Glycerol Release Research Articles

Rhubarb hydroxyanthraquinones act as antiobesity agents to inhibit adipogenesis and enhance lipolysis

Rhubarb as an herbal medicine has been shown to exhibit antiadipogenic activity. This study evaluated and compared the lipid-lowering activity of five rhubarb hydroxyanthraquinones (HAQs), including chrysophanol, aloe emodin, emodin, physcion, and rhein, aiming to identify candidate compounds for obesity treatment. Examination of the antiobesity effects of HAQs in 3T3-L1 adipocytes and high-fat diet (HFD)-induced obese rats showed that these anthraquinone compounds inhibited lipid accumulation in 3T3-L1 cells before and after differentiation. Emodin and rhein showed greater inhibition than the other compounds; dosage at 50 μM reduced intracellular triglyceride (TG) by about 30% in the differentiated adipocytes. Both compounds also revealed lipolytic effects to increase glycerol release from adipocytes. Adipokine overexpression induced by differentiation was downregulated by emodin and rhein through mitogen-activated protein kinase (MAPK) signaling. Despite their structural similarity, emodin and rhein exhibited different mechanisms on adipogenesis and lipid metabolism. Rhein restrained lipid deposition by controlling adipogenic transcriptional factors and lipolytic lipases during differentiation. The lipid-lowering effects of emodin did not use these pathways but reduced levels of lipogenic enzymes. HFD consumption in rats significantly increased body weight, visceral fat mass and adipocyte size, which were attenuated by intraperitoneal delivery of emodin or rhein. Rhein showed greater amelioration of obesity than emodin, decreasing plasma cholesterol by 29% and 14%, respectively. HAQs also suppressed cytokine upregulation in the liver and adipose tissues of obese rats. Rhein is a potential antiobesity agent through its ability to regulate obesity-associated adipogenesis, lipolysis and inflammation.

Anti-obesity activity of polyherbal formulation Divya-Medohar-Vati by inhibition of pancreatic lipase activity and triglyceride translocation through enterocytes

BackgroundObesity is a modern-day epidemic affecting human lifestyle worldwide and originates from high fat-content food consumption. Traditional Ayurveda-based polyherbal medicine ‘Divya-Medohar-Vati’ (DMV) has been recommended as an anti-obesity formulation. MethodsIn the present study, we explored the anti-obesity activity of DMV using biochemical assay, simulated gastrointestinal tract (GIT) digestion model, and Caco-2-derived enterocyte model. We further correlated the observed biological activity of DMV with its phytochemical profile determined using the high-performance-liquid-chromatography method. ResultsPhytochemicals such as Gallic acid, Ellagic acid, Chebulic acid, Picroside I, and Arjungenin were identified in DMV. DMV and ‘Orlistat’ (positive control) showed half-maximal inhibitory concentrations (IC50) of 647.88 and 21.80 µg/ ml, respectively against porcine pancreatic lipase activity. Addition of DMV and Orlistat to full cream during simulated GIT digestion reduced the hydrolysis of triglyceride into long-chain Palmitic acids and glycerol molecules in obtained digestae. At the same time, both DMV and Orlistat stimulated the release of medium-chained Capric acids in full cream digestae. Treatment of enterocytes with DMV-induced a loss of cell viability (IC50 at 308.5 μg/ml). Translocation of re-esterified triglycerides by enterocytes grown on transwell plate following treatment with full cream digestae was inhibited after the addition of DMV and Orlistat. ConclusionsTaken together, DMV showed good inhibition for pancreatic lipase activity, and hydrolysis of triglycerides modulating the release of free glycerol and dietary fatty acid (Capric acid and Palmitic acid) contents. DMV also reduces the translocation of triglycerides through the basal region of enterocytes. Hence, Divya-Medohar-Vati was found to be an effective anti-obesity polyherbal formulation.

Lipopolysaccharide induces lipolysis and insulin resistance in adipose tissue from dairy cows

Intense and protracted adipose tissue (AT) fat mobilization increases the risk of metabolic and inflammatory periparturient diseases in dairy cows. This vulnerability increases when cows have endotoxemia-common during periparturient diseases such as mastitis, metritis, and pneumonia-but the mechanisms are unknown. Fat mobilization intensity is determined by the balance between lipolysis and lipogenesis. Around parturition, the rate of lipolysis surpasses that of lipogenesis, leading to enhanced free fatty acid release into the circulation. We hypothesized that exposure to endotoxin (ET) increases AT lipolysis by activation of classic and inflammatory lipolytic pathways and reduction of insulin sensitivity. In experiment 1, subcutaneous AT (SCAT) explants were collected from periparturient (n = 12) Holstein cows at 11 ± 3.6 d (mean ± SE) before calving, and 6 ± 1 d and 13 ± 1.4 d after parturition. Explants were treated with the endotoxin lipopolysaccharide (LPS; 20 µg/mL; basal = 0 µg/mL) for 3 h. The effect of LPS on lipolysis was assessed in the presence of the β-adrenergic agonist and promoter of lipolysis isoproterenol (ISO; 1 µM; LPS+ISO). In experiment 2, SCAT explants were harvested from 24 nonlactating, nongestating multiparous Holstein dairy cows and exposed to the same treatments as in experiment 1 for 3 and 7 h. The effect of LPS on the antilipolytic responses induced by insulin (INS = 1 µL/L, LPS+INS) was established during ISO stimulation [ISO+INS, LPS+ISO+INS]. The characterization of lipolysis included the quantification of glycerol release and the assessment of markers of lipase activity [adipose triglyceride lipase (ATGL), hormone-sensitive lipase (HSL), and phosphorylated HSL Ser563 (pHSL)], and insulin pathway activation (AKT, pAKT) using capillary electrophoresis. Inflammatory gene networks were evaluated by real-time quantitative PCR. In periparturient cows, LPS increased AT lipolysis by 67 ± 12% at 3 h across all time points compared with basal. In nonlactating cows, LPS was an effective lipolytic agent at 3 h and 7 h, increasing glycerol release by 115 ± 18% and 68.7 ± 16%, respectively, relative to basal. In experiment 2, LPS enhanced ATGL activity with minimal HSL activation at 3 h. In contrast, at 7 h, LPS increased HSL phosphorylation (i.e., HSL activity) by 123 ± 11%. The LPS-induced HSL lipolytic activity at 7 h coincided with the activation of the MEK/ERK inflammatory pathway. In experiment 2, INS reduced the lipolytic effect of ISO (ISO+INS: -63 ± 18%) and LPS (LPS+INS: -45.2 ± 18%) at 3 h. However, the antilipolytic effect of INS was lost in the presence of LPS at 7 h (LPS+INS: -16.3 ± 16%) and LPS+ISO+INS at 3 and 7 h (-3.84 ± 23.6% and -21.2 ± 14.6%). Accordingly, LPS reduced pAKT:AKT (0.11 ± 0.07) compared with basal (0.18 ± 0.05) at 7 h. Our results indicated that exposure to LPS activated the classic and inflammatory lipolytic pathways and reduced insulin sensitivity in SCAT. These data provide evidence that during endotoxemia, dairy cows may be more susceptible to lipolysis dysregulation and loss of adipocyte sensitivity to the antilipolytic action of insulin.

Myostatin suppresses adipogenic differentiation and lipid accumulation by activating crosstalk between ERK1/2 and PKA signaling pathways in porcine subcutaneous preadipocytes.

The current study was undertaken to determine the effect of myostatin (MSTN) on lipid accumulation in porcine subcutaneous preadipocytes (PSPAs) and to further explore the potential molecular mechanisms. PSPAs isolated from Meishan weaned piglets were added with various concentrations of MSTN recombinant protein during the entire period of adipogenic differentiation process. Results showed that MSTN treatment significantly reduced the lipid accumulation, intracellular triglyceride (TG) content, glucose consumption, and glycerol phosphate dehydrogenase activity, while increased glycerol and free fatty acid release. Consistent with above results, the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway was obviously activated and thus key adipogenic transcription factors peroxisome proliferator-activated receptor-gamma (PPAR-γ), CCAAT/enhancer-binding protein-alpha (C/EBP-α), and their downstream enzymes fatty acid synthase and acetyl-CoA carboxylase were all inhibited. However, chemical inhibition of ERK1/2 signaling pathway by PD98059 markedly reversed the decreased TG content by increasing PPAR-γ expression. In addition, MSTN activated the cyclic AMP/protein kinase A (cAMP/PKA) pathway and stimulated lipolysis by reducing the expression of antilipolytic gene perilipin, thus elevated key lipolytic enzymes adipose triglyceride lipase and hormone-sensitive lipase (HSL) expression and enzyme activity. On the contrary, pretreatment with PKA inhibitor H89 significantly reversed TG accumulation by increasing PPAR-γ expression and thus inhibiting ERK1/2, perilipin, and HSL phosphorylation, supporting the crosstalk between PKA and ERK1/2 pathways in both the anti-adipogenic and pro-lipolytic effects. In summary, our results suggested that MSTN suppressed adipogenesis and stimulated lipolysis, which was mainly mediated by activating crosstalk of ERK1/2 and PKA signaling pathways, and consequently decreased lipid accumulation in PSPAs, our findings may provide novel insights for further exploring MSTN as a potent inhibitor of porcine subcutaneous lipid accumulation.

Glucose enhances catecholamine-stimulated lipolysis via increased glycerol-3-phosphate synthesis in 3T3-L1 adipocytes and rat adipose tissue.

During lipolysis, triglyceride (TG) are hydrolyzed into a glycerol and fatty acids in adipocyte. A significant portion of the fatty acids are re-esterificated into TG, and this is a critical step in promoting lipolysis. Although glycerol-3-phosphate (G3P) is required for triglyceride synthesis in mammalian cell, the substrate for G3P synthesis during active lipolysis is not known. A recent study showed that the inhibition of glucose uptake reduces catecholamine-stimulated lipolysis, suggesting that glucose availability is important in lipolysis in adipocytes. We hypothesized that glucose might play an essential role in generating G3P and thereby promoting catecholamine-stimulated lipolysis in adipocytes. Therefore, we determined the effect of glucose availability on catecholamine-stimulated lipolysis in 3T3-L1 adipocytes and rat adipose tissue. 3T3-L1 adipocytes and rat epididymal fat pads were cultured in a medium with/without glucose during stimulation by isoproterenol. Glycerol release was higher when adipocytes were cultured in a glucose-containing medium than that in a medium without glucose. Measurement of glucose uptake during catecholamine-stimulated lipolysis showed a slight, but significant increase in glucose uptake. We also compared glucose metabolism-related protein, such as glucose transporter 4, hexokinase, glycerol-3-phosphate dehydrogenase and lipase contents between fat tissues that play a critical role in active lipolysis. Epididymal fat exhibited higher lipolytic activity than inguinal fat because of higher lipase and glucose metabolism-related protein contents. We demonstrated that catecholamine-stimulated lipolysis is enhanced in the presence of glucose, and suggests that glucose is one of the primary substrates for G3P in adipocytes during active lipolysis.

Characterization of β-adrenergic receptors in bovine intramuscular and subcutaneous adipose tissue: comparison of lubabegron fumarate with β-adrenergic receptor agonists and antagonists.

Chinese hamster ovary cell constructs expressing either the β 1-, β 2- or β 3-adrenergic receptor (AR) were used to determine whether a novel β-AR modulator, lubabegron fumarate (LUB; Experior, Elanco Animal Health) might exert greater potency for a specific β-AR subtype. EC50 values calculated based on cAMP accumulation in dose response curves indicate that LUB is highly selective for the β 3-AR subtype, with an EC50 of 6 × 10–9 M, with no detectible agonistic activity at the β 2-AR. We hypothesized that the accumulation of lipolytic markers would reflect the agonist activity at each of the β-receptor subtypes of the specific ligand; additionally, there would be differences in receptor subtype expression in subcutaneous (s.c.) and intrmuscular (i.m.) adipose tissues. Total RNA was extracted from adipose tissue samples and relative mRNA levels for β 1-, β2-, and β 3-AR were measured using real-time quantitative polymerase chain reaction. Fresh s.c. and i.m. adipose tissue explants were incubated with isoproterenol hydrochloride (ISO; β-AR pan-agonist), dobutamine hydrochloride (DOB; specific β 1-AA), salbutamol sulfate (SAL; specific β 2-AA), ractopamine hydrochloride (RAC), zilpaterol hydrochloride (ZIL), BRL-37344 (specific β 3-agonist), or LUB for 30 min following preincubation with theophylline (inhibitor of phosphodiesterase). Relative mRNA amounts for β 1-, β 2-, and β 3-AR were greater (P < 0.05) in s.c. than in i.m. adipose tissue. The most abundant β-AR mRNA in both adipose tissues was the β 2-AR (P < 0.05), with the β 1- and β 3-AR subtypes being minimally expressed in i.m. adipose tissue. ISO, RH, and ZH stimulated the release of glycerol and nonesterified fatty acid (NEFA) from s.c. adipose tissue, but these β-AR ligands did not alter concentrations of these lipolytic markers in i.m. adipose tissue. LUB did not affect glycerol or NEFA concentrations in s.c. or i.m. adipose tissue, but attenuated (P < 0.05) the accumulation of cAMP mediated by the β 1- and β 2-AR ligands DOB and SAL in s.c. adipose tissue. Collectively, these data indicate that bovine i.m. adipose tissue is less responsive than s.c. adipose tissue to β-adrenergic ligands, especially those that are agonists at the β 1- and β3-receptor subtypes. The minimal mRNA expression of the β 1- and β 3 subtypes in i.m. adipose tissue likely limits the response potential to agonists for these β-AR subtypes.

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.

Enhanced Inhibition of Adipogenesis by Chrysin via Modification in Redox Balance, Lipogenesis, and Transcription Factors in 3T3-L1 Adipocytes in Comparison with Hesperidin

Objective The present study was conducted to elucidate the in-vitro anti-oxidant and anti-adipogenic effect of the flavone, chrysin in comparison with the citrus bioflavonoid, hesperidin during adipogenic differentiation in 3T3-L1 mouse preadipocytes. Methods The effect of chrysin and hesperidin on adipogenic differentiation was evaluated using Oil red-O staining, triglyceride estimation, free glycerol release, and ROS accumulation. The expression of adipogenesis-related genes was evaluated in real time-polymerase chain reaction. Results 50 µmol chrysin or hesperidin did not affect the cell viability of 3T3-L1 preadipocytes and adipocytes, but significantly reduced preadipocyte clonal population, accumulation of intracellular lipid and ROS and consequently increased lipolysis and antioxidant enzyme defence. It also decreased the expression of major adipogenic transcription factors, CCAAT/enhancer-binding protein-β, peroxisome proliferator activated receptor-γ, sterol regulatory element binding protein 1c, fatty acid synthase and hormone sensitive lipase. Conclusion(s) Herein we have indicated, for the first time, the effective anti-adipogenic mechanism of chrysin by down-regulating adipogenesis, lipogenesis and ROS and up-regulating lipolysis and antioxidant enzyme in differentiated 3T3-L1 adipocytes. As a nutritional bioflavonoid, chrysin with its more effective inhibition on adipogenesis than hesperidin has the potential to be developed as an anti-adipogenic nutraceutical agent.

Anti-Obesity and Lipid Lowering Activity of Bauhiniastatin-1 is Mediated Through PPAR-γ/AMPK Expressions in Diet-Induced Obese Rat Model

Objective: To evaluate the therapeutic efficacy and underlying molecular mechanisms of Bauhiniastatin-1 (BSTN1) to alleviate adiposity in diet-induced obese rodent model and in 3T3-L1 cells. Methods: BSTN1 was purified and confirmed through HPLC. In-vitro experiments such as MTT assay, Oil Red-O (ORO) stain, cellular lipid content, glycerol release and RT-PCR analysis were performed in 3T3-L1 cells in the presence and absence of BSTN1. In animal experiments, rats were divided into Group-I: normal pellet diet-fed, Group-II: HFD-fed, Groups-III, IV and V: HFD-fed BSTN1 (1.25, 2.5, and 5 mg/kg.b.wt./day/rat)-treated and Group-VI: HFD-fed Orlistat-treated. The rats were fed either normal diet or high fat diet (HFD) for 18 weeks and water ad-libitum. BSTN1 was orally administered from 13th week onwards to the selected HFD-fed groups. Body composition parameters, biochemical assays, histopathology examination and western blot analysis were performed to identify the predicted targets related to obesity. Molecular docking studies threw light on the binding interactions of BSTN1 against PPAR-γ, FAS and AMPK. Results: BSTN1 at 20 μM significantly (p < 0.001) inhibited adipocyte differentiation and lipid accumulation in 3T3-L1 cells. A conspicuous down-regulation in the mRNA expression levels of PPAR-γ, FAS and SREBP1 was observed but AMPK expression remained unchanged in BSTN1 treated 3T3-L1 cells. A substantial decrease in body weight gain, fat percent, total body fat, serum and liver lipid profile (except high-density lipoprotein), glucose, insulin and insulin resistance in BSTN1 treated rats was noticed in a dose dependent manner. In BSTN1 (5 mg/kg.b.wt.)-treated groups significantly (p < 0.01) elevated plasma adiponectin level but reduced leptin level as well as fall in serum AST and ALT were noticed. Further, the disturbed structural integrity and architecture of adipose and hepatic tissues due to high fat diet feeding were considerably recovered with BSTN1 treatment. Down-regulation in the protein expression level of PPAR-γ and activation of AMPK through phosphorylation was observed in BSTN1 treated rats than the untreated. Molecular docking studies revealed strong binding interactions of BSTN1 against PPAR-γ and AMPK and thus supported the experimental results. Conclusion: Taken together, the results suggest that BSTN1 could be a promising pharmacological molecule in the treatment of obesity and dyslipidemia.

Pharmacopuncture of Taraxacum platycarpum extract reduces localized fat by regulating the lipolytic pathway

Localized fat deposits are associated with health and aesthetic problems that mainly affect a large proportion of individuals. Recently, bioactive constituents of TP have been reported to affect lipid metabolism. In this study, we performed a network pharmacological analysis to assume potential lipolytic effects of TP and investigated the actual lipolytic effects of TP extract injection on local body fat and its underlying mechanism. Using the genes related to active compounds of TP, the network was constructed. Through the Functional Enrichment Analysis, Lipid Metabolism and Fatty Acid Metabolism were expected to be affiliated with the network, which implied possible lipolytic effects of TP. On the comparison between TP network and Obesity-related Gene Sets, about three-fourths of elements were in common with the gene sets, which indicated a high relevance between TP and obesity. Based on the genes in lipolysis-related pathways, Perilipin, CGI-58, ATGL, HSL and MGL were selected to identify the actual lipolytic effects of TP. TP injection reduced the inguinal fat weight. Also, the diameter of the adipocytes was decreased by the TP treatment in HFD-induced obese mice. In addition, TP suppressed lipid accumulation in differentiated 3T3-L1 adipocytes. Moreover, because the expression of Perilipin was increased, CGI-58, ATGL, HSL and MGL were markedly decreased. Furthermore, glycerol release was down-regulated by the TP treatment. TP exerted its lipolytic effects by regulating the lipolysis machinery through stimulation of lipases. Based on the present findings, TP is expected to be a potent component of injection lipolysis for removing localized body fat.

The beneficial effect of glycerophosphocholine to local fat accumulation: a comparative study with phosphatidylcholine and aminophylline.

Injection lipolysis or mesotherapy gained popularity for local fat dissolve as an alternative to surgical liposuction. Phosphatidylcholine (PPC) and aminophylline (AMPL) are commonly used compounds for mesotherapy, but their efficacy and safety as lipolytic agents have been controversial. Glycerophosphocholine (GPC) is a choline precursor structurally similar to PPC, and thus introduced in aesthetics as an alternative for PPC. This study aimed to evaluate the effects of GPC on adipocytes differentiation and lipolysis and compared those effects with PPC and AMPL using in vitro and in vivo models. Adipogenesis in 3T3-L1 was measured by Oil Red O staining. Lipolysis was assessed by measuring the amount of glycerol released in the culture media. To evaluate the lipolytic activity of GPC on a physiological condition, GPC was subcutaneously injected to one side of inguinal fat pads for 3 days. Lipolytic activity of GPC was assessed by hematoxylin and eosin staining in adipose tissue. GPC significantly suppressed adipocyte differentiation of 3T3-L1 in a concentration-dependent manner (22.3% inhibition at 4 mM of GPC compared to control). Moreover, when lipolysis was assessed by glycerol release in 3T3-L1 adipocytes, 6 mM of GPC stimulated glycerol release by two-fold over control. Subcutaneous injection of GPC into the inguinal fat pad of mice significantly reduced the mass of fat pad and the size of adipocytes of injected site, and these effects of GPC were more prominent over PPC and AMPL. Taken together, these results suggest that GPC is the potential therapeutic agent as a local fat reducer.

Oxytocin stimulates lipolysis, prostaglandin E2 synthesis, and leptin secretion in 3T3-L1 adipocytes

A model of oxytocin in the regulation of metabolic status has described one of oxytocin synthesis and release from the neurohypophysis in response to leptin, to suppress further leptin release. In addition, a lipogenic role for oxytocin has been suggested, consistent with an insulinergic action. This model, however, may be incorrect. Oxytocin reduces fat mass in the absence of either leptin or leptin receptor signalling, thereby challenging the interdependence between leptin and oxytocin. An oxytocin induced production of the anti-lipolytic prostaglandin E2 (PGE2) might account for this. Media from 3T3-L1 differentiated adipocytes treated with oxytocin (0–50 nmol.L−1) for 24 hrs were assayed for PGE2, leptin, adiponectin, and glycerol. Harvested cells were analysed for lipid droplet triglyceride and cytosolic free fatty acid (FFA) by flow cytometry, and for altered expression of lipolytic and lipogenic associated gene ontology transcripts by cDNA array. Both PGE2 and leptin secretion were significantly increased by oxytocin treatment whilst adiponectin secretion was not. A significant increase in cytosolic FFA was detected following oxytocin treatment, similar to that determined following treatment with isoproterenol (positive control). A significant increase in glycerol release to the culture media confirmed a lipolytic effect. No enrichment of lipolytic and lipogenic associated gene ontology transcripts was determined, but significant overrepresentation of chemosensory olfactory transcripts was. In conclusion, oxytocin stimulates lipolysis in 3T3-L1 adipocytes, mediated by autocrine/paracrine actions of PGE2 and leptin. To confirm that this response is mediated solely by the oxytocin receptor, further experiments would require those effects being blocked by a specific oxytocin antagonist.

High-throughput drug screening models of mature adipose tissues which replicate the physiology of patients’ Body Mass Index (BMI)

Obesity is a complex and incompletely understood disease, but current drug screening strategies mostly rely on immature in vitro adipose models which cannot recapitulate it properly. To address this issue, we developed a statistically validated high-throughput screening model by seeding human mature adipocytes from patients, encapsulated in physiological collagen microfibers. These drop tissues ensured the maintenance of adipocyte viability and functionality for controlling glucose and fatty acids uptake, as well as glycerol release. As such, patients’ BMI and insulin sensitivity displayed a strong inverse correlation: the healthy adipocytes were associated with the highest insulin-induced glucose uptake, while insulin resistance was confirmed in the underweight and severely obese adipocytes. Insulin sensitivity recovery was possible with two type 2 diabetes treatments, rosiglitazone and melatonin. Finally, the addition of blood vasculature to the model seemed to more accurately recapitulate the in vivo physiology, with particular respect to leptin secretion metabolism.

Restoration of energy homeostasis by SIRT6 extends healthy lifespan

Aging leads to a gradual decline in physical activity and disrupted energy homeostasis. The NAD+-dependent SIRT6 deacylase regulates aging and metabolism through mechanisms that largely remain unknown. Here, we show that SIRT6 overexpression leads to a reduction in frailty and lifespan extension in both male and female B6 mice. A combination of physiological assays, in vivo multi-omics analyses and 13C lactate tracing identified an age-dependent decline in glucose homeostasis and hepatic glucose output in wild type mice. In contrast, aged SIRT6-transgenic mice preserve hepatic glucose output and glucose homeostasis through an improvement in the utilization of two major gluconeogenic precursors, lactate and glycerol. To mediate these changes, mechanistically, SIRT6 increases hepatic gluconeogenic gene expression, de novo NAD+ synthesis, and systemically enhances glycerol release from adipose tissue. These findings show that SIRT6 optimizes energy homeostasis in old age to delay frailty and preserve healthy aging.

Inhibition of Lipid Accumulation and Cyclooxygenase-2 Expression in Differentiating 3T3-L1 Preadipocytes by Pazopanib, a Multikinase Inhibitor.

Pazopanib is a multikinase inhibitor with anti-tumor activity. As of now, the anti-obesity effect and mode of action of pazopanib are unknown. In this study, we investigated the effects of pazopanib on lipid accumulation, lipolysis, and expression of inflammatory cyclooxygenase (COX)-2 in differentiating and differentiated 3T3-L1 cells, a murine preadipocyte. Of note, pazopanib at 10 µM markedly decreased lipid accumulation and triglyceride (TG) content during 3T3-L1 preadipocyte differentiation with no cytotoxicity. Furthermore, pazopanib inhibited not only expression of CCAAT/enhancer-binding protein-α (C/EBP-α), peroxisome proliferator-activated receptor-γ (PPAR-γ), and perilipin A but also phosphorylation of signal transducer and activator of transcription (STAT)-3 during 3T3-L1 preadipocyte differentiation. In addition, pazopanib treatment increased phosphorylation of cAMP-activated protein kinase (AMPK) and its downstream effector ACC during 3T3-L1 preadipocyte differentiation. However, in differentiated 3T3-L1 adipocytes, pazopanib treatment did not stimulate glycerol release and hormone-sensitive lipase (HSL) phosphorylation, hallmarks of lipolysis. Moreover, pazopanib could inhibit tumor necrosis factor (TNF)-α-induced expression of COX-2 in both 3T3-L1 preadipocytes and differentiated cells. In summary, this is the first report that pazopanib has strong anti-adipogenic and anti-inflammatory effects in 3T3-L1 cells, which are mediated through regulation of the expression and phosphorylation of C/EBP-α, PPAR-γ, STAT-3, ACC, perilipin A, AMPK, and COX-2.

Glycerol‐3‐phosphate phosphatase/ Pgp in Pancreatic ß‐cells Functions as a Glucose Excess Security Valve Preventing Oversecretion of Insulin Secretion and Glucotoxicity

Objective The recently identified enzyme in mammalian cells, glycerol-3-phosphate (Gro3P) phosphatase (G3PP), gene name Pgp, was proposed to regulate intermediary metabolism. G3PP was shown in in vitro studies to regulate metabolism and glucose stimulated insulin secretion (GSIS) in ß-cells. We now examined the in vivo role of G3PP in the control of insulin secretion and ß-cell glucotoxicity. Methods Glucose and insulin tolerance were studied in ß-cell specific G3PP-KO (BKO) mice. Body weight gain, fed glycemia and insulinemia were measured. Pancreatic islets were isolated for ex vivo GSIS and biochemical measurements. Results BKO mice show increased body weight gain, unaltered fed glycemia and insulinemia, and enhanced insulin secretion in response to glucose load in an intraperitoneal but not oral glucose tolerance test, reaching 21 and 14 mM glucose, respectively. Insulin sensitivity in vivo remains unchanged in the BKO mice. GSIS response ex vivo at 16 mM but not 8 mM glucose is higher in BKO mouse islets. BKO islets show reduced glucose-induced glycerol release and elevated O2 consumption and ATP production at high (16 mM) but not low (4 mM) glucose levels. Glucotoxicity at 30 mM glucose for 7 days led to increased apoptosis, reduced insulin content and expression of Pdx-1 and Ins-2 genes in BKO islets. Conclusion G3PP impacts insulin secretion in vivo and ex vivo only under conditions of high but not intermediate and low glucose levels. G3PP plays a role in preventing ß-cell glucotoxicity. These effect likely result from the capacity of G3PP to redirect the excess glucose carbons from intermediate metabolism to glycerol that exits ß-cells. We propose that G3PP acts as a glucose excess security valve to prevent excessive insulin secretion and ß-cell dysfunction when blood glucose reaches very high levels.

Novel antiadipogenic effect of menadione in 3T3-L1 cells

Inhibition of adipocyte differentiation can be used as a strategy for preventing adipose tissue expansion and, consequently, for obesity management. Since reactive oxygen species (ROS) have emerged as key modulators of adipogenesis, the effect of menadione (a synthetic form of vitamin K known to induce the increase of intracellular ROS) on 3T3-L1 preadipocyte differentiation was studied. Menadione (15 μM) increased ROS and lipid peroxidation, generating mild oxidative stress without affecting cell viability. Menadione drastically inhibited adipogenesis, accompanied by decreased intracellular lipid accumulation and diminished expression of the lipo/adipogenic markers peroxisome proliferator-activated receptor (PPAR)γ, fatty acid synthase (FAS), CCAAT/enhancer-binding protein (C/EBP) α, fatty acid binding protein (FABP) 4, and perilipin. Menadione treatment also increased lipolysis, as indicated by augmented glycerol release and reinforced by the increased expression of hormone-sensitive lipase (HSL). Additionally, menadione increased the inhibitory phosphorylation of acetyl-CoA-carboxylase (ACC), which results in the inhibition of fatty acid synthesis. As a consequence, triglyceride content was decreased. Menadione also inhibited the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. Further, treatment with increased concentration of insulin, a potent physiological activator of the PI3K/Akt pathway, rescued the normal level of expression of PPARγ, the master regulator of adipogenesis, and overcame the restraining effect of menadione on the differentiation capacity of 3T3-L1 preadipocytes. Our study reveals novel antiadipogenic action for menadione, which is, at least in part, mediated by the PI3K/Akt pathway signaling and raises its potential as a therapeutic agent in the treatment or prevention of adiposity.

A bioactive fraction of Pterocarpus santalinus inhibits adipogenesis and inflammation in 3T3-L1 cells via modulation of PPAR-γ/SREBP-1c and TNF-α/IL-6.

Pterocarpus santalinus has huge demand owing to its commercial and medicinal value. However, there are limited research studies on its therapeutic activity against obesity and obesity-induced inflammation and underlying mechanism of action. Therefore, in the present study, chloroform bioactive fraction of P. santalinus (CFP) was isolated and evaluated for its activity against adipogenesis and adipogenesis-induced inflammation in 3T3-L1 cell culture model. LC-MS/MS analysis of CFP was performed to identify the compounds present. CFP-treated 3T3-L1 cells (50, 100 and 200μg/ml) have significantly (p < 0.01 or < 0.05) enhanced glycerol release and adiponectin level, but reduced lipid accumulation and leptin, and MTT assay demonstrated CFP was non-toxic till a dose of 300µg/ml at 24 and 48h. A considerable reduction in tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) levels was witnessed in lipopolysaccharide (LPS)-induced 3T3-L1 cells with CFP treatment in dose-dependent manner. Gene expression studies demonstrated down-regulation of mRNA expression of peroxisome proliferator-activated receptor-γ (PPAR-γ), sterol regulatory element-binding protein-1c (SREBP-1c), leptin, TNF-α and IL-6 but up-regulation of adiponectin and uncoupling protein-1 (UCP-1) and the same trend was observed in protein expression also. In conclusion, it is suggested that CFP could be beneficial to treat obesity and associated inflammation.

Alisol A 24-acetate stimulates lipolysis in 3\u2009T3-L1 adipocytes

BackgroundAlisol A 24-acetate (AA-24-a), one of the main active triterpenes isolated from the well-known medicinal plant Alisma orientale (Sam.) Juz., exhibits multiple biological activities including hypolipidemic activity. However, its effect on lipid metabolism in adipocytes remains unclear. The present study aimed to clarify the effect of AA-24-a on adipocyte lipolysis and to determine its potential mechanism of action using 3 T3-L1 cells.MethodsWe assayed the release of glycerol into culture medium of 3 T3-L1 cells under treatment with AA-24-a. Protein and mRNA expression and phosphorylation levels of the main lipases and kinases involved in lipolysis regulation were determined by quantitative polymerase chain reaction and western blotting. Specific inhibitors of protein kinase A (PKA; H89) and extracellular signal-regulated kinase (ERK; PD98059), which are key enzymes in relevant signaling pathways, were used to examine their roles in AA-24-a-stimulated lipolysis.ResultsAA-24-a significantly stimulated neutral lipolysis in fully differentiated adipocytes. To determine the underlying mechanism, we assessed the changes in mRNA and protein levels of key lipolysis-related genes in the presence or absence of H89 and PD98059. Both inhibitors reduced AA-24-a-induced lipolysis. Moreover, pretreatment with H89 attenuated AA-24-a-induced phosphorylation of hormone-sensitive lipase at Ser660, while pretreatment with PD98059 attenuated AA-24-a-induced downregulation of peroxisome proliferator-activated receptor-γ and perilipin A.ConclusionsOur results indicate that AA-24-a promoted neutral lipolysis in 3 T3-L1 adipocytes by activating PKA-mediated phosphorylation of hormone-sensitive lipase and ERK- mediated downregulation of expression of perilipin A.

A Holistic View of the Genetic Factors Involved in Triggering Hydrolytic and Oxidative Rancidity of Rice Bran Lipids

ABSTRACT The bran fraction of rice grains is used for extracting nutritious edible oil known as rice bran oil (RBO). In spite of its nutritional quality, bran lipids have a lower shelf-life due to the hydrolytic and oxidative rancidity. During the rice grain milling, endogenous lipases come in contact with storage lipids or triacylglycerols (TAGs) in the ruptured bran layers. The fatty acid ester bonds in the TAGs are hydrolyzed by the lipases causing the release of free fatty acids (FFAs) and glycerol. Further oxidation of these FFAs, especially the linoleic acid (18:2), are further oxidized by the action of lipoxygenases (LOXs). This reaction produces conjugated hydroperoxides of 18:2, which decompose and form secondary oxidation products responsible for the strong undesirable stale off-flavors. The outcome is a deterioration of bran lipids and significant loss of TAGs during RBO refining process. Therefore, this review aims to provide a holistic view on the current understanding of the catalog of genes and the underlying molecular mechanisms involved in triggering bran lipid rancidity. Generated knowledge about the individual genes will be crucial for developing advanced genome editing strategies to regulate rice bran lipid quality and stability.