Globular Adiponectin Research Articles

Full-Length Adiponectin Attenuates Insulin Signaling and Inhibits Insulin-Stimulated Amino Acid Transport in Human Primary Trophoblast Cells

OBJECTIVEMaternal adiponectin levels are reduced and placental nutrient transporters are upregulated in obesity and gestational diabetes mellitus; however, the effects of adiponectin on placental function are unknown. We hypothesized that adiponectin regulates placental amino acid transport.RESEARCH DESIGN AND METHODSHuman primary trophoblast cells were cultured and incubated with globular adiponectin (gAd) or full-length adiponectin (fAd) alone or in combination with insulin. System A and L amino acid transport and SNAT1, SNAT2, and SNAT4 isoform expression was measured. The activity of the AMP-activated protein kinase (AMPK), phosphatidylinositol 3 kinase–AKT, and peroxisome proliferator–activated receptor-α (PPARα) signaling pathways was determined.RESULTSIn the absence of insulin, gAd stimulated AMPK Thr172 phosphorylation, SNAT2 protein expression, and system A activity. This effect appeared to be mediated by interleukin-6 release and signal transducer and activator of transcription 3 (STAT3) signaling because gAd failed to stimulate system A in cells in which STAT3 had been silenced using small interfering RNA. fAd alone had no effect on system A activity or SNAT expression. Insulin increased AKT and insulin receptor substrate 1 (IRS-1) phosphorylation, system A activity, and SNAT2 expression. When combined with insulin, gAd did not affect system A activity or SNAT expression. In contrast, fAd abolished insulin-stimulated AKT Thr308 and IRS-1 Tyr612 phosphorylation, system A activity, and SNAT2 expression. Furthermore, fAd increased PPARα expression and PPARα (Ser21) phosphorylation.CONCLUSIONSIn contrast to the insulin-sensitizing actions of adiponectin in liver and muscle reported in the literature, fAd attenuates insulin signaling in primary human trophoblast cells. As a result, fAd inhibits insulin-stimulated amino acid transport, which may have important implications for placental nutrient transport and fetal growth in pregnancy complications associated with altered maternal adiponectin levels.

Globular Adiponectin as a Complete Mesoangioblast Regulator: Role in Proliferation, Survival, Motility, and Skeletal Muscle Differentiation

Mesoangioblasts are progenitor endowed with multipotent mesoderm differentiation ability. Despite the promising results obtained with mesoangioblast transplantation in muscle dystrophy, an improvement of their efficient engrafting and survival within damaged muscles, as well as their ex vivo activation/expansion and commitment toward myogenic lineage, is highly needed and should greatly increase their therapeutic potential. We show that globular adiponectin, an adipokine endowed with metabolic and differentiating functions for muscles, regulates vital cues of mesoangioblast cell biology. The adipokine drives mesoangioblasts to entry cell cycle and strongly counteracts the apoptotic process triggered by growth factor withdrawal, thereby serving as an activating and prosurvival stem cell factor. In addition, adiponectin provides a specific protection against anoikis, the apoptotic death due to lack of anchorage to extracellular matrix, suggesting a key protective role for these nonresident stem cells after systemic injection. Finally, adiponectin behaves as a chemoattractive factor toward mature myotubes and stimulates their differentiation toward the skeletal muscle lineage, serving as a positive regulator in mesoangioblast homing to injured or diseased muscles. We conclude that adiponectin exerts several advantageous effects on mesoangioblasts, potentially valuable to improve their efficacy in cell based therapies of diseased muscles.

The anti‐inflammatory effects of adiponectin are mediated via a heme oxygenase‐1–dependent pathway in rat Kupffer cells†

Altered expression and activity of immunomodulatory cytokines plays a major role in the pathogenesis of alcoholic liver disease. Chronic ethanol feeding increases the sensitivity of Kupffer cells, the resident hepatic macrophage, to lipopolysaccharide (LPS), leading to increased tumor necrosis factor alpha (TNF-alpha) expression. This sensitization is normalized by treatment of primary cultures of Kupffer cells with adiponectin, an anti-inflammatory adipokine. Here we tested the hypothesis that adiponectin-mediated suppression of LPS signaling in Kupffer cells is mediated via an interleukin-10 (IL-10)/heme oxygenase-1 (HO-1) pathway after chronic ethanol feeding. Knockdown of IL-10 expression in primary cultures of Kupffer cells with small interfering RNA (siRNA) prevented the inhibitory effect of globular adiponectin (gAcrp) on LPS-stimulated TNF-alpha expression. gAcrp increased IL-10 mRNA and protein expression, as well as expression of the IL-10 inducible gene, HO-1; expression was higher in Kupffer cells from ethanol-fed rats compared with pair-fed controls. Although IL-10 receptor surface expression on Kupffer cells was not affected by ethanol feeding, IL-10-mediated phosphorylation of STAT3 and expression of HO-1 was higher in Kupffer cells after ethanol feeding. Inhibition of HO-1 activity, either by treatment with the HO-1 inhibitor zinc protoporphyrin or by siRNA knockdown of HO-1, prevented the inhibitory effect of gAcrp on LPS-stimulated TNF-alpha expression in Kupffer cells. LPS-stimulated TNF-alpha expression in liver was increased in mice after chronic ethanol exposure. When mice were treated with cobalt protoporphyrin to induce HO-1 expression, ethanol-induced sensitivity to LPS was ameliorated. gAcrp prevents LPS-stimulated TNF-alpha expression in Kupffer cells through the activation of the IL-10/STAT3/HO-1 pathway. Kupffer cells from ethanol-fed rats are highly sensitive to the anti-inflammatory effects of gAcrp; this sensitivity is associated with both increased expression and sensitivity to IL-10.

Abstract 5039: Globular and Full-length Adiponectin Induce NO-Dependent Vasodilation in Resistance Arteries of ZL but Not ZDF Rats

Adiponectin increases NO-production in endothelial cell cultures and is reduced in the circulation of obese and diabetic patients, but it is not known whether adiponectin has a functional effect on small resistance arteries. In order to address this question, we assessed the direct vasodilatory response of mesenteric resistance arteries of Zucker diabetic fatty (ZDF) rat and the Zucker lean (ZL) rats to globular (gAd) and full-length adiponectin (fAd) in a small vessel myograph after precontraction with norepinephrine. Serum adiponectin levels measured by ELISA were significantly higher in ZL than in ZDF rats (ZL 4.0±0.1 μ g/l vs ZDF 2.9±0.2 μ g/l; p<0.001). Both gAd and fAd induced a relevant dose dependent vasodilation in ZL, but not ZDF rats (max. vasodilation to gAd: ZL 21.0±0.9% vs ZDF 2.9±0.9%; p<0.001. max. vasodilation to fAd: ZL 23.8±1.2% vs ZDF 1.4±1.1%; p<0.001). This vasodilatory effect in ZL rats could totally be blunted by preincubation with L-NAME (p<0.001) indicating that it is exclusively mediated by NO. ZDF rats showed a significantly lower endothelium-dependent vasodilation response to acetylcholine (max. vasodilation: ZL 96.7±5.5% vs ZDF 60.1±6.0%; p<0.001). The additional administration of low dose gAd or fAd could not improve the vasodilatory response to ACh. The gene expressions of AdipoR1 and AdipoR2 quantified by real-time RT-PCR were not significantly different in both animal models, AdipoR2 only tended to be higher expressed in ZDF rats (ZL 0.62±0.03 vs ZDF 0.68±0.06; p=0.061). But AdipoR1 was significantly more abundant expressed than AdipoR2 in resistance arteries of both ZL and ZDF rats (ZL: 1.06±0.04 vs 0.62±0.03; p<0.001. ZDF: 1.06±0.08 vs 0.68±0.06; p<0.001). In conclusion, we showed that adiponectin exerts a relevant NO-mediated vasodilation in resistance arteries of normal control rats. Reduced adiponectin serum levels and a blunted response to adiponectin in diabetic animals may contribute to endothelial dysfunction and thus play a role in the pathogenesis of atherosclerosis. In diabetes mellitus, adiponectin receptors are not downregulated in resistance arteries indicating a dysfunction downstream of the receptors.

Involvement of mTOR in globular adiponectin-induced generation of reactive oxygen species

Globular adiponectin (gAd) induces the generation of reactive oxygen species (ROS) and nitric oxide (NO) in the murine macrophage cell line RAW 264. This study investigated the role of the mammalian target of rapamycin (mTOR) in gAd-induced ROS and NO generation. gAd stimulation induced phosphorylation of mTOR, which peaked at 20 min and dissolved rapidly. Inhibition of phosphatidylinositol 3-kinase activity with wortmannin suppressed gAd-induced phosphorylation of Akt and mTOR. Administration of rapamycin partially reduced gAd-induced generation of intracellular and mitochondrial ROS, but not release of NO. To further confirm the role of mTOR in gAd stimulation, the effect of the activators of AMP-activated protein kinase (AMPK) on gAd-induced mTOR phosphorylation was examined. Pre-treatment with three kinds of AMPK activators, AICAR, 2-deoxy-D-glucose and A-769662, suppressed gAd-induced mTOR phosphorylation. Furthermore, these AMPK activators significantly reduced gAd-evoked intracellular and mitochondrial ROS generation and NO release.

Effects of globular adiponectin and glucose on the expression of PPARα,carnitine palmitoyltransferase I and acyl-CoA oxidase in INS-1 cells

INS-1 cells were treated with 3,11,20 mmol/L glucose or 2.5 mg/L globular adiponectin at the concentration of 5.5 mmol/L glucose for 24 h.The levels of PPARα,carnitine palmitoyhransferase Ⅰ (CPT1) and acyl-CoA oxidase (ACO) mRNA were detected by RT-PCR.The results showed that PPARα,PPT1 and ACO mRNA expressions were down-regulated with the increase of glucose concentration(all P＜0.05).Globular adiponectin increased PPARα (0.945±0.153 vs 0.749 ± 0.069),CPT1 (0.590±0.029 vs 0.388± 0.037) and ACO (1.553±0.131 vs 1.287±0.224)mRNA expressions(all P＜0.05),which were inhibited by AMP-activated protein kinase inhibitor adenine 9-β-D-arabinfuranoside (0.360±0.113,0.248±0.037 and 0.942±0.303,respectively,all P＜0.05). Key words: INS-1 cells; Glucose; Globular adiponectin; AMP-activated protein kinase

The effects of adiponectin on interleukin-6 and MCP-1 secretion in lipopolysaccharide-treated 3T3-L1 adipocytes: Role of the NF-κB pathway

It was recently suggested that the transcription nuclear factor-kappaB (NF-kappaB) plays an important role in controlling the inflammation and metabolic alterations associated with obesity. In endothelial and monocytic cells, adiponectin acts as a modulator of the inflammatory response, suppressing NF-kappaB activation. The aim of this study was to assess the ability of different forms of adiponectin to modulate the inflammatory response in adipocytes. 3T3-L1 preadipocytes were cultured according to standard conditions. Fully differentiated adipocytes were stimulated with 1 microg/ml lipopolysaccharides (LPS) for 16 h, with or without pre-treatment with 10 microg/ml of globular (AdG) or full-length (AdFl) adiponectin. Both AdG and AdFl significantly suppressed LPS-induced expression of IL-6 mRNA in adipocytes and reduced the concentration of IL-6 in culture media. Adiponectin pre-treatment significantly reduced the increase in MCP-1 mRNA in adipocytes exposed to LPS. In culture media, the increase in MCP-1 detected after LPS stimulation was significantly attenuated after pre-treatment with AdG. In 3T3-L1, AdG and AdFl reduced NF-kappaB activity by 50 and 40%, respectively compared to the NF-kappaB activation induced by LPS alone. Moreover, both forms of adiponectin significantly attenuated IkappaB-alpha as well as IKK gene expression. Pre-treatment of adipocytes with AdG or AdFl significantly increased PPARgamma mRNA levels, taking its expression back to the basal level. Both AdG and AdFl exert anti-inflammatory activity suppressing IL-6 and MCP-1 production from inflamed adipocytes. This anti-inflammatory action may be mediated through inhibition of NF-kappaB activity as well as through increased PPARgamma expression.

Adiponectin directly improves endothelial dysfunction in obese rats through the AMPK–eNOS Pathway

Hypoadiponectinemia has been proved to be closely related to endothelial dysfunction in peripheral arteries and is thought to be an independent risk factor for cardiovascular disease. The objective of this study was to investigate whether adiponectin might independently improve endothelial dysfunction in aorta isolated from high-fat-diet-induced obese. Sprague-Dawley rat and to study the mechanism involved. Male Sprague-Dawley rats were fed with a regular or a high-fat diet for 6 weeks. The aorta was isolated, and vascular segments were incubated with vehicle or the globular adiponectin (globular domain (gAD); 2 mg ml(-1)) for 2 h. The effect of gAD on endothelial function and nitric oxide (NO) production was determined. Human aortic endothelial cells in primary culture were treated with vehicle or gAD (4 mg ml(-1)). The effect of gAD on the level of phosphorylation of endothelial nitric oxide synthase (eNOS) at Ser1177, AMPK at Thr176 and Akt at Ser473 in endothelial cells were determined. Severe endothelial dysfunction was observed in high-fat diet fed rat aortic segments. After gAd incubation, the endothelium-dependent relaxation was partly improved and total production of nitric oxide as result of enhanced eNOS activity was also increased. In the cultured endothelial cell line HUVEC, globular adiponectin increased the activity of eNOS through activating AMPK by stimulating its phosphorylation at Thr176 but not Akt. The demonstration in the current study that adiponectin reverses endothelial dysfunction through increasing NO production by eNOS phosphorylation, and decreasing NO inactivation by blocking superoxide production provides a new direction in the prevention of vascular injury in the obesity population.

Globular adiponectin inhibits angiotensin II‐induced nuclear factor κB activation through AMP‐activated protein kinase in cardiac hypertrophy

Activation of nuclear factor kappaB (NF-kappaB) has been found necessary for cardiac hypertrophic growth in vivo and in vitro experiments. Adiponectin, an adipocyte-derived polypeptide, suppresses cardiac hypertrophy in response to pressure overload. Here we investigated the potential effect of adiponectin on NF-kappaB activation in hypertrophic neonatal rat ventricular myocytes (NRVMs) and related signal transduction pathway. We treated NRVMs with globular adiponectin (gAd) before angiotensin II (AngII) stimulation. Pretreating cells with gAd reduced the increased incorporation of [(3)H]-leucine and the mRNA levels of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) stimulated by AngII, indicating gAd inhibited AngII-induced cardiac hypertrophic signaling. Moreover, gAd pretreatment suppressed inhibitory protein kappaB (I-kappaB) phosphorylation and decreased p65 nuclear translocation, DNA-binding and transcription activity of NF-kappaB. Meanwhile, gAd promoted AMP-activated protein kinase (AMPK) phosphorylation, which is a downstream signaling mediator of adiponectin. Pharmacological activator of AMPK could inhibit AngII-induced NF-kappaB translocation, and inhibitor of AMPK or a dominant-negative AMPK adenovirus suppressed gAd-mediated inhibition of I-kappaB phosphorylation and NF-kappaB activation. When AMPK was inhibited, the suppressive effect of gAd on ANP mRNA expression was reduced. Our data indicate that gAd inhibits cardiac hypertrophic signaling through AMPK mediated suppression of NF-kappaB activation.

Induction of gene expression in response to globular adiponectin in vascular endothelial cells

Aims Adiponectin is an adipocyte-specific protein that plays an important regulatory role in the development or prevention of diabetes and atherosclerosis. Main methods In the present study, we examined the effect of a proteolytic cleavage product of adiponectin, known as globular adiponectin (gAd), on induction of gene expression and activation of various signaling pathways in vascular endothelial cells. Key findings We showed that gAd induces the expression of a number of genes using PCR arrays, including MCP-1, VCAM-1, E-selectin, IL-6, and IL-8, all of which have been previously shown to be associated with adiponectin, as well as SOD2, PAI-1, and CSF2, which is a new finding. We also demonstrated that gAd activates AMPK, Akt, and NF-kB, as well as various MAPKs, including ERK1/2, JNK, and p38MAPK. Significance Upstream regulation of gene expression might involve two or more activated pathways which interact with one another.

Anti-inflammatory activity of a globular adiponectin function on RAW 264 cells stimulated by lipopolysaccharide fromAggregatibacter actinomycetemcomitans

Adiponectin is an adipokine with potent anti-inflammatory properties. We previously reported that a globular adiponectin (gAd) suppresses Aggregatibacter actinomycetemcomitans lipopolysaccharide-induced nuclear factor-kappaB activity, suggesting an anti-inflammatory effect of gAd. In this study, we investigated whether gAd is able to modulate the effect of A. actinomycetemcomitans lipopolysaccharide on cytokine induction in a murine macrophage cell line (RAW 264). The phosphorylation of p38 mitogen-activated protein kinase, c-Jun N-terminal kinase, extracellular signal-regulated kinase, and IkappaB kinase alpha/beta and the degradation of IkappaB, which were induced by A. actinomycetemcomitans lipopolysaccharide intoxication, were clearly reduced in gAd-pretreated RAW 264 cells compared with the untreated cells. Expression levels of tumor necrosis factor (TNF)-alpha and interleukin-10 (IL-10) mRNA were assessed by real-time PCR. Cell-free supernatants were collected after 12 h of stimulation and analyzed by enzyme-linked immunosorbent assay for TNF-alpha and IL-10. Pretreatment with gAd significantly inhibited the A. actinomycetemcomitans lipopolysaccharide-induced TNF-alpha mRNA expression and protein secretion. In contrast, pretreatment with gAd significantly enhanced the A. actinomycetemcomitans lipopolysaccharide-induced IL-10 mRNA expression and protein secretion. These data suggest a mechanism for the anti-inflammatory activity of gAd in local inflammatory lesions, such as periodontitis.

Leptin prevents the metabolic effects of adiponectin in L6 myotubes

Adiponectin and leptin are negatively and positively correlated with human obesity respectively, and have both been shown to regulate energy metabolism in skeletal muscle. However, little is known about their signalling and functional crosstalk. Here we investigated the effects of leptin on metabolic actions of (1) globular adiponectin (gAd) and (2) full-length adiponectin (fAd) in L6 cells. Glucose uptake was measured upon gAd and fAd treatment after incubation with different doses (0.3, 0.6, 3, 6, 60 nmol/l) of leptin for 6, 12 and 24 h. We also measured adiponectin receptor (ADIPOR) expression and stimulation of downstream signalling by gAd and fAd using co-immunoprecipitation and western blotting following leptin pretreatment, as well as analysis of fatty acid uptake and oxidation using radiolabelled tracers. Leptin attenuated the stimulation of glucose uptake by gAd and fAd in a dose- and time-dependent manner, a finding correlated with decreased levels of ADIPOR1 and ADIPOR2. gAd and fAd increased palmitate uptake via activation of AMP protein kinase (T172), enhanced expression of the fatty acid transporter CD36, phosphorylated acetyl-CoA carboxylase (S79) and enhanced palmitate oxidation, all of which were attenuated by leptin pretreatment. Adiponectin can also enhance insulin sensitivity via direct signalling crosstalk; here we show that enhanced insulin-stimulated IRS-1 (Y612) and Akt (T308) phosphorylation in response to fAd was attenuated by leptin. APPL1 was recently identified as a critical mediator of adiponectin action in skeletal muscle. We demonstrated that leptin attenuated binding of APPL1 to LKB1, a downstream target leading to AMPK phosphorylation. The direct metabolic and insulin-sensitising effects of adiponectin were attenuated in the presence of leptin.

Effect of globular adiponectin on the function of NIT-I cells under high glucose medium

The effect of globular adiponectiin (gAd)on the function of NIT-1 cells under high glucose medium was investigated. The results showed that gad could completely block the increase of NADPH oxidase components p47phox expression and recover mRNA expression of pancreatic duodenal homeobox-I ,paired box gene 6,glucose transpoter 2,and glucokinase except neurogenic differentiation factor 1 (P<0.05 or P<0.01). Whereas,impaired insulin secretion and mRNA expression at high glucose concentration were not significantly improved by gAd. Key words: NIT-I cell lines; Global adiponectin; NAD(P) H oxidase

Adiponectin decreases pyruvate dehydrogenase kinase 4 gene expression in obese‐ and diabetic‐derived myotubes

To investigate the effects of globular adiponectin (gAd) on gene expression and whether these effects are mediated through 3',5'-cyclic monophosphate-activated protein kinase in skeletal muscle myotubes obtained from lean, obese and obese diabetic individuals. Rectus abdominus muscle biopsies were obtained from surgical patients to establish primary skeletal muscle cell cultures. Three distinct primary cell culture groups were established (lean, obese and obese diabetic; n = 7 in each group). Once differentiated, these cultures were then exposed to gAd or 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR) for 6 h. Stimulation with gAd decreased pyruvate dehydrogenase kinase 4 (PDK4) gene expression in the obese and diabetic samples (p < or = 0.05) and increased cytochrome c oxidase (COX) subunit 4 (COXIV) gene expression in the myotubes derived from lean individuals only (p < 0.05). AICAR treatment also decreased PDK4 gene expression in the obese- and diabetic-derived myotubes (p < or = 0.05) and increased the gene expression of the mitochondrial gene, COXIII, in the lean-derived samples only (p < 0.05). This study demonstrated distinct disparity between myotubes derived from lean compared with obese and obese diabetic individuals following gAd and AICAR treatment. Further understanding of the regulation of PDK4 in obese and diabetic skeletal muscle and its interaction with adiponectin signalling is required as this appears to be an important early molecular event in these disease states that may improve blood glucose control and metabolic flux.

Adiponectin Promotes Endotoxin Tolerance in Macrophages by Inducing IRAK-M Expression

High levels of plasma adiponectin are associated with low levels of inflammatory markers and cardioprotection. The mechanism via which adiponectin exerts its anti-inflammatory effect is yet unknown. In the present study, we demonstrate that globular adiponectin (gAd) induces the expression of the inactive isoform of IL-1R-associated kinases (IRAK), IRAK-M. Homologous deletion of IRAK-M in IRAK-M(-/-) mice abolished the tolerogenic properties of gAd because pretreatment of IRAK-M(-/-) macrophages with gAd did not suppress LPS-induced proinflammatory cytokine production. GAd activated the MAPKs MEK1/2 and ERK1/2 in macrophages via their upstream regulator Tpl2. Activation of ERK1/2 via Tpl2 appeared necessary for the induction of IRAK-M because gAd did not induce IRAK-M in Tpl2(-/-) macrophages or in macrophages pretreated with the MEK1/2 inhibitor UO126. In addition, activation of PI3K and Akt1 also appeared necessary for the induction of IRAK-M by gAd, because treatment of Akt1(-/-) macrophages or pretreatment of macrophages with the PI3K inhibitor wortmannin abolished gAd-induced IRAK-M expression. Analysis of IRAK-M expression in human peripheral blood cells confirmed that serum adiponectin was negatively associated with IRAK-M and responsiveness to LPS. In conclusion, our data demonstrate that IRAK-M is a major mediator of gAd-induced endotoxin tolerance in primary macrophages, expression of which depends on the activation of Tpl2/ERK and PI3K/Akt1 signaling pathways.

Adiponectin induces interleukin‐6 production and activates STAT3 in adult mouse cardiac fibroblasts

APN (adiponectin), an adipocyte-derived cytokine highly presented in serum, which exerts antidiabetic, anti-atherosclerotic and cardioprotective actions, also enhances CFB (cardiac fibroblast) proliferation and protects against cardiac fibrosis. STAT3 (signal transducer and activator of transcription 3), a major mediator in the gp130/JAK2 (Janus kinase 2)/STATs signalling pathway, plays a critical role in cardioprotective events. Almost two-thirds of total myocardial cells are CFBs; however, whether APN regulates STAT3 signalling pathway has not been clarified yet in CFBs. In the present study, we investigated the effect of recombinant globular APN on the STAT3 activity in adult mouse CFBs and explored the possible signalling transduction mechanism. In cultured CFBs, APN (10 microg/ml) can significantly induce delayed STAT3 Tyr(705) phosphorylation time-dependently, up to 60 min, and mediate STAT3 translocation from cytoplasm to nucleus. Transfection of siRNA (small interfering RNA) specific for AdipoR1 (APN receptor 1), but not AdipoR2, obviously inhibited APN-induced STAT3 Tyr(705) phosphorylation, indicating that AdipoR1, not AdipoR2, is required for STAT3 phosphorylation. Both inhibition of gp130 by anti-gp130 neutralizing antibody and JAK2 by AG490 (a specific inhibitor for JAK2) can inhibit APN-induced STAT3 phosphorylation and STAT3 transcription activity detected using 2 x pAPRE-Luc (APRE reporter) assay. Furthermore, we found that the IL (interleukin)-6 level in culture medium was significantly increased after stimulation with APN and the IL-6 mRNA level was also markedly increased in CFBs, which can be reversed by siRNA for AdipoR1, but not for AdipoR2, and that anti-IL-6 neutralizing antibody can significantly inhibit APN-induced STAT3 Tyr(705) phosphorylation. APN induces IL-6 production mediated by AdipoR1, not AdipoR2, in adult mouse CFBs, which leads to the stimulation of the gp130/JAK signalling pathway, and as a result causes STAT3 activation.

Globular adiponectin induces differentiation and fusion of skeletal muscle cells

The growing interest in skeletal muscle regeneration is associated with the opening of new therapeutic strategies for muscle injury after trauma, as well as several muscular degenerative pathologies, including dystrophies, muscular atrophy, and cachexia. Studies focused on the ability of extracellular factors to promote myogenesis are therefore highly promising. We now report that an adipocyte-derived factor, globular adiponectin (gAd), is able to induce muscle gene expression and cell differentiation. gAd, besides its well-known ability to regulate several metabolic functions in muscle, including glucose uptake and consumption and fatty acid catabolism, is able to block cell cycle entry of myoblasts, to induce the expression of specific skeletal muscle markers such as myosin heavy chain or caveolin-3, as well as to provoke cell fusion into multinucleated syncytia and, finally, muscle fibre formation. gAd exerts its pro-differentiative activity through redox-dependent activation of p38, Akt and 5'-AMP-activated protein kinase pathways. Interestingly, differentiating myoblasts are autocrine for adiponectin, and the mimicking of pro-inflammatory settings or exposure to oxidative stress strongly increases the production of the hormone from differentiating cells. These data suggest a novel function of adiponectin, directly coordinating the myogenic differentiation program and serving an autocrine function during skeletal myogenesis.

The Cardioprotective Effects of Adiponectin in the ZDF Heart Following Ischemia Are Associated with a Significant Attenuation of Myocardial ICAM‐1 Expression

Ischemic heart disease (IHD) is significantly enhanced in type 2 diabetes (T2D) and inflammation is associated with IHD and T2D. The adipocytokine, adiponectin (APN), which has anti‐inflammatory actions, is chronically reduced in type 2 diabetes. APN may link inflammation, T2D and IHD. We examined if a globular form of adiponectin was cardioprotective in the Zucker Diabetic Fatty (ZDF) rodent model of T2D and if the cardioprotection was associated with myocardial expression of ICAM‐1, a key adhesion molecule and marker of coronary microvascular dysfunction. ZDF (n=14) rats and their lean controls ZLC (n=13) were subjected to 30 minutes of LAD occlusion and 120 minutes of reperfusion. Animals were treated with either PBS or recombinant globular adiponectin (1.0 μg/g). At R120, the extent of myocardial infarction was measured. Myocardial ICAM‐1 expression was also quantified immunohistochemically. We found that adiponectin treatment significantly attenuated the enhanced myocardial infarction in the ZDF model of T2D (p&lt;0.05). The reduced infarction was associated with a significant reduction in myocardial ICAM‐1 expression (p&lt;0.05). These findings suggest that adiponectin treatment protects against I/R injury in the T2D heart. The cardioprotective action may be related to reduced coronary microvascular inflammation enhancing PMN sequestration and PMN‐mediated tissue injury. Supported by NIH HL 58859.

Involvement of Ca2+ in globular adiponectin-induced reactive oxygen species

Globular adiponectin (gAd) induces the generation of reactive oxygen species (ROS) and nitric oxide (NO) in the murine macrophage cell line RAW 264. We investigated the role of Ca2+ in gAd-induced ROS and NO generation. Pretreatment with BAPTA-AM, a selective chelator of intracellular Ca2+ ([Ca2+]i), partially reduced gAd-induced generation of ROS and NO in gAd-treated RAW 264 cells. The lowest [Ca2+]i occurred 30min after gAd treatment, after which [Ca2+]i increased continually and exceeded the initial level. The mitochondrial Ca2+ ([Ca2+]m) detected by Rhod-2 fluorescence started to increase at 6h after gAd treatment. Pretreatment with a NAD(P)H oxidase inhibitor, diphenyleneiodonium, prevented the reduction of [Ca2+]i in the early phase after gAd treatment. Calcium depletion by BAPTA-AM had no effect on the gAd-induced [Ca2+]m oscillation. The administration of a specific calmodulin inhibitor, calmidazolium, significantly suppressed gAd-induced ROS and NO generation and NOS activity.

Globular adiponectin-induced RAW 264 apoptosis is regulated by a reactive oxygen species-dependent pathway involving Bcl-2

Globular adiponectin (gAd), a truncated form of adipocyte-derived cytokine, stimulates RAW 264 cells to produce reactive oxygen species (ROS), which trigger an apoptotic cascade. In this study, we investigated the generation of intracellular and mitochondrial ROS in gAd-stimulated RAW 264 cells. Treatment with gAd efficiently induced the generation of intracellular and mitochondrial ROS, as detected by dichlorodihydrofluorescein diacetate and MitoSOX fluorescence, respectively. Furthermore, gAd treatment significantly increased 8-oxoguanine, a specific indicator of oxidative DNA damage. The transfection of RAW 264 cells with iNOS- and gp91 phox-specific small interfering RNA reduced markedly the generation of intracellular, but not mitochondrial, ROS. Quantitative PCR revealed that the expression ratio of Bcl-2 to Bax was reduced in a time-dependent manner in gAd-treated RAW 264 cells. The overexpression of Bcl-2 markedly inhibited gAd-induced apoptosis in RAW 264 cells and also reduced both the intracellular and the mitochondrial ROS generation induced by gAd treatment. Moreover, the overexpression of Bcl-2 significantly suppressed gAd-induced NO secretion and NOS activity. In addition, the inhibition of NOS activity partially reduced the oxidative DNA damage induced by gAd. Taken together, these results demonstrate that the gAd-induced apoptotic pathway acting via ROS/RNS generation involves Bcl-2.