (4) Insulin did not affect gene and protein variation pattern of adipophilin and perilipin. Taken together, this study provides evidence that ASP-evoked changes in gene and protein expression of adipophilin and perilipin correlate with ASP-stimulated TGS acceleration, and adipophilin and perilipin are involved in the molecular mechanism of ASP-induced adipogenesis and LD formation.
3T3-L1 Cells ; Adipocytes ; cytology ; Animals ; Carrier Proteins ; metabolism ; Cell Differentiation ; Complement C3a ; pharmacology ; Gene Expression ; Insulin ; pharmacology ; Membrane Proteins ; metabolism ; Mice ; Perilipin-1 ; Perilipin-2 ; Phosphoproteins ; metabolism

Adipose tissue is the main energy reserve of the body. When energy is required, adipocyte triglycerides stored in lipid droplets (LDs) are broken down by lipase, and free fatty acids are released to supply the physiological need. Intracellular LDs are active metabolic organelles in mammalian cells, particularly in adipocytes. The present study was aimed to investigate the morphological changes of LDs and the alternation of LD-associated perilipin family proteins during long-term lipolysis stimulated by forskolin. Primary differentiated adipocytes derived from epididymal fat pads of Sprague-Dawley (SD) rats were incubated in the presence or absence of 1 μmol/L forskolin for 24 h. Content of glycerol released to the culture medium was determined by a colorimetric assay and served as an index of lipolysis. Morphological changes of LDs were observed by Nile red staining. The mRNA level of perilipin family genes was detected by quantitative real-time PCR. The protein level and subcellular localization were examined by immunoblotting and immunofluorescence staining, respectively. The results showed that forskolin induced sustained lipolysis in differentiated adipocytes. The morphology of LDs changed in a time-dependent manner. Large clustered LDs became gradually smaller in size and eventually disappeared; in contrast, peripheral micro-LDs increased gradually in number until the cytoplasm was filled with numerous micro-LDs. The protein level of the perilipin family proteins showed obvious alternation. Mature adipocytes physiologically expressed a very low level of Plin2 protein, whereas in adipocytes stimulated with lipolytic forskolin, the protein and mRNA levels of Plin2 were significantly increased, and the increased Plin2 was specifically bound to the surface of LDs. During chronic stimulation of forskolin, the mRNA level of Plin3 was unchanged, but the mRNA levels of Plin1, Plin4 and Plin5 were significantly decreased. These results suggest that the morphology of LDs and perilipin family proteins on the surface of LDs are significantly altered during long-term lipolysis stimulated by forskolin, representing a dynamic process of the remodeling of LDs.
Adipocytes ; drug effects ; Animals ; Cells, Cultured ; Colforsin ; pharmacology ; Lipid Droplets ; Lipolysis ; Perilipin-2 ; metabolism ; Perilipins ; metabolism ; Rats ; Rats, Sprague-Dawley

Humans ; Diabetes Mellitus, Type 2/genetics* ; Hyperlipidemias ; Mutation ; Insulin Resistance/genetics* ; Metabolic Diseases ; Proto-Oncogene Proteins c-akt ; Perilipin-1/genetics*

Atherosclerosis is a chronic, inflammatory disorder characterized by the deposition of excess lipids in the arterial intima. The formation of macrophage-derived foam cells in a plaque is a hallmark of the development of atherosclerosis. Lipid homeostasis, especially cholesterol homeostasis, plays a crucial role during the formation of foam cells. Recently, lipid droplet-associated proteins, including PAT and CIDE family proteins, have been shown to control the development of atherosclerosis by regulating the formation, growth, stabilization and functions of lipid droplets in macrophage-derived foam cells. This review focuses on the potential mechanisms of formation of macrophage-derived foam cells in atherosclerosis with particular emphasis on the role of lipid homeostasis and lipid droplet-associated proteins. Understanding the process of foam cell formation will aid in the future discovery of novel therapeutic interventions for atherosclerosis.
Acyltransferases ; metabolism ; Apoptosis Regulatory Proteins ; metabolism ; Atherosclerosis ; metabolism ; pathology ; Cholesterol ; metabolism ; Foam Cells ; cytology ; metabolism ; Humans ; Lipid Metabolism ; physiology ; Macrophages ; cytology ; immunology ; Membrane Proteins ; metabolism ; Perilipin-2 ; Peroxisome Proliferator-Activated Receptors ; metabolism ; Sterol Regulatory Element Binding Proteins ; metabolism

OBJECTIVETo investigate the effects of hypoxia on lipid metabolism in the normal human hepatic cell line L02 and to investigate the underlying molecular mechanisms.

METHODSL02 cells were exposed to hypoxic conditions (experimental groups: at 1% O2, 5% CO2, 94% N2 for 3, 6, 12, 24, or 48 hours) or normoxic conditions (control group: at 21% O2). Lipid droplet accumulation and triglyceride content were measured in each group by oil red O staining and biochemical assay, respectively. The mRNA expression levels of hypoxia-inducible factor (HIF)-2a and sterol regulatory element binding protein (SREBP)-1c were detected by reverse transcription-polymerase chain reaction. Protein expression levels of HIF-2a, adipose differentiation-related protein (ADRP), and Fas were tested by Western blot analysis.

RESULTSLipid droplet accumulation and the triglyceride content were significantly higher in the hypoxia group than the normoxia group. In addition, the hypoxia groups had significantly down-regulated mRNA expression of SREBP-1c (12h: 0.236+/-0.043, 24 h: 0.287+/-0.044, 48 h: 0.342+/-0.049 vs. normoxia: 0.503+/-0.037; F = 28.37, P less than 0.01) and FAS protein (12 h: 0.562+/-0.054, 24 h: 0.674+/-0.062, 48 h: 0.682+/-0.057 vs normoxia: 0.857+/-0.069; F = 16.08, P less than 0.01). In normoxic cells, little or no expression of HIF-2a protein was detected by Western blot. In hypoxic cells, HIF-2a protein expression peaked at 6h (0.973+/-0.067). ADRP protein expression was significantly higher in hypoxia groups than in the normoxia group (12 h: 0.319+/-0.043, 24 h: 0.732+/-0.056 and 48 h: 0.873+/-0.066 vs. 0.211+/-0.019; all, P less than 0.05.

CONCLUSIONExposure to hypoxic conditions might induce lipidosis in normal human hepatic cells by stimulating HIF-2a and ADRP expression.

Basic Helix-Loop-Helix Transcription Factors ; metabolism ; Cell Hypoxia ; Cell Line ; Down-Regulation ; Hepatocytes ; metabolism ; Humans ; Lipid Metabolism ; Membrane Proteins ; metabolism ; Oxygen ; metabolism ; Perilipin-2 ; Sterol Regulatory Element Binding Protein 1 ; metabolism

OBJECTIVETo observe the effects of puerarin on ADRP gene mRNA expression in fatty tissue of type 2 diabetes mellitus rats (T2DM).

METHODWiastar rats of T2DM model were made by feeding with high glucose and fat diet and injecting with small dose of streptozocin (25 mg x kg(-1)). 40 model rats were randomly divided into model control group and three puerarin groups (40, 80, 160 mg x kg(-1)), another 10 rats were selected as normal control group. FBG and FINS were measured to calculate IR after rats were injected consecutively for 6 weeks. The level of ADRP gene mRNA in fatty tissue was determined by RT-PCR after rats were injected eight weeks.

RESULTCompared with model control group, high and middle dosage of puerarin can decreased ADRP gene mRNA expression in fatty tissue obviously, FBG, IR level in each puerarin group and FINS in high and middle dosage puerarin groups decreased obviously.

CONCLUSIONPuerarin can decrease the blood glucose level of T2DM by downregulating ADRP mRNA expression and depressing the insulin resistance.

Adipose Tissue ; drug effects ; metabolism ; Animals ; Blood Glucose ; drug effects ; Diabetes Mellitus, Experimental ; drug therapy ; genetics ; Female ; Gene Expression Regulation ; drug effects ; In Vitro Techniques ; Isoflavones ; pharmacology ; Male ; Membrane Proteins ; genetics ; Perilipin-2 ; Rats ; Rats, Wistar ; Reverse Transcriptase Polymerase Chain Reaction ; Vasodilator Agents ; pharmacology