To explore the effect of chronic high dose of insulin on lipolysis in porcine adipocytes and the underlying molecular regulation mechanisms, we cultured primary porcine adipocytes and incubated them with different concentrations of insulin (0, 200, 400, 800, 1600 nmol/L) for 24-96 h in the absence or presence of specific protein kinase A (PKA) inhibitor or extracellular signal-related kinase (ERK) inhibitor. Then, we measured the glycerol release into the culture media as an indicator of the lipolysis, and observed the lipid accumulation morphology by phase-contrast microscopy. Further, we analyzed the gene expressions of perilipin A and peroxisome proliferator-activated receptor-gamma 2 (PPAR gamma 2) with semi-quantitative RT-PCR and Western blotting, respectively. The results showed that chronic high dose of insulin stimulated lipolysis in differentiated porcine adipocytes in a dose- and time-dependent manner, and significantly attenuated the lipolytic response to isoprenaline. Meanwhile, the protein and mRNA expressions of PPAR gamma 2 and perilipin A were significantly reduced. In addition, both PKA and ERK inhibitors significantly suppressed insulin-stimulated lipolysis, however, only ERK inhibitor reversed the insulin-induced down-regulation of perilipin A. These findings imply that chronic high dose of insulin stimulates lipolysis in porcine adipocytes by repressing perilipin A, which is involved in ERK pathway.
Adipocytes ; cytology ; drug effects ; metabolism ; Animals ; Carrier Proteins ; Dose-Response Relationship, Drug ; Down-Regulation ; drug effects ; Insulin ; pharmacology ; Lipolysis ; drug effects ; Perilipin-1 ; Phosphoproteins ; metabolism ; Swine

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

Growth Hormone (GH) is an important and powerful metabolic hormone that is secreted in a pulsatile pattern from cells in the anterior pituitary, influenced by several normal and pathophysiological conditions. Human GH was first isolated in the 1950s and human derived cadaveric GH was initially used to treat patients with GH deficiency. However, synthetic recombinant GH has been widely available since the mid-1980s and the advent of this recombinant GH boosted the abuse of GH as a doping agent. Doping with GH is a well-known problem among elite athletes and among people training at gyms, but is forbidden for both medical and ethical reasons. It is mainly the anabolic and, to some extent, the lipolytic effects of GH that is valued by its users. Even though GH's rumour as an effective ergogenic drug among athletes, the effectiveness of GH as a single doping agent has been questioned during the last few years. There is a lack of scientific evidence that GH in supraphysiological doses has additional effects on muscle exercise performance other than those obtained from optimised training and diet itself. However, there might be synergistic effects if GH is combined with, for example, anabolic steroids, and GH seems to have positive effect on collagen synthesis. Regardless of whether or not GH doping is effective, there is a need for a reliable test method to detect GH doping. Several issues have made the development of a method for detecting GH doping complicated but a method has been presented and used in the Olympics in Athens and Turin. A problem with the method used, is the short time span (24-36 hours) from the last GH administration during which the test effectively can reveal doping. Therefore, out-of-competition testing will be crucial.However, work with different approaches to develop an alternative, reliable test is ongoing.
Body Composition ; Bone and Bones ; drug effects ; Doping in Sports ; Growth Hormone ; adverse effects ; pharmacology ; physiology ; Humans ; Lipolysis ; Muscle, Skeletal ; drug effects ; physiology

Leptin, a cytokine predominantly secreted from fat tissue, plays an important role in regulating organism energy balance. Leptin can stimulate lipolysis, but the mechanism is unclear. In order to study the molecular mechanism of leptin stimulating lipolysis, we systemically studied the mRNA expression of key lipolytic enzymes. Morphological observation, Oil Red O staining and RT-PCR were used to identify pig primary adipocytes; commercial kits were used to measure the glycerol and FFA release; Semiquantitative RT-PCR was used to detect the mRNA expression of key lipolytic enzymes. The results showed that 100 nmol/L leptin up-regulated the mRNA expression of ATGL, TGH-2, HSL, MGL and LPL (P<0.01), but down-regulated the Perilipin mRNA expression (P<0.01). At the same time, leptin promoted the glycerol release in a dose dependent manner (P<0.01), but had no effect on the FFA release (P>0.05). These indicate that leptin may mainly stimulate lipolysis in pig primary adipocytes by up-regulating the expression of ATGL, MGL, LPL and down-regulating the expression of Perilipin. The unchanged FFA release may be resulted from Leptin promoting UCPs mRNA expression and increasing FFA expenditure.
Adipocytes ; cytology ; enzymology ; metabolism ; Animals ; Animals, Newborn ; Cells, Cultured ; Leptin ; pharmacology ; Lipase ; genetics ; metabolism ; Lipolysis ; drug effects ; Male ; Monoacylglycerol Lipases ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Swine

Hydroxypropyl methylcellulose (HPMC) propels self-emulsifying drug delivery systems (SEDDS) to achieve the supersaturated state in gastrointestinal tract, which possesses important significance to enhance oral absorption for poorly water-soluble drugs. This study investigated capacities and mechanisms of HPMC with different viscosities (K4M, K15M and K100M) to inhibit drug precipitation of SEDDS in the simulated gastrointestinal tract environment in vitro. The results showed that HPMC inhibited drug precipitation during the dispersion of SEDDS under gastric conditions by inhibiting the formation of crystal nucleus and the growth of crystals. HPMC had evident effects on the rate of SEDDS lipolysis and benefited the distribution of drug molecules across into the aqueous phase and the decrease of drug sediment. The mechanisms were related to the formed network of HPMC and its viscosities and molecular weight. These results offered a reference for selecting appropriate type of HPMC as the precipitation inhibitor of supersaturatable SEDDS.
Caprylates ; chemistry ; Chemical Precipitation ; drug effects ; Crystallization ; Drug Delivery Systems ; methods ; Emulsifying Agents ; chemistry ; Emulsions ; Ethylene Glycols ; chemistry ; Glycerides ; chemistry ; Hypromellose Derivatives ; administration & dosage ; chemistry ; pharmacology ; Indomethacin ; administration & dosage ; chemistry ; Lipolysis ; drug effects ; Molecular Weight ; Polyethylene Glycols ; chemistry ; Viscosity

PURPOSE: Ascorbic acid has been reported to have an adipogenic effect on 3T3-L1 preadipocytes, while evidence also suggests that ascorbic acid reduces body weight in humans. In this study, we tested the effects of ascorbic acid on adipogenesis and the balance of lipid accumulation in ovariectomized rats, in addition to long-term culture of differentiated 3T3-L1 adipocytes. MATERIALS AND METHODS: Murine 3T3-L1 fibroblasts and ovariectomized rats were treated with ascorbic acid at various time points. In vitro adipogenesis was analyzed by Oil Red O staining, and in vivo body fat was measured by a body composition analyzer using nuclear magnetic resonance. RESULTS: When ascorbic acid was applied during an early time point in 3T3-L1 preadipocyte differentiation and after bilateral ovariectomy (OVX) in rats, adipogenesis and fat mass gain significantly increased, respectively. However, lipid accumulation in well-differentiated 3T3-L1 adipocytes showed a significant reduction when ascorbic acid was applied after differentiation (10 days after induction). Also, oral ascorbic acid administration 4 weeks after OVX in rats significantly reduced both body weight and subcutaneous fat layer. In comparison to the results of ascorbic acid, which is a well-known cofactor for an enzyme of collagen synthesis, and the antioxidant ramalin, a potent antioxidant but not a cofactor, showed only a lipolytic effect in well-differentiated 3T3-L1 adipocytes, not an adipogenic effect. CONCLUSION: Taking these results into account, we concluded that ascorbic acid has both an adipogenic effect as a cofactor of an enzymatic process and a lipolytic effect as an antioxidant.
3T3-L1 Cells ; Adipocytes/drug effects ; Adipocytes/metabolism ; Adipogenesis/drug effects ; Animals ; Antioxidants/pharmacology ; Ascorbic Acid/pharmacology ; Body Composition/drug effects ; Body Weight/drug effects ; Cell Differentiation/drug effects ; Female ; Fibroblasts/drug effects ; Fibroblasts/metabolism ; Lipolysis/drug effects ; Mice ; Ovariectomy ; Rats, Sprague-Dawley

OBJECTIVEIn order to investigate the possible mechanism of its function to degrade lipid, we detect the effects of hawthorn flavanone to the influence on blood-fat levels and adipogenesis genes transcription expression in fat and muscle tissue of hyperlipoidemia mouse.

METHODIn this experiment, a total of 48 mouse were randomised to four groups and irrigated with two different concentrations (1.5 g kg(-1) body weight and 3.0 g kg(-1) body weight) of hawthorn flavanone, and killed in 0 h, 1 h, 2 h and 4 h. To estimate the content of TC, TG and HCL-C in blood: Total RNA was isolated from adipose and muscle, Real-time RT-PCR was used to analyze expression changes of adipogenesis genes (SREBP-1c, FAS, HSL and TGH) with time series; to analyze the correlation between TG in blood and some kinds of adipogenesis genes and the ratio of FAS/HARMEAN (HSL, TGH) mRNA in adipose.

RESULTHawthorn flavanone was able to cut down the level ofTC, TG and HDL significantly in blood and achieved the lowest level at 1 h. In adipose tissue, hawthorn flavanone up-regulated FAS, HSL and TGH, and achieved the level of significance (P<0.05), the expression level of FAS and TGH was ascend after 1 h, but HSL descend. The expression level of SREBP-1c was descend rapidly and achieved the level of significance after treating with hawthorn flavanone at 1 h (P<0.05), after that it rise again to even higher than the level of before treatment. After treating with hawthorn flavanone, the ratio of FAS/HARMEAN (HSL, TGH) in adipose was significantly descend and achieved the lowest level at 1 h (P<0.01), but it was descendsubsequently. In muscle tissue, hawthorn flavanone was able to significantly up-regulated the expression of FAS and HSL and lower dose group showed greater increasing, the change of SREBP-1c was similar in adipose tissue except the more heavily upgrade.

CONCLUSIONHawthorn flavanone had the function of depressing the concentration of blood-fat, it co-adjusted lipid metabolism of animal by regulating the transcription expression of FAS, HSL, TGH and SREBP-1c especially HSL and SREBP-1c transcription level.

Adipose Tissue ; drug effects ; metabolism ; Animals ; Crataegus ; chemistry ; Flavanones ; pharmacology ; Gene Expression Regulation ; drug effects ; Hyperlipidemias ; blood ; genetics ; Lipids ; blood ; Lipogenesis ; drug effects ; Lipolysis ; drug effects ; genetics ; Male ; Mice ; RNA, Messenger ; genetics ; metabolism ; Sterol Regulatory Element Binding Protein 1 ; genetics ; Triglycerides ; blood ; Up-Regulation ; drug effects ; fas Receptor ; genetics

This study is to evaluate the therapeutic effect of fibroblast growth factor 21 (FGF21) on NAFLD in MSG-IR mice and to provide mechanism insights into its therapeutic effect. The MSG-IR mice with insulin resistance were treated with high dose (0.1 micromol.kg-1d-1) and low dose (0.025 micromol.kg-1d-1) of FGF21 once a day for 5 weeks. Body weight was measured weekly. At the end of the experiment, serum lipids, insulin and aminotransferases were measured. Hepatic steatosis was observed. The expression of key genes regulating energy metabolism were detected by real-time PCR. The results showed that after 5 weeks treatment, both doses of FGF21 reduced body weight (P<0.01), corrected dyslipidemia (P<0.01), reversed steatosis and restored the liver morphology in the MSG model mice and significantly ameliorated insulin resistance. Additionally, real-time PCR showed that FGF21 significantly reduced transcription levels of fat synthetic genes, decreased fat synthesis and promoted lipolysis and energy metabolism by up-regulating key genes of lipolysis, thereby liver fat accumulation was reduced and liver function was restored to normal levels. In conclusion, FGF21 significantly reduces body weight of the MSG-IR mice, ameliorates insulin resistance, reverses hepatic steatosis. These findings provide a theoretical support for clinical application of FGF21 as a novel therapeutics for treatment of NAFLD.
Animals ; Body Weight ; drug effects ; Dose-Response Relationship, Drug ; Dyslipidemias ; metabolism ; Energy Metabolism ; drug effects ; Fatty Liver ; chemically induced ; complications ; Female ; Fibroblast Growth Factors ; administration & dosage ; pharmacology ; therapeutic use ; Insulin Resistance ; Lipolysis ; drug effects ; Liver ; metabolism ; pathology ; Male ; Mice ; Non-alcoholic Fatty Liver Disease ; drug therapy ; Sodium Glutamate

PURPOSE: To investigate the effects of topical prostaglandin analogue drugs on the differentiation of adipocytes. METHODS: Differentiation of 3T3-L1 preadipocytes was induced with isobutylmethylxanthine, dexamethasone, and insulin. 3T3-L1 cells were exposed to 0.008, 0.08, 0.2 microM of latanoprost and travoprost. Reverse transcription polymerase chain reaction for mRNA expression of lipoprotein lipase and peroxisome proliferator-activated receptor gamma 2 (PPARgamma2), and glycerol-3-phosphate dehydrogenase (G3PDH) assays were performed to examine the effects on early and late differentiation, respectively. Also, glycerol assays were done to evaluate the effect of prostaglandin analogues on lipolysis after differentiation. RESULTS: Both prostaglandin analogues inhibited differentiation of preadipocytes. Topical prostaglandin analogues significantly decreased G3PDH activity, a marker of late differentiation. However, topical prostaglandin analogues did not change mRNA expressions of lipoprotein lipase and PPARgamma2, markers of early differentiation. The activities of the early markers of differentiation were not changed significantly before and after growth arrest. Compared to latanoprost, travoprost decreased G3PDH activity more significantly (p < 0.05). Both prostaglandin analogues did not affect the lipolysis of differentiated adipocytes (p > 0.05). CONCLUSIONS: Prostaglandin analogues display an inhibitory effect on the differentiation of adipocytes when the cells start to differentiate especially in the late stage of differentiation. Thus, commercial topical prostaglandin analogues may decrease the fat contents of eyelids.
3T3-L1 Cells ; Adipocytes/drug effects/*pathology ; Animals ; Antihypertensive Agents/administration & dosage ; Cell Differentiation/drug effects ; Disease Models, Animal ; Glaucoma/*drug therapy/pathology ; Lipolysis/*drug effects ; Mice ; Neuroprotective Agents/administration & dosage ; Ophthalmic Solutions/administration & dosage ; Prostaglandins F, Synthetic/*administration & dosage ; Prostaglandins, Synthetic/*administration & dosage

OBJECTIVETo investigate the therapeutic effects of Ping-tang Recipe (, PTR) on high-fat diet (HFD)-induced insulin resistance and non-alcoholic fatty liver disease (NAFLD), and to elucidate the underlying mechanisms.

METHODSForty male SD rats were included in the study. Ten rats were fed on normal diet as normal control, and thirty rats were fed on HFD for 8 weeks to induce obesity, followed with low dose (0.42 g/kg) or high dose (0.84 g/kg) of PTR or vehicle for 8 weeks with 10 animals for each group. Glucose metabolism and insulin sensitivity were evaluated by oral glucose tolerance test and insulin tolerance test. Hepatic steatosis was measured by immunohistochemistry. Liver lipid metabolic genes were analyzed by quantitative real-time polymerase chain reaction, while AMP-activated protein kinase (AMPK) expression was examined by Western blot.

RESULTSRats fed on HFD developed abdominal obesity, insulin resistance and NAFLD. PTR treatment reduced visceral fat (peri-epididymal and peri-renal) accumulation, improved glucose metabolism, and attenuated hepatic steatosis. The expressions of the key lipolytic regulating genes, including peroxisome proliferators-activated receptor γ co-activator 1α (PGC-1α), peroxisome proliferator-activated receptor γ (PRAR-γ) and α (PRAR-α), were up-regulated (P<0.05 or P<0.01), while the expressions of lipogenic genes such as sterol regulatory element-binding protein 1c (SREBP-1c), fatty acid synthase (FAS) and liver fatty acid-binding protein (L-FABP) were down-regulated (P<0.05 or P<0.01). In addition, PTR activated AMPK and promoted acetyl-CoA carboxylase phosphorylation in the liver.

CONCLUSIONSPTR improves insulin resistance and reverse hepatic steatosis in the rat model of HFD-induced obesity through promotion of lipolysis and reduction of lipogenesis, which involves the AMPK signaling pathway, thus representing a new therapeutic intervention for obesity related insulin resistance and NAFLD.

AMP-Activated Protein Kinases ; metabolism ; Animals ; Body Weight ; drug effects ; Diet, High-Fat ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Fatty Liver ; blood ; complications ; prevention & control ; Gene Expression Regulation ; drug effects ; Glucose ; metabolism ; Glucose Tolerance Test ; Insulin Resistance ; Intra-Abdominal Fat ; drug effects ; pathology ; Lipogenesis ; drug effects ; Lipolysis ; drug effects ; Liver ; drug effects ; enzymology ; pathology ; Male ; Obesity ; blood ; complications ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Triglycerides ; metabolism