Isoproterenol stimulates monocyte chemoattractant protein-1 expression and secretion in 3T3-L1 adipocytes

Recently, monocyte chemoattractant protein (MCP)-1 has been characterized as a novel adipocytokine upregulated in obesity and insulin resistance which impairs insulin signaling in muscle and fat in vitro. Growing evidence, on the other hand, suggests that increased activity of the sympathetic nervous system is an integral part in the development of insulin resistance. In the current study, the impact of the beta-adrenergic agonist isoproterenol on MCP-1 mRNA synthesis and secretion was determined in 3T3-L1 adipocytes. Interestingly, isoproterenol increased MCP-1 secretion 3-fold. Furthermore, 10 microM isoproterenol acutely induced MCP-1 mRNA by up to 5.3-fold in a time-dependent fashion with significant stimulation seen at concentrations as low as 0.3 microM effector. Studies using pharmacological inhibitors suggested that basal and isoproterenol-induced MCP-1 expressions are mediated via beta-adrenergic receptors and protein kinase A. Moreover, acute activation of adenylyl cyclase by forskolin was sufficient to mimic the effects of isoproterenol. Taken together, our results demonstrate that isoproterenol induces MCP-1 expression and secretion via a classical GS-protein-coupled pathway and support the notion that MCP-1 might be an interesting novel candidate linking obesity and insulin resistance.     

Effects of adrenal hormones on the expression of adiponectin and adiponectin receptors in adipose tissue, muscle and liver

Background

Adiponectin, an insulin-sensitive hormone that is primarily synthesized in adipose tissue, exerts its effects by binding to two receptors, adipoR1 and adipoR2. Little is known regarding the effects of glucocorticoids on the expression of adiponectin receptors.

Methods

Male Wistar rats were bilaterally adrenalectomized and treated with dexamethasone (0.2 mg/100 g) twice daily for 3 days. To analyze the potential effects of glucocorticoids, rats received two daily injections of the glucocorticoid receptor antagonist (RU-486, 5.0 mg) over the course of 3 days. Additionally, 3T3-L1 adipocytes and C2C12 myotubes were treated with dexamethasone, adrenaline or RU-486. The gene expression of adiponectin, adipoR1 and adipoR2 was determined by real-time PCR, and protein secretion was examined by Western blotting using lysates from retroperitoneal, epididymal and subcutaneous adipose tissue depots, liver and muscle.

Results

In rats, excess glucocorticoids increased the levels of insulin in serum and decreased serum adiponectin concentrations, whereas adrenalectomy decreased the mRNA expression of adiponectin (3-fold) and adipoR2 (7-fold) in epididymal adipose tissue and increased adipoR2 gene expression in muscle (3-fold) compared to control group sham-operated. Dexamethasone treatment did not reverse the effects of adrenalectomy, and glucocorticoid receptor blockade did not reproduce the effects of adrenalectomy. In 3T3-L1 adipocytes, dexamethasone and adrenaline both increased adipoR2 mRNA levels, but RU-486 reduced adipoR2 gene expression in vitro.

Conclusion

Dexamethasone treatment induces a state of insulin resistance but does not affect adiponectin receptor expression in adipose tissue. However, the effects of catecholamines on insulin resistance may be due to their effects on adipoR2.     

Pioglitazone upregulates adiponectin receptor 2 in 3T3-L1 adipocytes

AimsPioglitazone, a full peroxisome proliferator-activated receptor (PPAR)-γ agonist, improves insulin sensitivity by increasing circulating adiponectin levels. However, the molecular mechanisms by which pioglitazone induces insulin sensitization are not fully understood. In this study, we investigated whether pioglitazone improves insulin resistance via upregulation of either 2 distinct receptors for adiponectin (AdipoR1 or AdipoR2) expression in 3T3-L1 adipocytes.Main methodsGlucose uptake was evaluated by 2-[³H] deoxy-glucose uptake assay in 3T3-L1 adipocytes with pioglitazone treatment. AdipoR1 and AdipoR2 mRNA expressions were analyzed by qRT–PCR.Key findingsWe first confirmed that pioglitazone significantly increased insulin-induced 2-deoxyglucose (2-DOG) uptake in 3T3-L1 adipocytes. Next, we investigated the mRNA expression and regulation of AdipoR1 and AdipoR2 after treatment with pioglitazone. Interestingly, pioglitazone significantly induced AdipoR2 expression but it did not affect AdipoR1 expression. In addition, adenovirus-mediated PPARγ expression significantly enhanced the effects of pioglitazone on insulin-stimulated 2-DOG uptake and AdipoR2 expression in 3T3-L1 adipocytes. These data suggest that pioglitazone enhances adiponectin's autocrine and paracrine actions in 3T3-L1 adipocytes via upregulation of PPARγ-mediated AdipoR2 expression. Furthermore, we found that pioglitazone significantly increased AMP-activated protein kinase (AMPK) phosphorylation in insulin-stimulated 3T3-L1 adipocytes, but it did not lead to the phosphorylation of IRS-1, Akt, or protein kinase Cλ/ζ.SignificanceOur results suggest that pioglitazone increases insulin sensitivity, at least partly, by PPARγ-AdipoR2-mediated AMPK phosphorylation in 3T3-L1 adipocytes. In conclusion, the upregulation of AdipoR2 expression may be one of the mechanisms by which pioglitazone improves insulin resistance in 3T3-L1 adipocytes.     

Lipoic acid inhibits adiponectin production in 3T3-L1 adipocytes

Lipoic acid (LA) is a naturally occurring compound with antioxidant properties. Recent attention has been focused on the potential beneficial effects of LA on obesity and related metabolic disorders. Dietary supplementation with LA prevents insulin resistance and upregulates adiponectin, an insulin-sensitizing adipokine, in obese rodents. The aim of this study was to investigate the direct effects of LA on adiponectin production in cultured adipocytes, as well as the potential signaling pathways involved. For this purpose, fully differentiated 3T3-L1 adipocytes were treated with LA (1–500 μM) during 24 h. The amount of adiponectin secreted to media was detected by ELISA, while adiponectin mRNA expression was determined by RT-PCR. Treatment with LA induced a dose-dependent inhibition on adiponectin gene expression and protein secretion. Pretreatment with the PI3K inhibitor LY294002 inhibited adiponectin secretion and mRNA levels, and significantly potentiated the inhibitory effect of LA on adiponectin secretion. The AMPK activator AICAR also reduced adiponectin production, but surprisingly, it was able to reverse the LA-induced inhibition of adiponectin. The JNK inhibitor SP600125 and the MAPK inhibitor PD98059 did not modify the inhibitory effect of LA on adiponectin. In conclusion, our results revealed that LA reduces adiponectin secretion in 3T3-L1 adipocytes, which contrasts with the stimulation of adiponectin described after in vivo supplementation with LA, suggesting that an indirect mechanism or some in vivo metabolic processing is involved.     

Effects of oxidative stress on adiponectin secretion and lactate production in 3T3-L1 adipocytes

Obesity is an increasing nutritional disorder in developed countries, and oxidative stress has been identified as a key factor in numerous pathologies such as diabetes, inflammation, and atherosclerosis, which are favored by obesity. The objective of the present study was to investigate the effects of oxidative stress in 3T3-L1 adipose cells on two parameters involved in metabolic complications associated with obesity, namely adiponectin secretion and lactate production. Differentiated 3T3-L1 adipose cells were exposed to increasing concentrations of glucose oxidase. 4-Hydroxynonenal (4-HNE), a relevant lipid peroxidation by-product which may affect several metabolic processes in making covalent adducts with various molecules; adiponectin secretion; and lactate production were measured in response to glucose oxidase exposure. Results show an inhibition of adiponectin mRNA expression by glucose oxidase and a significant inverse correlation between 4-HNE formation and adiponectin secretion. Furthermore, 4-HNE alone inhibits adiponectin production by 3T3-L1. On the other hand, glucose oxidase and 4-HNE significantly stimulated lactate production by 3T3-L1 adipocytes. These results demonstrate that adipose cells are highly sensitive to oxidative stress, with subsequent decreased adiponectin secretion and increased lactate production, two events involved in the development of insulin resistance.     

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.     

Apelin stimulates glucose uptake through the PI3K/Akt pathway and improves insulin resistance in 3T3-L1 adipocytes

Apelin, a cytokine mainly secreted by adipocytes, is closely related with insulin resistance. The underlying molecular mechanisms of how apelin affects insulin resistance, however, are poorly understood. This study aimed to investigate the effect of apelin on glucose metabolism and insulin resistance in 3T3-L1 adipocytes. After 10 ng/ml TNF-α treatment for 24 h, insulin-stimulated glucose uptake was reduced by 47% in 3T3-L1 adipocytes. Apelin treatment improved glucose uptake in a time- and dose-dependent manner. Treatment of 1,000 nM apelin for 60 min maximally augmented glucose uptake in insulin-resistant 3T3-L1 adipocytes. Furthermore, apelin pre-incubation also increased adipocytes' insulin-stimulated glucose uptake, and PI3K/Akt pathway were involved in these effects. In addition, immunocytochemistry staining and western blotting analysis indicated that apelin could increase glucose transporter 4 translocation from the cytoplasm to the plasma membrane. Apelin also increased the anti-inflammatory adipokine adiponectin mRNA expression while reducing that of pro-inflammatory adipokine interleukin-6 in insulin-resistant 3T3-L1 adipocytes. These results suggest that apelin stimulates glucose uptake through the PI3K/Akt pathway, promotes GLUT4 translocation from the cytoplasm to the plasma membrane, and modulates inflammatory responses in insulin-resistant 3T3-L1 adipocytes.     

Biglycan Deletion Alters Adiponectin Expression in Murine Adipose Tissue and 3T3-L1 Adipocytes

Obesity promotes increased secretion of a number of inflammatory factors from adipose tissue. These factors include cytokines and very lately, extracellular matrix components (ECM). Biglycan, a small leucine rich proteoglycan ECM protein, is up-regulated in obesity and has recently been recognized as a pro-inflammatory molecule. However, it is unknown whether biglycan contributes to adipose tissue dysfunction. In the present study, we characterized biglycan expression in various adipose depots in wild-type mice fed a low fat diet (LFD) or obesity-inducing high fat diet (HFD). High fat feeding induced biglycan mRNA expression in multiple adipose depots. Adiponectin is an adipokine with anti-inflammatory and insulin sensitizing effects. Due to the importance of adiponectin, we examined the effect of biglycan on adiponectin expression. Comparison of adiponectin expression in biglycan knockout (bgn^−/0) and wild-type (bgn^+/0) reveals higher adiponectin mRNA and protein in epididymal white adipose tissue in bgn^−/0 mice, as well higher serum concentration of adiponectin, and lower serum insulin concentration. On the contrary, knockdown of biglycan in 3T3-L1 adipocytes led to decreased expression and secretion of adiponectin. Furthermore, treatment of 3T3-L1 adipocytes with conditioned medium from biglycan treated macrophages resulted in an increase in adiponectin mRNA expression. These data suggest a link between biglycan and adiponectin expression. However, the difference in the pattern of regulation between in vivo and in vitro settings reveals the complexity of this relationship.     

Long-term regulation of cyclic nucleotide phosphodiesterase type 3B and 4 in 3T3-L1 adipocytes

Phosphodiesterase 3B (PDE3B), is known to play an important role in acute insulin and cAMP-mediated regulation of lipid metabolism, and PDE4 are the main PDE types expressed in adipocytes. Here, we show that members of all PDE4 isoforms are expressed in 3T3-L1 and primary mouse adipocytes. Long-term treatment of 3T3-L1 adipocytes with insulin induced up-regulation of PDE3B and PDE4D in a phosphatidylinositol 3-kinase-dependent manner whereas long-term treatment with beta-adrenergic agonists induced down-regulation of PDE3B and up-regulation of PDE4D. Thus, PDE3B and PDE4D can be added to the list of genes regulated by insulin and cAMP-increasing hormones. Altered expression of PDE3B and PDE4D in response to long-term treatment with insulin and catecholamines may contribute to altered regulation of metabolism in diabetes.     

Possible involvement of pertussis toxin-sensitive GTP-binding protein(s) in c-fos expression during differentiation of 3T3-L1 fibroblasts to adipocytes.

Following the differentiation of 3T3-L1 fibroblasts by insulin/dexamethasone/methylisobutylxanthine, marked increases in cAMP levels by isoproterenol but not forskolin and in 2-deoxyglucose uptake by insulin occurred. Pertussis toxin-pretreatment prior to addition of insulin/dexamethasone/methylisobutylxanthine and exposure of cells to pertussis toxin during differentiation attenuated glycerophosphate dehydrogenase activity as a differentiation marker enzyme and the responses to isoproterenol and insulin by approximately 50% of those in pertussis toxin-untreated cells. On the other hand, insulin/dexamethasone/methylisobutylxanthine caused induction of c-fos proto-oncogene in confluent 3T3-L1 fibroblasts. This induction was also reduced in pertussis toxin-pretreated cells. These results suggested that pertussis toxin-sensitive GTP-binding protein(s) is involved in expression of c-fos mRNA accompanied by differentiation. In addition, accumulation of c-fos mRNA by insulin/dexamethasone/methylisobutylxanthine was enhanced in protein kinase C-depleted cells pretreated with phorbol 12-myristate 13-acetate, indicating that protein kinase C may negatively regulate c-fos expression induced by insulin/dexamethasone/methylisobutylxanthine.     

Gene expression profiling of 3T3-L1 adipocytes exposed to phloretin

Adipocyte dysfunction plays a major role in the outcome of obesity, insulin resistance and related cardiovascular complications. Thus, considerable efforts are underway in the pharmaceutical industry to find molecules that target the now well-documented pleiotropic functions of adipocyte. We previously reported that the dietary flavonoid phloretin enhances 3T3-L1 adipocyte differentiation and adiponectin expression at least in part through PPARγ activation. The present study was designed to further characterize the molecular mechanisms underlying the phloretin-mediated effects on 3T3-L1 adipocytes using microarray technology. We show that phloretin positively regulates the expression of numerous genes involved in lipogenesis and triglyceride storage, including GLUT4, ACSL1, PEPCK1, lipin-1 and perilipin (more than twofold). The expression of several genes encoding adipokines, in addition to adiponectin and its receptor, is positively or negatively regulated in a way that suggests a possible reduction in systemic insulin resistance and obesity-associated inflammation. Improvement of insulin sensitivity is also suggested by the overexpression of genes associated with insulin signal transduction, such as CAP, PDK1 and Akt2. Many of these genes are PPARγ targets, confirming the involvement of PPARγ pathway in the phloretin effects on adipocytes. In light of these microarray data, it is reasonable to assume that phloretin may be beneficial for reducing insulin resistance, in a similar way to the thiazolidinedione class of antidiabetic drugs.     

Effect of PPAR-delta agonist on the expression of visfatin, adiponectin, and resistin in rat adipose tissue and 3T3-L1 adipocytes

It has been recently reported that activation of PPAR-delta, by specific agonists or genetic manipulation, alleviates dyslipidemia, hyperglycemia, and insulin resistance in animal models of obesity and type 2 diabetes. The purpose of the present study was to determine whether the PPAR-delta agonist has a direct effect on adipokines in visceral adipose tissue of rats and in cultured adipocytes. We examined the expression of visfatin, adiponectin, and resistin mRNA in visceral adipose tissue of Wistar rats fed a high-fat diet and 3T3-L1 adipocytes treated with PPAR-delta agonist (L-165041). Body weight and biochemical measurements were performed. Rats fed a high-fat diet showed a greater increase in body weight than those fed a standard diet (P<0.05), and treatment with L-165041 (10 mg/kg/day) significantly decreased weight gain (P<0.05). The concentration of total cholesterol was lower, and HDL cholesterol was higher in L-165041-treated rats (P<0.05). In the visceral adipose tissue of L-165041-treated rats, visfatin and adiponectin mRNA levels significantly increased compared to those of the untreated rats (P<0.05). However, the expression of resistin decreased in the L-165041-treated rats. Furthermore, in cultured 3T3-L1 adipocytes, the level of visfatin and adiponectin mRNA was up-regulated in response to L-165041 treatment for nine days. By contrast, resistin mRNA levels were down-regulated by L-165041 treatment. The present study provides a novel evidence to suggest that the PPAR-delta agonist has regulatory effects on a variety of adipokines, and these effects might explain some of their metabolic function.