Tomoya Sakamoto

Triiodothyronine induces UCP-1 expression and mitochondrial biogenesis in human adipocytes

Uncoupling protein (UCP)-1 expressed in brown adipose tissue plays an important role in thermogenesis. Recent data suggest that brown-like adipocytes in white adipose tissue (WAT) and skeletal muscle play a crucial role in the regulation of body weight. Understanding of the mechanism underlying the increase in UCP-1 expression level in these organs should, therefore, provide an approach to managing obesity. The thyroid hormone (TH) has profound effects on mitochondrial biogenesis and promotes the mRNA expression of UCP in skeletal muscle and brown adipose tissue. However, the action of TH on the induction of brown-like adipocytes in WAT has not been elucidated. Thus we investigate whether TH could regulate UCP-1 expression in WAT using multipotent cells isolated from human adipose tissue. In this study, triiodothyronine (T(3)) treatment induced UCP-1 expression and mitochondrial biogenesis, accompanied by the induction of the CCAAT/enhancer binding protein, peroxisome proliferator-activated receptor-γ coactivator-1α, and nuclear respiratory factor-1 in differentiated human multipotent adipose-derived stem cells. The effects of T(3) on UCP-1 induction were dependent on TH receptor-β. Moreover, T(3) treatment increased oxygen consumption rate. These findings indicate that T(3) is an active modulator, which induces energy utilization in white adipocytes through the regulation of UCP-1 expression and mitochondrial biogenesis. Our findings provide evidence that T(3) serves as a bipotential mediator of mitochondrial biogenesis.

Bixin regulates mRNA expression involved in adipogenesis and enhances insulin sensitivity in 3T3-L1 adipocytes through PPARγ activation

Insulin resistance is partly due to suppression of insulin-induced glucose uptake into adipocytes. The uptake is dependent on adipocyte differentiation, which is controlled at mRNA transcription level. The peroxisome proliferator-activated receptor (PPAR), a ligand-regulated nuclear receptor, is involved in the differentiation. Many food-derived compounds serve as ligands to activate or inactivate PPAR. In this study, we demonstrated that bixin and norbixin (annatto extracts) activate PPARgamma by luciferase reporter assay using GAL4-PPAR chimera proteins. To examine the effects of bixin on adipocytes, 3T3-L1 adipocytes were treated with bixin or norbixin. The treatment induced mRNA expression of PPARgamma target genes such as adipocyte-specific fatty acid-binding protein (aP2), lipoprotein lipase (LPL), and adiponectin in differentiated 3T3-L1 adipocytes and enhanced insulin-dependent glucose uptake. The observations indicate that bixin acts as an agonist of PPARgamma and enhances insulin sensitivity in 3T3-L1 adipocytes, suggesting that bixin is a valuable food-derived compound as a PPAR ligand to regulate lipid metabolism and to ameliorate metabolic syndrome.

Inflammation induced by RAW macrophages suppresses UCP1 mRNA induction via ERK activation in 10T1/2 adipocytes

Recently, it has been demonstrated that uncoupling protein-1 (UCP1)-expressing white adipocytes (brown-like adipocytes) are important for energy expenditure in white adipose tissue (WAT), in which energy expenditure decreases under obese conditions. However, the relationship between the induction of brown-like adipocytes and the decrease in energy expenditure in obese WAT remains to be elucidated. Here, we show that proinflammatory cytokines derived from activated macrophages suppress the induction of UCP1 promoter activity and mRNA expression via an extracellular signal-related kinase (ERK) in white adipocytes. The coculture with RAW264.7 (RAW) macrophages suppressed the induction of UCP1 mRNA expression by isoproterenol (ISO), a typical β-adrenergic receptor agonist, in C3H10T1/2 (10T1/2) adipocytes. A conditioned medium derived from lipopolysaccharide (LPS)-activated macrophages and tumor necrosis factor-α (TNF-α) also suppressed the induction of UCP1 mRNA but did not affect its mRNA stability. By using a luciferase reporter assay system, the conditioned medium and TNF-α also suppressed the activity of the UCP1 promoter and transcriptional factors binding to the cAMP response element (CRE). Importantly, PD98059, an ERK inhibitor, partially abrogated the suppression of UCP1 promoter activation and mRNA induction. These results indicate that ERK is an important factor in the suppression of UCP1 transcriptional activation in the interaction between white adipocytes and activated macrophages. This report suggests a possible mechanism of the UCP1 transcriptional suppression in white adipocytes associated with obese and diabetic conditions.

Activation of peroxisome proliferator-activated receptor-α enhances fatty acid oxidation in human adipocytes.

Peroxisome proliferator-activated receptor-α (PPARα) is a key regulator for maintaining whole-body energy balance. However, the physiological functions of PPARα in adipocytes have been unclarified. We examined the functions of PPARα using human multipotent adipose tissue-derived stem cells as a human adipocyte model. Activation of PPARα by GW7647, a potent PPARα agonist, increased the mRNA expression levels of adipocyte differentiation marker genes such as PPARγ, adipocyte-specific fatty acid-binding protein, and lipoprotein lipase and increased both GPDH activity and insulin-dependent glucose uptake level. The findings indicate that PPARα activation stimulates adipocyte differentiation. However, lipid accumulation was not changed, which is usually observed when PPARγ is activated. On the other hand, PPARα activation by GW7647 treatment induced the mRNA expression of fatty acid oxidation-related genes such as CPT-1B and AOX in a PPARα-dependent manner. Moreover, PPARα activation increased the production of CO(2) and acid soluble metabolites, which are products of fatty acid oxidation, and increased oxygen consumption rate in human adipocytes. The data indicate that activation of PPARα stimulates both adipocyte differentiation and fatty acid oxidation in human adipocytes, suggesting that PPARα agonists could improve insulin resistance without lipid accumulation in adipocytes. The expected effects of PPARα activation are very valuable for managing diabetic conditions accompanied by obesity, because PPARγ agonists, usually used as antidiabetic drugs, induce excessive lipid accumulation in adipocytes in addition to improvement of insulin resistance.

Proinflammatory cytokine interleukin-1β suppresses cold-induced thermogenesis in adipocytes

In this study, we investigated the effects of interleukin-1β (IL-1β), a typical proinflammatory cytokine on the β-adrenoreceptor-stimulated induction of uncoupling protein 1 (UCP1) expression in adipocytes. IL-1β mRNA expression levels were upregulated in white adipose tissues of obese mice and in RAW264.7 macrophages under conditions designed to mimic obese adipose tissue. Isoproterenol-stimulated induction of UCP1 mRNA expression was significantly inhibited in C3H10T1/2 adipocytes by conditioned medium from lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages in comparison with control conditioned medium. This inhibition was significantly attenuated in the presence of recombinant IL-1 receptor antagonist and IL-1β antibody, suggesting that activated macrophage-derived IL-1β is an important cytokine for inhibition of β-adrenoreceptor-stimulated UCP1 induction in adipocytes. IL-1β suppressed isoproterenol-induced UCP1 mRNA expression in C3H10T1/2 adipocytes, and this effect was partially but significantly abrogated by inhibition of extracellular signal-regulated kinase (ERK). IL-1β also suppressed the isoproterenol-induced activation of the UCP1 promoter and transcription factors binding to the cAMP response element. Moreover, intraperitoneal administration of IL-1β suppressed cold-induced UCP1 expression in adipose tissues. These findings suggest that IL-1β upregulated in obese adipose tissues suppresses β-adrenoreceptor-stimulated induction of UCP1 expression through ERK activation in adipocytes.

Bixin activates PPARα and improves obesity-induced abnormalities of carbohydrate and lipid metabolism in mice.

Peroxisome proliferator-activated receptor α (PPARα) is a ligand-activated transcription factor that regulates the expression of the genes involved in fatty acid oxidation. PPARα activators induce fatty acid oxidation in the liver, thereby improving lipid and carbohydrate metabolism in obese mice. In this study, the dietary cis-carotenoids bixin and norbixin, which are commonly used in the food coloring industry, were found to activate PPARα by luciferase reporter assays using GAL4/PPARα chimeric and full-length PPARα systems. Treatment with bixin and norbixin induced the mRNA expression of PPARα target genes involved in fatty acid oxidation in PPARα-expressing HepG2 hepatocytes. In obese KK-Ay mice, bixin treatment suppressed the development of hyperlipidemia and hepatic lipid accumulation. In the livers of bixin-treated mice, the mRNA levels of PPARα target genes related to fatty acid oxidation were up-regulated. Moreover, bixin treatment also improved obesity-induced dysfunctions of carbohydrate metabolism, such as hyperglycemia, hyperinsulinemia, and hypoadiponectinemia. Glucose tolerance test and insulin tolerance test revealed that glucose intolerance and insulin resistance in KK-Ay obese mice were attenuated by the treatment with bixin. These results indicate that bixin acts as a food-derived agonist of PPARα, and bixin treatment is useful for the management of obesity-induced metabolic dysfunctions in mice.

Dehydroabietic acid, a diterpene, improves diabetes and hyperlipidemia in obese diabetic KK-Ay mice

Terpenoids, which are contained in a large number of dietary and herbal plants, have many biological effects. In this study, the effects of dehydroabietic acid (DAA), a diterpene, on glucose and lipid metabolism were examined using obese diabetic KK‐Ay mice. We showed here that DAA treatment decreased not only plasma glucose and insulin levels but also plasma triglyceride (TG) and hepatic TG levels. To examine the mechanism underlying the effects of DAA, the production of inflammatory cytokines was measured. It was shown that the DAA treatment suppressed the production of monocyte chemoattractant protein‐1 (MCP‐1) and tumor necrosis factor‐α (TNFα) (proinflammatory cytokines) and increased that of adiponectin (an anti‐inflammatory cytokine). As a result of the changes in the production of inflammatory cytokines caused by the DAA treatment, the accumulation of macrophages in adipose tissues was reduced. These results indicate that treatment with DAA improves the levels of plasma glucose, plasma insulin, plasma TG, and hepatic TG through the decrease in the macrophage infiltration into adipose tissues, suggesting that DAA is a useful food‐derived compound for treating obesity‐related diseases.

Macrophage infiltration into obese adipose tissues suppresses the induction of UCP1 level in mice.

Emergence of thermogenic adipocytes such as brown and beige adipocytes is critical for whole body energy metabolism. Promoting the emergence of these adipocytes, which increase energy expenditure, could be a viable strategy in treating obesity and its related diseases. However, little is known regarding the mechanisms that regulate the emergence of these adipocytes in obese adipose tissue. Here, we demonstrated that classically activated macrophages (M1 Mϕ) suppress the induction of thermogenic adipocytes in obese adipose tissues of mice. Cold exposure significantly induced the expression levels of uncoupling protein-1 (UCP1), which is a mitochondrial protein unique in thermogenic adipocytes, in C57BL/6 mice fed a normal diet. However, UCP1 induction was significantly suppressed in adipose tissues of C57BL/6 mice fed a high-fat diet, into which M1 Mϕ infiltrated. Depletion of M1 Mϕ using clodronate liposomes eliminated the suppressive effect and markedly reduced the mRNA level of tumor necrosis factor-α (TNFα) in the adipose tissues. Importantly, consistent with the observed changes in the expression levels of marker genes for thermogenic adipocytes, combination treatment of clodronate liposome and cold exposure resulted in metabolic benefits such as lowered body weight and blood glucose level in obese mice. Moreover, intraperitoneal injection of recombinant TNFα protein suppressed UCP1 induction in lean adipose tissues of mice. Collectively, our data indicate that infiltrated M1 Mϕ suppress the induction of thermogenic adipocytes in obese adipose tissues via TNFα. This report suggests that inflammation induced by infiltrated Mϕ could cause not only insulin resistance but also reduction of energy expenditure in adipose tissues.

Auraptene suppresses inflammatory responses in activated RAW264 macrophages by inhibiting p38 mitogen-activated protein kinase activation.

SCOPE Inflammation plays a key role in obesity-related pathologies such as insulin resistance and type 2 diabetes. Hypertrophied adipocytes trigger the enhancement of macrophage infiltration and the release of various proinflammatory factors in obese adipose tissue. In this study, we examined whether auraptene, a citrus-fruit-derived compound, could suppress the production of inflammatory factors that mediate the interaction between adipocytes and macrophages. METHODS AND RESULTS Experiments using a co-culture system of 3T3-L1 adipocytes and RAW264 macrophages showed that auraptene reduced the production of nitric oxide and tumor necrosis factor-α. In RAW264 macrophages, auraptene also suppressed the inflammation induced by either LPS or the conditioned medium derived from 3T3-L1 adipocytes. In addition, auraptene inhibited the phosphorylation of the p38 mitogen-activated protein kinase and suppressed the production of proinflammatory mediators in activated macrophages. CONCLUSION Our findings indicate that auraptene exhibits anti-inflammatory properties by suppressing the production of inflammatory factors that mediate the interaction between adipocytes and macrophages, suggesting that auraptene is a valuable food-derived compound with a potential to attenuate chronic inflammation in adipose tissue and to improve obesity-related insulin resistance.

Tomato extract suppresses the production of proinflammatory mediators induced by interaction between adipocytes and macrophages.

Obese adipose tissue is characterized by enhanced macrophage infiltration. A loop involving monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor-α (TNFα) between adipocytes and macrophages establishes a vicious cycle that augments inflammatory changes and insulin resistance in obese adipose tissue. Tomatoes, one of the most popular crops worldwide, contain many beneficial phytochemicals that improve obesity-related diseases such as diabetes. Some of them have also been reported to have anti-inflammatory properties. In this study, we focused on the potential protective effects of phytochemicals in tomatoes on inflammation. We screened fractions of tomato extract using nitric oxide (NO) assay in lipopolysaccharide (LPS)-stimulated RAW264 macrophages. One fraction, RF52, significantly inhibited NO production in LPS-stimulated RAW264 macrophages. Furthermore, RF52 significantly decreased MCP-1 and TNFα productions. The coculture of 3T3-L1 adipocytes and RAW264 macrophages markedly enhanced MCP-1, TNFα, and NO productions compared with the control cultures; however, the treatment with RF52 inhibited the production of these proinflammatory mediators. These results suggest that RF52 from tomatoes may have the potential to suppress inflammation by inhibiting the production of NO or proinflammatory cytokines during the interaction between adipocytes and macrophages. Graphical Abstract Tomato extract suppresses the production of proinflammatory mediators induced by interaction between adipocytes and macrophages.