Perilipin membrane integration determines lipid droplet heterogeneity in differentiating adipocytes.

The storage of fat within lipid droplets (LDs) of adipocytes is critical for whole-body health. Acute fatty acid (FA) uptake by differentiating adipocytes leads to the formation of at least two LD classes marked by distinct perilipins (PLINs). How this LD heterogeneity arises is an important yet unresolved cell biological problem. Here, we show that an unconventional integral membrane segment (iMS) targets the adipocyte specific LD surface factor PLIN1 to the endoplasmic reticulum (ER) and facilitates high-affinity binding to the first LD class. The other PLINs remain largely excluded from these LDs until FA influx recruits them to a second LD population. Preventing ER targeting turns PLIN1 into a soluble, cytoplasmic LD protein, reduces its LD affinity, and switches its LD class specificity. Conversely, moving the iMS to PLIN2 leads to ER insertion and formation of a separate LD class. Our results shed light on how differences in organelle targeting and disparities in lipid affinity of LD surface factors contribute to formation of LD heterogeneity.

Amber Extract Reduces Lipid Content in Mature 3T3-L1 Adipocytes by Activating the Lipolysis Pathway.

Amber-the fossilized resin of trees-is rich in terpenoids and rosin acids. The physiological effects, such as antipyretic, sedative, and anti-inflammatory, were used in traditional medicine. This study aims to clarify the physiological effects of amber extract on lipid metabolism in mouse 3T3-L1 cells. Mature adipocytes are used to evaluate the effect of amber extract on lipolysis by measuring the triglyceride content, glucose uptake, glycerol release, and lipolysis-related gene expression. Our results show that the amount of triacylglycerol, which is stored in lipid droplets in mature adipocytes, decreases following 96 h of treatment with different concentrations of amber extract. Amber extract treatment also decreases glucose uptake and increases the release of glycerol from the cells. Moreover, amber extract increases the expression of lipolysis-related genes encoding perilipin and hormone-sensitive lipase (HSL) and promotes the activity of HSL (by increasing HSL phosphorylation). Amber extract treatment also regulates the expression of other adipocytokines in mature adipocytes, such as adiponectin and leptin. Overall, our results indicate that amber extract increases the expression of lipolysis-related genes to induce lipolysis in 3T3-L1 cells, highlighting its potential for treating various obesity-related diseases.

Impact of adrenomedullin blockage on lipid metabolism in female mice exposed to high-fat diet.

PURPOSE: Adrenomedullin (ADM) levels are elevated in gestational and type 2 diabetic patients. ADM also stimulates lipolysis in vitro. Disturbed lipid metabolism has been implicated in the pathogenesis of diabetes. Here, we explore whether blockade of ADM is beneficial for metabolic homeostasis in a diabetic mouse model. METHODS: C57BL/6J female mice were placed on either a control or a high fat high sucrose (HFHS) diet for 8 weeks. At week 4, osmotic mini-pumps were implanted for constant infusion of either saline or ADM antagonist, ADM22-52. Glucose tolerance tests were performed prior to infusion and 4 weeks after infusion began. Animals were then sacrificed and visceral adipose tissue collected for further analysis. RESULTS: Mice fed HFHS displayed glucose intolerance, increased mRNA expressions in VAT for Adm and its receptor components, Crlr. HFHS fed mice also had increased basal and isoprenaline-induced glycerol release by VAT explants. ADM22-52 did not significantly affect glucose intolerance. ADM22-52 did suppress basal and isoprenaline-induced glycerol release by VAT explants. This alteration was associated with enhanced mRNA expression of insulin signaling factors Insr and Glut4, and adipogenic factor Pck1. CONCLUSIONS: HFHS diet induces glucose intolerance and enhances ADM and its receptor expressions in VAT in female mice. ADM22-52 treatment did not affect glucose intolerance in HFHS mice, but reduced both basal and isoprenaline-induced lipolysis, which is associated with enhanced expression of genes involved in adipogenesis. These results warrant further research on the effects of ADM blockade in improving lipid homeostasis in diabetic patients.

Extract of Paecilomyces hepiali mycelia induces lipolysis through PKA-mediated phosphorylation of hormone-sensitive lipase and ERK-mediated downregulation of perilipin in 3T3-L1 adipocytes.

BACKGROUND: Cordyceps sinensis has been used for centuries in China as one of the most valued herbal medicine and tonic food. Paecilomyces hepiali, a fungal strain isolated from natural C. sinensis, has been used widely as a substitute of C. sinensis in medicine and health food. P. hepiali has been reported to have various pharmaceutical benefits, including triglyceride-lowing activity. However, its effects on triglyceride metabolism in adipocytes remain unknown. The purpose of the present study was to evaluate the effect of P. hepiali mycelia on adipocyte lipolysis and to clarify the underlying mechanisms. METHODS: The fully differentiated 3T3-L1 adipocytes were treated with methanol extract of Paecilomyces hepiali mycelia (PHME). Contents of glycerol released into the culture medium and intracellular triglyceride were measured as indices of lipolysis using glycerol assay kit and Oil red O staining, respectively. Then, effects of PHME on the main lipases or kinases involved in lipolysis regulation were investigated. Protein expression of adipose triglyceride lipase (ATGL) and perilipin, as well as phosphorylation of hormone-sensitive lipase (HSL), AMP-activated protein kinase (AMPK), and mitogen-activated protein kinases (MAPKs) were determined by western blotting. Moreover, nucleosides, important constituents of PHME, were analyzed using high performance liquid chromatography (HPLC). RESULTS: Treatment with PHME led to a significant increase in glycerol release thereby reduced intracellular triglyceride accumulation in fully differentiated adipocytes. PHME upregulated protein kinase (PK) A-mediated phosphorylation of HSL at serine residues of 563 and 660. Meanwhile, PHME treatment also upregulated phosphorylation of extracellular signal-regulated kinase (ERK), and downregulated the protein level of perilipin. Pretreatment with the PKA inhibitor, H89, blunted the PHME-induced lipolysis and the phosphorylation of HSL (Ser 563 and 660). Moreover, pretreatment with ERK inhibitor, PD98059, weakened the PHME-caused glycerol release and downregulation of perilipin expression. HPLC analysis indicated there were adenosine, cordycepin, uridine and vernine in PHME. CONCLUSIONS: Our results showed that PHME significantly induced lipolysis in 3T3-L1 adipocytes, which is mainly mediated by activation of HSL through PKA pathway and by downregulation of perilipin through activation of ERK pathway.

An ethanolic extract of Artemisia scoparia inhibits lipolysis in vivo and has antilipolytic effects on murine adipocytes in vitro.

An ethanolic extract of Artemisia scoparia (SCO) has metabolically favorable effects on adipocyte development and function in vitro and in vivo. In diet-induced obese mice, SCO supplementation significantly reduced fasting glucose and insulin levels. Given the importance of adipocyte lipolysis in metabolic health, we hypothesized that SCO modulates lipolysis in vitro and in vivo. Free fatty acids and glycerol were measured in the sera of mice fed a high-fat diet with or without SCO supplementation. In cultured 3T3-L1 adipocytes, the effects of SCO on lipolysis were assessed by measuring glycerol and free fatty acid release. Microarray analysis, qPCR, and immunoblotting were used to assess gene expression and protein abundance. We found that SCO supplementation of a high-fat diet in mice substantially reduces circulating glycerol and free fatty acid levels, and we observed a cell-autonomous effect of SCO to significantly attenuate tumor necrosis factor-α (TNFα)-induced lipolysis in cultured adipocytes. Although several prolipolytic and antilipolytic genes were identified by microarray analysis of subcutaneous and visceral adipose tissue from SCO-fed mice, regulation of these genes did not consistently correlate with SCO's ability to reduce lipolytic metabolites in sera or cell culture media. However, in the presence of TNFα in cultured adipocytes, SCO induced antilipolytic changes in phosphorylation of hormone-sensitive lipase and perilipin. Together, these data suggest that the antilipolytic effects of SCO on adipose tissue play a role in the ability of this botanical extract to improve whole body metabolic parameters and support its use as a dietary supplement to promote metabolic resiliency.

Green tea supplementation upregulates uncoupling protein 3 expression in severe obese women adipose tissue but does not promote weight loss.

This study aims (i) to verify expression of the UCPs, PLIN1, PPARG2, and ADRB3 genes in the abdominal subcutaneous adipose tissue of obese women at baseline and after 8 weeks of supplementation with decaffeinated green tea extract, and (ii) to associate findings with clinical parameters. This is a longitudinal study during which 11 women with obesity grade III were submitted to supplementation with 450 mg of (-)-epigallocatechin gallate (EGCG) (intervention group); the control group consisted of 10 eutrophic women. Anthropometric parameters [weight, height, and body mass index (BMI)], resting metabolic rate (RMR, measured by indirect calorimetry), and gene expression (measured by real-time PCR, RT-qPCR) were determined before and after supplementation. After 8 weeks, clinical parameters and UCP1, PLIN1, PPARG2, and ADRB3 expression remained unaltered in the intervention group (p > .05). Genetic analysis also showed that the UCP3 gene was upregulated (p = .026), but its upregulation did not promote weight loss.

Low Neonatal Plasma n-6/n-3 PUFA Ratios Regulate Offspring Adipogenic Potential and Condition Adult Obesity Resistance.

Adipose tissue expansion progresses rapidly during postnatal life, influenced by both prenatal maternal factors and postnatal developmental cues. The ratio of omega-6 (n-6) relative to n-3 polyunsaturated fatty acids (PUFAs) is believed to regulate perinatal adipogenesis, but the cellular mechanisms and long-term effects are not well understood. We lowered the fetal and postnatal n-6/n-3 PUFA ratio exposure in wild-type offspring under standard maternal dietary fat amounts to test the effects of low n-6/n-3 ratios on offspring adipogenesis and adipogenic potential. Relative to wild-type pups receiving high perinatal n-6/n-3 ratios, subcutaneous adipose tissue in 14-day-old wild-type pups receiving low n-6/n-3 ratios had more adipocytes that were smaller in size; decreased Pparγ2, Fabp4, and Plin1; several lipid metabolism mRNAs; coincident hypermethylation of the PPARγ2 proximal promoter; and elevated circulating adiponectin. As adults, offspring that received low perinatal n-6/n-3 ratios were diet-induced obesity (DIO) resistant and had a lower positive energy balance and energy intake, greater lipid fuel preference and non-resting energy expenditure, one-half the body fat, and better glucose clearance. Together, the findings support a model in which low early-life n-6/n-3 ratios remodel adipose morphology to increase circulating adiponectin, resulting in a persistent adult phenotype with improved metabolic flexibility that prevents DIO.

Melatonin promotes triacylglycerol accumulation via MT2 receptor during differentiation in bovine intramuscular preadipocytes.

Melatonin (N-acetyl-5-methoxytryptamine) is a derivative of tryptophan which is produced and secreted mainly by the pineal gland and regulates a variety of important central and peripheral actions. To examine the potential effects of melatonin on the proliferation and differentiation of bovine intramuscular preadipocytes (BIPs), BIPs were incubated with different concentrations of melatonin. Melatonin supplementation at 1 mM significantly increased peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer-binding protein (C/EBP) ß, and C/EBPα expression and promoted the differentiation of BIPs into adipocytes with large lipid droplets and high cellular triacylglycerol (TAG) levels. Melatonin also significantly enhanced lipolysis and up-regulated the expression of lipolytic genes and proteins, including hormone sensitive lipase (HSL), adipocyte triglyceride lipase (ATGL), and perilipin 1 (PLIN1). Moreover, melatonin reduced intracellular reactive oxygen species (ROS) levels by increasing the expression levels and activities of superoxide dismutase 1 (SOD1) and glutathione peroxidase 4 (GPX4). Finally, the positive effects of melatonin on adipogenesis, lipolysis, and redox status were reversed by treatment with luzindole, anantagonist of nonspecific melatonin receptors 1 (MT1) and 2 (MT2), and 4-phenyl-2-propionamidotetraline (4P-PDOT), a selective MT2 antagonist. These results reveal that melatonin promotes TAG accumulation via MT2 receptor during differentiation in BIPs.

Downregulation of Hepatic De Novo Lipogenesis and Adipogenesis in Adipocytes by Pinus densiflora Bark Extract.

Korean red pine (Pinus densiflora) bark extract, PineXol (PX), was investigated for its potential antioxidant and anti-inflammation effects in vitro. It was hypothesized that PX treatment (25-150 µg/ml) would reduce the lipid synthesis in HepG2 hepatocytes as well as lipid accumulation in 3T3-L1 adipocytes. Hepatocytes' intracellular triglycerides and cholesterol were decreased in the PX 150 µg/ml treatment group compared with the control (p < 0.05). Consequently, de novo lipogenic proteins (acetyl-CoA carboxylase 1, stearoyl-CoA desaturase 1, elongase of very long chain fatty acids 6, glycerol-3-phosphate acyltransferase 1, and sterol regulatory element-binding protein 1) were significantly decreased in hepatocytes by PX 150 µg/ml treatment compared with the control (p < 0.05). In differentiated 3T3-L1 adipocytes, the lipid accumulation was significantly attenuated by all PX treatments (p < 0.01). Regulators of adipogenesis, including CCAAT-enhancer-binding proteins alpha, peroxisome proliferator-activated receptor gamma, and perilipin, were decreased in PX 100 µg/ml treatment compared with the control (p < 0.05). In conclusion, PX might have anti-obesity effects by blocking hepatic lipogenesis and by inhibiting adipogenesis in adipocytes.

Grape seed proanthocyanidin supplementation reduces adipocyte size and increases adipocyte number in obese rats.

OBJECTIVES: White adipose tissue (WAT) expands through hypertrophy (increased adipocyte size) and/or hyperplasia (increased adipocyte number). Hypertrophy has been associated with insulin resistance and dyslipidemia independently of body composition and fat distribution. In contrast, hyperplasia protects against metabolic alterations. Proanthocyanidins, which are the most abundant flavonoids in the human diet, improve metabolic disturbances associated with diet-induced obesity without reducing body weight or adiposity. The aim of this study was to determine whether grape seed proanthocyanidin extract (GSPE) can modulate WAT expandability. Because GSPE also contains gallic acid, we also studied the capacity of gallic acid to remodel WAT. DESIGN: Male Wistar rats were fed a standard chow diet (n=6) or a cafeteria diet (CAF) for 11 weeks. After 8 weeks, the CAF-fed animals were supplemented with 25 mg GSPE/kg body weight (n=6), 7 mg gallic acid/kg body weight (n=6) or the vehicle (n=6) for 3 weeks. Histological analyses were performed in the retroperitoneal (rWAT) and inguinal (iWAT) WAT to determine adipocyte size and number. Specific markers for adipogenesis and WAT functionality were analysed in rWAT using quantitative RT-PCR. RESULTS: GSPE or gallic acid supplementation did not reduce weight gain or reverse and adiposity. However, GSPE reduced adipocyte size significantly in rWAT and moderately in iWAT and tripled the adipocyte number in rWAT. Gallic acid slightly reduced adipocyte size in rWAT and iWAT and doubled the adipocyte number in both WATs. In accordance with this adipogenic activity, Pref-1 and PPARγ tended to be overexpressed in rWAT of rats supplemented with GSPE. Moreover, GSPE supplementation increased Plin1 and Fabp4 expression and restored adiponectin expression completely, indicating a better functionality of visceral WAT. CONCLUSIONS: GSPE supplementation has anti-hypertrophic and hyperplasic activities in rats with established obesity, mainly in visceral WAT inducing a healthier expansion of WAT to match the surplus energy provided by the cafeteria diet.

Pinus Densiflora Bark Extract (PineXol) Decreases Adiposity in Mice by Down-Regulation of Hepatic De Novo Lipogenesis and Adipogenesis in White Adipose Tissue.

PineXol, extracted from Korean red pine bark, has beneficial effects, such as antioxidant, antiinflammatory, and antilipogenic activities in vitro. We tested the hypothesis that PineXol supplementation could have anti-obesity effects on mice fed a high-fat diet (HFD). Four-week-old male C57BL/6 mice were fed normal chow (18% kcal from fat) or a HFD (60% kcal from fat). HFD-fed animals were also subjected to PineXol treatment at a dose of 10 or 50 mg/kg body weight (BW) (PX10 or PX50, respectively) body weight. The body weight and body fat mass in the PX50 group were statistically lower than those in the HFD group (p < 0.05 and p < 0.001, respectively). The concentration of hepatic triglycerides, total cholesterol, and low-density lipoprotein cholesterol were reduced in the PX50 group compared with the HFD group (p < 0.01). Acetyl CoA carboxylase (p < 0.01), elongase of very long chain fatty acids 6 (p < 0.01), stearoyl CoA desaturase 1 (p < 0.05), microsomal triglyceride transfer protein (p < 0.01), and sterol regulatory element-binding protein 1 (p < 0.05) were significantly decreased in the PX50 group compared with that in the HFD group. In white adipose tissue, CCAATenhancer-binding protein alpha (p < 0.05), peroxisome proliferator-activated receptor gamma (p < 0.001), and perilipin (p < 0.01) were decreased in the PX50 group compared with those in the HFD group. Therefore, the current study implies the potential of PineXol for the prevention and/or amelioration of obesity, in part by inhibition of both hepatic lipid synthesis and adipogenesis in white adipose tissue.

Exposure to Tumescent Solution Significantly Increases Phosphorylation of Perilipin in Adipocytes.

BACKGROUND: Lidocaine and epinephrine could potentially decrease adipocyte viability, but these effects have not been substantiated. The phosphorylation status of perilipin in adipocytes may be predictive of cell viability. Perilipin coats lipid droplets and restricts access of lipases; phospho-perilipin lacks this protective function. OBJECTIVES: The authors investigated the effects of tumescent solution containing lidocaine and epinephrine on the phosphorylation status of perilipin in adipocytes. METHODS: In this in vitro study, lipoaspirates were collected before and after tumescence from 15 women who underwent abdominoplasty. Fat samples were fixed, sectioned, and stained for histologic and immunohistochemical analyses. Relative phosphorylation of perilipin was inferred from pixel intensities of immunostained adipocytes observed with confocal microscopy. RESULTS: For adipocytes collected before tumescent infiltration, 10.08% of total perilipin was phosphorylated. In contrast, 30.62% of total perilipin was phosphorylated for adipocytes collected from tumescent tissue (P < .01). CONCLUSIONS: The tumescent technique increases the relative phosphorylation of perilipin in adipocytes, making these cells more vulnerable to lipolysis. Tumescent solution applied for analgesia or hemostasis of the donor site should contain the lowest possible concentrations of lidocaine and epinephrine. LEVEL OF EVIDENCE 5.

Ethanol extracts of chickpeas alter the total lipid content and expression levels of genes related to fatty acid metabolism in mouse 3T3-L1 adipocytes.

Desi-type chickpeas, which have long been used as a natural treatment for diabetes, have been reported to lower visceral adiposity, dyslipidemia and insulin resistance induced by a chronic high-fat diet in rats. In this study, in order to examine the effects of chickpeas of this type in an in vitro system, we used the 3T3-L1 mouse cell line, a subclone of Swiss 3T3 cells, which can differentiate into cells with an adipocyte-like phenotype, and we used ethanol extracts of chickpeas (ECP) instead of chickpeas. Treatment of the 3T3-L1 cells with ECP led to a decrease in the lipid content in the cells. The desaturation index, defined as monounsaturated fatty acids (MUFAs)/saturated fatty acids (SFAs), was also decreased by ECP due to an increase in the cellular content of SFAs and a decrease in the content of MUFAs. The decrease in this index may reflect a decreased reaction from SFA to MUFA, which is essential for fat storage. To confirm this hypothesis, we conducted a western blot analysis, which revealed a reduction in the amount of stearoyl-CoA desaturase 1 (SCD1), a key enzyme catalyzing the reaction from SFA to MUFA. We observed simultaneous inactivations of enzymes participating in lipogenesis, i.e., liver kinase B1 (LKB1), acetyl-CoA carboxylase (ACC) and AMPK, by phosphorylation, which may lead to the suppression of reactions from acetyl-CoA to SFA via malonyl-CoA in lipogenesis. We also investigated whether lipolysis is affected by ECP. The amount of carnitine palmitoyltransferase 1 (CPT1), an enzyme important for the oxidation of fatty acids, was increased by ECP treatment. ECP also led to an increase in uncoupling protein 2 (UCP2), reported as a key protein for the oxidation of fatty acids. All of these results obtained regarding lipogenesis and fatty acid metabolism in our in vitro system are consistent with the results previously shown in rats. We also examined the effects on SCD1 and lipid contents of ethanol extracts of Kabuli-type chickpeas, which are used worldwide. The effects were similar, but of much lesser magnitude compared to those of ECP described above. Thus, Desi-type chickpeas may prove to be effective for the treatment of diabetes, as they can alter the lipid content, thus reducing fat storage.