Liver X receptor preferentially activates de novo lipogenesis in human preadipocytes

The liver X receptor (LXR) was demonstrated to play a key role in cholesterol metabolism in liver, intestine and macrophage. However, its function on the regulation of preadipocyte differentiation remains unclear since contradictory results were reported. The objective of the present study was to unravel the functionality of LXR in human preadipocytes. We show that the LXR agonist T0901317 strongly stimulated the expression of SREBP-1c and the lipogenic enzymes ACC-1, FAS and SCD-1 in both the human preadipose cell line Chub-S7 as well as human primary stromal vascular fraction (SVF) cells. The effects on gene expression were associated with the stimulation of de novo lipogenesis in both cell models, resulting in the induction of lipid accumulation. In contrast with a PPARgamma agonist (BRL49653), T0901317 enhanced only slightly the expression of PPARgamma dependent genes (PPARgamma, aP2 and adiponectin) in Chub-S7 cells and failed to change their expression in human SVF cells. These results show that LXR stimulated preferentially triglyceride accumulation in human preadipocytes via the induction of de novo lipogenesis, rather than activating the differentiation process through PPARgamma activation.     

2-Formyl-komarovicine promotes adiponectin production in human mesenchymal stem cells through PPARγ partial agonism

Adiponectin is a major adipocytokine secreted from mammalian adipocytes. Relatively low expression of adiponectin is associated with various human metabolic diseases and some cancers. Adiponectin-secreting compounds have therapeutic potential for these diseases. Adipogenesis of human bone marrow-mesenchymal stem cells (hBM-MSCs) has been used as a phenotypic assay to find adiponectin secreting compounds. In a phytochemical library screen, 2-formyl-komarovicine, 1-(quinolin-8-yl)-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indole-2-carbaldehyde, isolated from Nitraria komarovii was identified as a potential adiponectin-secreting compound. To validate the results of the impure phytochemical, we synthesized 2-formyl-komarovicine. The synthetic 2-formyl-komarovicine significantly promoted adiponectin production during adipogenesis in hBM-MSCs. In a target identification experiment, 2-formyl-komarovicine bound to peroxisome proliferator-activated receptor γ (PPARγ) in a concentration-dependent manner. Notably, 2-formyl-komarovicine competitively inhibited the adiponectin-promoting activity of a full PPARγ agonist, troglitazone, in hBM-MSCs, which is a pharmacological feature of a partial agonist. The ligand-docking model showed that 2-formyl-komarovicine interacted with the hydrophobic pocket of the PPARγ ligand-binding domain, but lacked an interaction to stabilize helix H12, which is one of the major binding themes of PPARγ partial agonists. We concluded that 2-formyl-komarovicine provides a novel pharmacophore for PPARγ partial agonists to increase adiponectin production.     

Eicosapentaenoic Acid and Rosiglitazone Increase Adiponectin in an Additive and PPARγ‐Dependent Manner in Human Adipocytes

Adiponectin, an anti‐inflammatory and insulin‐sensitizing protein secreted from adipose tissue, may be modulated by dietary fatty acids, although the mechanism is not fully known. Our objective was to investigate the effect of long‐chain n‐3 polyunsaturated fatty acids (PUFAs) on adiponectin in cultured human adipocytes, and to elucidate the role of peroxisome proliferator‐activated receptor‐γ (PPARγ) in this regulation. Isolated human adipocytes were cultured for 48 h with 100 µmol/l eicosapentaenoic acid (C20:5n‐3, EPA), docosahexaenoic acid (C22:6n‐3, DHA), palmitic acid (C16:0), 100 µmol/l EPA plus 100 µmol/l DHA, or bovine serum albumin (control). Additionally, adipocytes were treated for 48 h with a PPARγ antagonist (BADGE) or agonist (rosiglitazone) in isolation or in conjunction with either EPA or DHA. At 48 h, EPA and DHA increased (P < 0.05) adiponectin secretion by 88 and 47%, respectively, while EPA, but not DHA, also increased (136%, P < 0.001) cellular adiponectin protein. Interestingly, PPARγ antagonism completely abolished the DHA‐mediated increase in secreted adiponectin, but only partially attenuated the EPA‐mediated response. Thus, EPA's effects on adiponectin do not appear to be entirely PPARγ mediated. Rosiglitazone increased (P < 0.001) the secreted and cellular adiponectin protein (90 and 582%, respectively). Finally, the effects of EPA and rosiglitazone on adiponectin secretion were additive (+230% at 48 h combined, compared to 121 and 124% by EPA or rosiglitazone alone, respectively). Overall, our findings emphasize the therapeutic importance of long‐chain n‐3 PUFA alone, or in combination with a PPARγ agonist, as a stimulator of adiponectin, a key adipokine involved in obesity and related diseases.     

Dietary docosahexaenoic acid alters pregnant rat reproductive tissue prostaglandin and matrix metalloproteinase production

Shortened gestation is a major cause of infant mortality and morbidity. Evidence from both human and animal studies suggests that essential fatty acids of the n-6 and n-3 series play important and modifiable roles in gestational duration. We examined the influence of linolenic acid (LnA) vs. docosahexaenoic acid (DHA) on rat reproductive tissue prostaglandin (PG) and matrix metalloproteinase (MMP) indices of gestational duration. By varying the oil source of the diet, AIN-93G diets were constructed to provide either 0.7 energy % (en%) LnA, the current US intake of n-3 fatty acids, or 0.7 en% DHA. In addition, enhanced levels of 2.0 en% LnA or 2.0 e% DHA diets were also constructed. All diets contained approximately 6.0 en% linoleic acid (LA), the current US intake of LA. Four groups of 10 female rats were time-mated and fed the respective diets from conception through Day 20 of gestation. Day 20 uterus and placenta DHA were significantly increased by 160-180% by the 0.7 en% DHA diet, and by 250-350% by the 2.0 en% DHA diets in comparison to 0.7 en% LnA diet. DHA diets also significantly reduced uterus and placenta arachidonic acid content. Day 20 placenta and uterus PGE(2) and placenta PGF(2alpha) production rates were significantly reduced by 27-47% in the 0.7 en% DHA group in comparison to 0.7 en% LnA. Increasing LnA to 2.0 en% was without effect. Providing DHA at the enhanced 2.0 en% did not significantly enhance the suppression of PG production. Placenta active MMP-2 and active MMP-9 (gelatinase) production was suppressed significantly by 30-43% in the 0.7 en% DHA group in comparison to the 0.7 en% LnA group, and 2.0 en% DHA did not enhance this suppression. Placenta collagenase activity comprising the sum of MMP-1, MMP-8 and MMP-13 was also suppressed by 60% in the 0.7 en% DHA diet group with no additional effect with 2.0 en% DHA provision. These results suggest that substituting DHA for LnA even at the current US n-3 fatty acid intake of 0.7 en% is effective in suppressing indices of premature delivery and shortened gestation. Increasing LnA intake by 3-fold to 2.0 en% is not effective. The form of dietary n-3 fatty acid, DHA vs. LnA, appears to be more important than the amount.     

Adiponectin secretion is regulated by SIRT1 and the endoplasmic reticulum oxidoreductase Ero1-L alpha

Adiponectin is secreted from adipose tissue in response to metabolic effectors in order to sensitize the liver and muscle to insulin. Reduced circulating levels of adiponectin that usually accompany obesity contribute to the associated insulin resistance. The molecular mechanisms controlling the production of adiponectin are essentially unknown. In this report, we demonstrate that the endoplasmic reticulum (ER) oxidoreductase Ero1-L alpha and effectors modulating peroxisome proliferator-activated receptor gamma (PPAR gamma) and SIRT1 activities regulate secretion of adiponectin from 3T3-L1 adipocytes. Specifically, adiponectin secretion and Ero1-L alpha expression are induced during the early phase of adipogenesis but are then down-regulated during the terminal phase, coincident with an increased expression of SIRT1. Suppression of SIRT1 or activation of PPAR gamma enhances Ero1-L alpha expression and stimulates secretion of high-molecular-weight complexes of adiponectin in mature adipocytes. Suppression of Ero1-L alpha through expression of a corresponding small interfering RNA reduces adiponectin secretion during the differentiation of 3T3-L1 preadipocytes. Moreover, ectopic expression of Ero1-L alpha in Ero1-L alpha-deficient 3T3 fibroblasts stimulates the secretion of adiponectin following their conversion into adipocytes and prevents the suppression of adiponectin secretion in response to activation of SIRT1 by exposure to resveratrol. These findings provide a framework to understand the mechanisms by which adipocytes regulate secretion of adiponectin in response to various metabolic states.     

Divergent effects of eicosapentaenoic and docosahexaenoic acid ethyl esters, and fish oil on hepatic fatty acid oxidation in the rat

The physiological activity of fish oil, and ethyl esters of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) affecting hepatic fatty acid oxidation was compared in rats. Five groups of rats were fed various experimental diets for 15 days. A group fed a diet containing 9.4% palm oil almost devoid of n-3 fatty acids served as a control. The test diets contained 4% n-3 fatty acids mainly as EPA and DHA in the form of triacylglycerol (9.4% fish oil) or ethyl esters (diets containing 4% EPA ethyl ester, 4% DHA ethyl ester, and 1% EPA plus 3% DHA ethyl esters). The lipid content of diets containing EPA and DHA ethyl esters was adjusted to 9.4% by adding palm oil. The fish oil diet and ethyl ester diets, compared to the control diet containing 9.4% palm oil, increased activity and mRNA levels of hepatic mitochondrial and peroxisomal fatty acid oxidation enzymes, though not 3-hydroxyacyl-CoA dehydrogenase activity. The extent of the increase was, however, much greater with the fish oil than with EPA and DHA ethyl esters. EPA and DHA ethyl esters, compared to the control diet, increased 3-hydroxyacyl-CoA dehydrogenase activity, but fish oil strongly reduced it. It is apparent that EPA and DHA in the form of ethyl esters cannot mimic the physiological activity of fish oil at least in affecting hepatic fatty acid oxidation in rat.     

Differential effects of eicosapentaenoic acid and docosahexaenoic acid in promoting the differentiation of 3T3-L1 preadipocytes

The objective of this study was to determine the effects of enrichment with n-3 fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), on the differentiation of 3T3-L1 preadipocytes. Enrichment with DHA but not EPA significantly increased the differentiation markers compared to control differentiated cells. DHA compared to EPA treatment led to a greater increase in adiponectin secretion and, conditioned media collected from DHA treated cells inhibited monocyte migration. Moreover, DHA treatment resulted in inhibition of pro-inflammatory signaling pathways. DHA treated cells predominantly accumulated DHA in phospholipids whereas EPA treatment led to accumulation of both EPA and its elongation product docosapentaenoic acid (DPA), an n-3 fatty acid. Of note, adding DPA to DHA inhibited DHA-induced differentiation. The differential effects of EPA and DHA on preadipocyte differentiation may be due, in part, to differences in their intracellular modification which could impact the type of n-3 fatty acids incorporated into the cells.     

Changes in fatty acids metabolism during differentiation of Atlantic salmon preadipocytes; effects of n-3 and n-9 fatty acids

Atlantic salmon (Salmo salar) preadipocytes, isolated from visceral adipose tissue, differentiate from an unspecialized fibroblast like cell type to mature adipocytes filled with lipid droplets in culture. The expression of the adipogenic gene markers peroxisome proliferated activated receptor (PPAR) alpha, lipoprotein lipase (LPL), microsomal triglyceride transfer protein (MTP), fatty acid transport protein (FATP) 1 and fatty acid binding protein (FABP) 3 increased during differentiation. In addition, we describe a novel alternatively spliced form of PPARgamma (PPARgamma short), the expression of which increased during differentiation. Eicosapentaenoic acid (20:5n-3, EPA) and docosahexaenoic acid (22:6n-3, DHA) lowered the triacylglycerol (TAG) accumulation in mature salmon adipocytes compared to oleic acid (18:1n-9, OA). This finding indicates that a reduced level of highly unsaturated n-3 fatty acids (HUFAs) in fish diets, when the traditional marine oil is exchanged for n-9 fatty acids (FAs) rich vegetable oils (VOs), may influence visceral fat deposition in salmonids. Moreover, major differences in the metabolism of EPA, DHA and OA at different stages during differentiation of adipocytes occur. Most of the EPA and DHA were oxidized in preadipocytes, while they were mainly stored in TAGs in mature adipocytes in contrast to OA which was primarily stored in TAGs at all stages of differentiation.     

Anti-adipogenic diarylheptanoids from Alnus hirsuta f. sibirica on 3T3-L1 cells

A new diarylheptanoid, (5S)-hydroxy-1-(3,4-dihydroxyphenyl)-7-(4-hydroxyphenyl)-hepta-1E-en-3-one (1), was isolated along with seventeen known diarylheptanoids (2–18) from the methanol extract of Alnus hirsuta f. sibirica leaves using bioactivity-guided fractionation. Among the isolated compounds, compounds 1 and 2 and 4–12 reduced lipid accumulation dose-dependently in 3T3-L1 preadipocytes. Of the compounds active in the present assay system, the most potent compound 7, platyphyllonol-5-O-β-d-xylopyranoside, significantly suppressed the induction of peroxisome proliferator activated receptor γ (PPARγ and CCAAT/enhancer binding protein α (C/EBPα) protein expression, and inhibited adipocyte differentiation induced by troglitazone, a PPARγ agonist. It was demonstrated that compound 7 has anti-adipogenic activity mediated by the regulation of PPARγ dependent pathways.     

Aldosterone perturbs adiponectin and PAI-1 expression and secretion in 3T3-L1 adipocytes

Aldosterone is considered as a new cardiovascular risk factor that plays an important role in metabolic syndrome; however, the underlying mechanism of these effects is not clear. Hypoadiponectinemia and elevated circulating concentration of plasminogen activator inhibitor-1 (PAI-1) are causally associated with obesity-related insulin resistance and cardiovascular disease. The aim of the present study is to investigate the effect of aldosterone on the production of adiponectin and PAI-1 in 3T3-L1 adipocytes. Northern and Western blot analyses revealed that aldosterone treatment inhibited adiponectin mRNA expression and secretion and simultaneously enhanced PAI-1 mRNA expression and secretion in a time- and dose-dependent manner. Rosiglitazone did not prevent aldosterone's effect on adiponectin or PAI-1 expression. In contrast, tumor necrosis factor (TNF)-α produced dramatic synergistic effects on adiponectin and PAI-1 expression when added together with aldosterone. Furthermore, the effects of aldosterone on adiponectin and PAI-1 expression appear to be mediated through glucocorticoid receptor (GR) but not mineralocorticoid receptor (MR). These results suggest that the effects of aldosterone on adiponectin and PAI-1 production are one of the underlying mechanisms linking it to insulin resistance, metabolic syndrome and cardiovascular disease.     

JD-5006 and JD-5037: Peripherally restricted (PR) cannabinoid-1 receptor blockers related to SLV-319 (Ibipinabant) as metabolic disorder therapeutics devoid of CNS liabilities

Analogs of SLV-319 (Ibipinibant), a CB1 receptor inverse agonist, were synthesized with functionality intended to limit brain exposure while maintaining the receptor affinity and selectivity of the parent compound. Structure activity relationships of this series, and pharmacology of two lead compounds, 16 (JD-5006) and 23 (JD-5037) showing little brain presence as indicated by tissue distribution and receptor occupancy studies, are described. Effects with one of these compounds on plasma triglyceride levels, liver weight and enzymes, glucose tolerance and insulin sensitivity support the approach that blockade of peripheral CB₁ receptors is sufficient to produce many of the beneficial metabolic effects of globally active CB₁ blockers. Thus, PR CB₁ inverse agonists may indeed represent a safer alternative to highly brain-penetrant agents for the treatment of metabolic disorders, including diabetes, liver diseases, dyslipidemias, and obesity.     

Advanced oxidation protein products inhibit differentiation and activate inflammation in 3T3‐L1 preadipocytes

Accumulation of advanced oxidation protein products (AOPPs) is prevalent in metabolic syndromes, a condition with impaired preadipocytes differentiation. In the present study, we tested the hypothesis that AOPPs disturb preadipocyte differentiation. Exposure of 3T3‐L1 preadipocytes to increased levels of AOPPs inhibited accumulation of intracellular triglyceride and decreased the expression of the essential markers of matured adipocytes, such as adipocyte fatty‐acid‐binding protein (aP2), CAAT/enhancer‐binding protein (C/EBP)‐α, and peroxisome proliferator‐activated receptor (PPAR)‐γ, in response to standard adipogenic induction. Inhibitory effects of AOPPs on preadipocytes differentiation was time sensitive, which occurred at the early stage of differentiation. In the presence of AOPPs, induction of preadipocytes differentiation resulted in upregulated expression of C/EBP homologous protein (CHOP) and CUG‐Triplet repeat‐binding protein (CUGBP), two important inhibitors of preadipocytes differentiation. In addition, treatment with AOPPs increased abundance of C/EBP‐β‐liver enriched inhibitory protein (C/EBP‐β‐LIP), a truncated C/EBP‐β isoform without adipogenic activity. Moreover, AOPPs‐treated preadipocytes expressed a macrophage marker F4/80 and overexpressed tumor necrosis factor‐α and interleukin‐6 via nuclear factor‐κB (NF‐κB)‐dependent pathway. However, blocking inflammation with NF‐κB inhibitor failed to improve AOPPs‐induced inhibition of preadipocytes differentiation. These data suggest that accumulation of AOPPs may inhibit differentiation of preadipocytes and activate inflammation in these cells. This information might have implication for understanding the impairment of preadipocytes differentiation and fat inflammation seen in metabolic syndrome. J. Cell. Physiol. 225: 42–51, 2010. © 2010 Wiley‐Liss, Inc.     

High-throughput screening for small-molecule adiponectin secretion modulators

Adiponectin is an adipokine secreted by adipocytes and plays a role in the suppression of metabolic disorders that can result in type 2 diabetes, obesity, and atherosclerosis. Several studies have shown that upregulation of adiponectin has a number of therapeutic benefits. Although peroxisome proliferator-activated receptor γ (PPARγ) agonists are known to increase adiponectin secretion both in cultured adipocytes and humans, they have several side effects, such as weight gain, congestive heart failure, and edema. Therefore, adiponectin secretion modulators that do not possess PPARγ agonistic activity seem to promising for a number of conditions. Here, the authors report on the development of a reporter-based high-throughput screening (HTS) assay using insulin-resistant-mimic 3T3-L1 adipocytes for discovery of adiponectin secretion modulators. They screened a library of approximately 100 000 small-molecule compounds using this model, performed several follow-up screens, and identified six hit compounds that increase adiponectin secretion without having PPARγ agonistic activity. These compounds may be useful drug candidates for diabetes, obesity, atherosclerosis, and other metabolic syndromes. This HTS assay might be applicable to screening for other adipokine modulators that can be useful for the treatment of other conditions.     

Adipose tissue as a secretory organ: from adipogenesis to the metabolic syndrome

Adipose tissue contains various types of cells that include preadipocytes and adipocytes. Studies have emphasized that (i) preadipocytes secrete factors involved in their own differentiation and (ii) adipocytes acquire the ability to communicate systemically with other organs (brain, liver, skeletal muscle) and locally with other cells (preadipocytes, endothelial cells and monocytes/macrophages). Adipocytes secrete proteins exhibiting either beneficial (leptin, adiponectin) or deleterious effects (angiotensinogen). Associated to the effect of secretory products from macrophages (cytokines), a disturbance in the balance between these various secreted factors leads to the development of a metabolic syndrome.     

Eicosapentaenoic and docosahexaenoic acid ethyl esters differentially enhance B-cell activity in murine obesity

EPA and DHA are not biologically equivalent; however, their individual activity on B cells is unknown. We previously reported fish oil enhanced murine B-cell activity in obesity. To distinguish between the effects of EPA and DHA, we studied the ethyl esters of EPA and DHA on murine B-cell function as a function of time. We first demonstrate that EPA and DHA maintained the obese phenotype, with no improvements in fat mass, adipose inflammatory cytokines, fasting insulin, or glucose clearance. We then tested the hypothesis that EPA and DHA would increase the frequency of splenic B cells. EPA and DHA differentially enhanced the frequency and/or percentage of select B-cell subsets, correlating with increased natural serum IgM and cecal IgA. We next determined the activities of EPA and DHA on ex vivo production of cytokines upon lipopolysaccharide stimulation of B cells. EPA and DHA, in a time-dependent manner, enhanced B-cell cytokines with DHA notably increasing IL-10. At the molecular level, EPA and DHA differentially enhanced the formation of ordered microdomains but had no effect on Toll-like receptor 4 mobility. Overall, the results establish differential effects of EPA and DHA in a time-dependent manner on B-cell activity in obesity, which has implications for future clinical studies.     

Promotion of adiponectin multimerization by emodin: A novel AMPK activator with PPARγ-agonist activity

Adiponectin is an important insulin‐sensitizing adipokine with multiple beneficial effects on obesity‐associated medical complications. It is secreted from adipocytes into circulation as high, medium, and low molecular weight forms (HMW, MMW, and LMW). Each oligomeric form of adiponectin exerts non‐overlapping biological functions, with the HMW oligomer possessing the most potent insulin‐sensitizing activity. In this study, we reported that emodin, a natural product and active ingredient of various Chinese herbs, activates AMPK in both 3T3‐L1 adipocytes and 293T cells. Activation of AMPK by emodin promotes the assembly of HMW adiponectin and increases the ratio of HMW adiponectin to total adiponectin in 3T1‐L1 adipocytes. Emodin might activate AMPK by an indirect mechanism similar to berberine. We also found that emodin activates PPARγ and promotes differentiation and adiponectin expression during differentiation of 3T3‐L1 preadipocytes. Therefore, emodin is a novel AMPK activator with PPARγ‐agonist activity. Our results demonstrate that the effects of emodin on adiponectin expression and multimerization are the ultimate effects resulting from both AMPK activation and PPARγ activation. The dual‐activity makes emodin or the derivatives potential drug candidates for the treatment of type 2 diabetes and other obesity‐related metabolic diseases. J. Cell. Biochem. 113: 3547–3558, 2012. © 2012 Wiley Periodicals, Inc.     

Antidiabetic features of AdipoAI,a novel AdipoR agonist

Adiponectin is an antidiabetic endogenous adipokine that plays a protective role against the unfavorable metabolic sequelae of obesity. Recent evidence suggests a sinister link between hypoadiponectinemia and development of insulin resistance/type 2 diabetes (T2D). Adiponectin's insulin-sensitizing property is mediated through the specific adiponectin receptors R1 and R2, which activate the AMP-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor (PPAR) α pathways. AdipoAI is a novel synthetic analogue of endogenous adiponectin with possibly similar pharmacological effects. Thus, there is a need of orally active small molecules that activate Adipoq subunits, and their downstream signaling, which could ameliorate obesity related type 2 diabetes. In the study we aim to investigate the effects of AdipoAI on obesity and T2D. Through in-vitro and in-vivo analyses, we investigated the antidiabetic potentials of AdipoAI and compared it with AdipoRON, another orally active adiponectin receptors agonist. Our results showed that in-vitro treatment of AdipoAI (0–5 µM) increased adiponectin receptor subunits AdipoR1/R2 with increase in AMPK and APPL1 protein expression in C2C12 myotubes. Similarly, in-vivo, oral administration of AdipoAI (25 mg/kg) observed similar effects as that of AdipoRON (50 mg/kg) with improved control of blood glucose and insulin sensitivity in diet-induced obesity (DIO) mice models. Further, AdipoAI significantly reduced epididymal fat content with decrease in inflammatory markers and increase in PPAR-α and AMPK levels and exhibited hepatoprotective effects in liver. Further, AdipoAI and AdipoRON also observed similar results in adipose tissue. Thus, our results suggest that low doses of orally active small molecule agonist of adiponectin AdipoAI can be a promising therapeutic target for obesity and T2D.