Induction of mitochondrial uncoupling enhances VEGF₁₂₀ but reduces MCP-1 release in mature 3T3-L1 adipocytes: possible regulatory mechanism through endogenous ER stress and AMPK-related pathways.

Although white adipocytes contain a larger number of mitochondria per cytoplasmic volume, adipocyte mitochondrial uncoupling to reduce the efficiency of ATP production on cellular function including secretory regulation of bioactive molecules such as VEGF and MCP-1 remains to be elucidated. Here we induce mitochondrial uncoupling under hypoxia-independent conditions in mature 3T3-L1 adipocytes using a metabolic uncoupler, dinitrophenol (DNP). MCP-1 release was significantly decreased by 26% (p<0.01) in 24h DNP (30 μmol/L)-treated adipocytes compared to control cells. In contrast, secreted VEGF(120) lacking a heparin-binding domain was markedly increased 2.0-fold (p<0.01). CHOP content in these cells also were augmented (p<0.01), but no significant increase of endogenous oxidative stress was observed. Treatment with thapsigargin, which can induce exogenous endoplasmic reticulum (ER) stress, clearly attenuated MCP-1 release (p<0.01), but exhibited no effects on VEGF(120) secretion. On the other hand, exogenous H(2)O(2) amplified both MCP-1 and VEGF(120) secretion (p<0.05). In addition, under chronic activation of AMPK by AICAR, MCP-1 release was significantly diminished (p<0.05) but VEGF(120) secretion was increased (p<0.01). JNK phosphorylation in mature adipocytes was decreased by treatment with either DNP or AICAR (p<0.01). Enhanced VEGF(120) secretion with either DNP or AICAR was markedly suppressed by PI3K inhibitor LY294002 (p<0.01). Thus, induced mitochondrial uncoupling in adipocytes can reduce MCP-1 release through induction of endogenous ER stress and by reduced JNK activities via chronic activation of AMPK. Under this condition, VEGF(120) secretion was increased through PI3K-dependent pathways, which were chronically activated by AMPK, and not through ER stress. Because the decrease of MCP-1 secretion under mitochondrial uncoupling might attenuate chronic low-grade inflammation by suppressing macrophages recruitment to adipose tissue, clarification of the mechanism might reveal novel therapeutic targets for ameliorating obesity-associated insulin resistance in metabolic syndrome and type 2 diabetes.

Miyokawa-Gorin K ，Takahashi K ，Handa K ，Kitahara A ，Sumitani Y ，Katsuta H ，Tanaka T ，Nishida S ，Yoshimoto K ，Ohno H ，Ishida H ... -  《-》

Ghrelin augments the expressions and secretions of proinflammatory adipokines, VEGF120 and MCP-1, in differentiated 3T3-L1 adipocytes.

Ghrelin is a physiological-active peptide with growth hormone-releasing activity, orexigenic activity, etc. In addition, the recent study has also suggested that ghrelin possesses the pathophysiological abilities related with type 2 diabetes. However, the ghrelin-direct-effects implicated in type 2 diabetes on peripheral tissues have been still unclear, whereas its actions on the central nervous system (CNS) appear to induce the development of diabetes. Thus, to assess its peripheral effects correlated with diabetes, we investigated the regulatory mechanisms about adipokines, which play a central role in inducing peripheral insulin resistance, secreted from mature 3T3-L1 adipocytes stimulated with ghrelin in vitro . The stimulation with 50 nmol/L ghrelin for 24 h resulted in the significant 1.9-fold increase on vascular endothelial growth factor-120 (VEGF(120)) releases (p < 0.01) and the 1.7-fold on monocyte chemoattractant protein-1 (MCP-1) (p < 0.01) from 3T3-L1 adipocytes, respectively, while ghrelin failed to enhance tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6, IL-10 and adiponectin secretions. In addition, Akt phosphorylation on Ser473 and c-Jun NH2 -terminal protein kinase (JNK) phosphorylation on Thr183/Tyr185 were markedly enhanced 1.4-fold (p < 0.01) and 1.6-fold (p < 0.01) in the ghrelin-stimulated adipocytes, respectively. Furthermore, the treatment with LY294002 (50 μmol/L) and Wortmannin (10nmol/L), inhibitors of phosphatidylinositol 3-kinase (PI3K), significantly decreased the amplified VEGF(120) secretion by 29% (p < 0.01) and 28% (p < 0.01) relative to the cells stimulated by ghrelin alone, respectively, whereas these inhibitors had no effects on increased MCP-1 release. On the other hand, JNK inhibitor SP600125 (10 μmol/L) clearly reduced the increased MCP-1, but not VEGF(120), release by 35% relative to the only ghrelin-stimulated cells (p < 0.01). In conclusion, ghrelin can enhance the secretions of proinflammatory adipokines, VEGF(120) and MCP-1, but fails to affect IL-10 and adiponectin which are considered to be anti-inflammatory adipokines. Moreover, this augmented VEGF(120) release is invited through the activation of PI3K pathways and the MCP-1 is through JNK pathways. Consequently, our results strongly suggest that ghrelin can induce the development of diabetes via its direct-action in peripheral tissues as well as via in CNS.

Kitahara A ，Takahashi K ，Moriya R ，Onuma H ，Handa K ，Sumitani Y ，Tanaka T ，Katsuta H ，Nishida S ，Sakurai T ，Inukai K ，Ohno H ，Ishida H ... -  《-》

Endogenous oxidative stress, but not ER stress, induces hypoxia-independent VEGF120 release through PI3K-dependent pathways in 3T3-L1 adipocytes.

Expressions of vascular endothelial growth factor (VEGF) are increased in obese adipocytes and is secreted from obese adipose tissue through hypoxia-independent pathways. Therefore, we investigated the hypoxia-independent mechanism underlying increased expression and release of VEGF in obese adipocytes. We compared signal transduction pathways regulating VEGF with those regulating monocyte chemoattractant protein-1 (MCP-1), which is increased in obese adipocytes, in an in vitro model of artificially hypertrophied 3T3-L1 adipocytes preloaded with palmitate, without the influence of hypoxia. Palmitate-preloaded cells exhibited significantly enhanced oxidative stress (P < 0.01) and showed increased VEGF120 and MCP-1 release (P < 0.01, respectively), while endoplasmic reticulum (ER) stress was not induced. Increased VEGF120 release was significantly decreased with PI3K inhibitor LY294002 (P < 0.01). In addition, antioxidant N-acetyl-cysteine (NAC) markedly diminished not only VEGF120 secretion (P < 0.01) but also augmented Akt phosphorylation on Ser473 (P < 0.01). In contrast, increased MCP-1 release was suppressed with JNK inhibitor SP600125 and p38 MAPK inhibitor SB203580 (P < 0.01). VEGF120 release from hypertrophied adipocytes can be enhanced through PI3K pathways activated by oxidative stress but not by ER stress, suggesting that VEGF120 secretion is regulated through oxidative stress-dependent pathways distinct from those involved in MCP-1 release through either JNK or p38 MAPK activation.

Takahashi K ，Miyokawa-Gorin K ，Handa K ，Kitahara A ，Moriya R ，Onuma H ，Sumitani Y ，Tanaka T ，Katsuta H ，Nishida S ，Yoshimoto K ，Ohno H ，Ishida H ... -  《-》

JNK- and IkappaB-dependent pathways regulate MCP-1 but not adiponectin release from artificially hypertrophied 3T3-L1 adipocytes preloaded with palmitate in vitro.

Takahashi K ，Yamaguchi S ，Shimoyama T ，Seki H ，Miyokawa K ，Katsuta H ，Tanaka T ，Yoshimoto K ，Ohno H ，Nagamatsu S ，Ishida H ... -  《AMERICAN JOURNAL OF PHYSIOLOGY-ENDOCRINOLOGY AND METABOLISM》

Simvastatin inhibits ox-LDL-induced inflammatory adipokines secretion via amelioration of ER stress in 3T3-L1 adipocyte.

Adipocytes behave as a rich source of pro-inflammatory cytokines including tumor necrosis factor-α (TNF-α) and monocyte chemoattractant protein 1 (MCP-1). Endoplasmic reticulum (ER) stress in adipocytes can alter adipokines secretion and induce inflammation. The aim of this study is to evaluate the effect of simvastatin on the ox-LDL-induced ER stress and expression and secretion of TNF-α and MCP-1 in 3T3-L1 adipocytes. Differentiated adipocytes were treated with various concentrations of ox-LDL (0-100 μg/ml) for 24h with or without simvastatin pre-treatment. The protein expressions of ER stress markers, glucose-regulated protein 78 (GRP78) and C/EBP homology protein (CHOP), were determined by Western blot analysis. The mRNA expressions of TNF-α and MCP-1 were measured by real-time PCR. The protein release of TNF-α and MCP-1 in culture medium were evaluated by ELISA. Ox-LDL treatment led to significant up-regulation of GRP78 and CHOP in dose-dependent manner. The expressions of TNF-α and MCP-1 were dose-dependently increased at mRNA and protein levels after ox-LDL intervention. The effects of ox-LDL on adipocytes were abolished by pre-treatment with 4-phenylbutyrate (4-PBA), a chemical chaperone known to ameliorate ER stress. Simvastatin could inhibit ox-LDL-induced ER stress and reduce the expression of TNF-α and MCP-1 at mRNA and protien level in dose dependent manner. In conclusion, ox-LDL can stimulate the expression and secretion of TNF-α and MCP-1 through its activation of ER stress in adipocytes. Simvastatin might exert direct anti-inflammatory effects in adipocytes through amelioration of ER stress.

Wu ZH ，Chen YQ ，Zhao SP 《-》