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Naomi Takeichi,
Sanae Midorikawa,
Atsushi Watanabe,
Banyar Than Naing,
Hideki Tamura,
Toshiko Wakakuri-Kano,
Akira Ishizaki,
Hitoshi Sugihara,
Sumiko Nissato,
Yuria Saito, [......],
Hisato Hara,
Tatsuhiko Ikeda,
Kazuo Shimizu,
Shinichi Suzuki,
Hitoshi Shimano,
Masashi Kawamoto,
Takashi Shimada,
Tsuyoshi Watanabe,
Shinichi Oikawa,
Kazuhiro Takekoshi
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ABSTRACT: Sunitinib is an oral multitargeted receptor tyrosine kinase inhibitor with antiangiogenic and antitumor activity that mainly targets vascular endothelial growth factor receptors, and recently, it has been shown to be an active agent for the treatment of malignant pheochromocytomas. Previously, we demonstrated that sunitinib directly inhibited mTORC1 signaling in rat pheochromocytoma PC12 cells. Although autophagy is a highly regulated cellular process, its relevance to cancer seems to be complicated. It is of note that inhibition of mTORC1 is a prerequisite for autophagy induction. Indeed, direct mTORC1 inhibition initiates ULK1/2 autophosphorylation and subsequent Atg13 and FIP200 phosphorylation, inducing autophagy. Here, we demonstrated that sunitinib significantly increased the levels of LC3-II, concomitant with a decrease of p62 in PC12 cells. Following sunitinib treatment, immunofluorescent imaging revealed a marked increased punctate LC3-II distribution. Furthermore, Atg13 knockdown significantly reduced its protein level, which in turn abolished sunitinib-induced autophagy. Moreover, inhibition of autophagy by siRNAs targeting Atg13 or by pharmacological inhibition with ammonium chloride, enhanced both sunitinib-induced apoptosis and anti-proliferation. Thus, sunitinib-induced autophagy is dependent on the suppression of mTORC1 signaling and the formation of ULK1/2-Atg13-FIP200 complexes. Inhibition of autophagy may be a promising therapeutic option for improving the anti-tumor effect of sunitinib.
Journal of Pharmacological Sciences 12/2012; · 2.08 Impact Factor
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ABSTRACT: Sunitinib is an oral, small molecule multitargeted receptor tyrosine kinase inhibitor with antiangiogenic and antitumor activity that primarily targets vascular endothelial growth factor receptors (VEGFRs). Although sunitinib is an active agent for the treatment of malignant pheochromocytomas, it is unclear whether sunitinib acts through only antiangiogenic mechanisms or also directly targets tumor cells. We previously showed that sunitinib directly induced apoptosis of PC-12 cells. To further confirm these direct effects, we examined the effects of sunitinib on tyrosine hydroxylase (TH) (the rate-limiting enzyme in catecholamine biosynthesis) activity and catecholamine secretion in PC-12 cells and the underlying mechanisms. Sunitinib inhibited TH activity in a dose-dependent manner, and decreased TH protein levels. Consistent with this finding, sunitinib decreased TH phosphorylation at Ser(31) and Ser(40) and significantly decreased catecholamine secretion. VEGFR-2 knockdown attenuated these effects, including inhibition of TH activity and catecholamine secretion, suggesting that they were mediated by VEGFR-2. Sunitinib significantly decreased phospholipase C (PLC)-γ phosphorylation and subsequent protein kinase C (PKC) activity. Because Ser(40) phosphorylation significantly affects TH activity and is known to be regulated by PKC, sunitinib may inhibit Ser(40) phosphorylation via the VEGFR-2/PLC-γ/PKC pathway. Additionally, sunitinib markedly decreased the activity of extracellular signal-regulated kinase (ERK), but not c-Jun NH(2)-terminal kinase or p38 mitogen-activated protein kinase. Therefore, sunitinib may reduce TH Ser(31) phosphorylation through inhibition of the VEGFR-2/PLC-γ/PKC/Raf/mitogen-activated protein kinase/extracellular signal-regulated kinase kinase/ERK pathway. Sunitinib also significantly reduced inositol 1,4,5-trisphosphate production. However, because PC-12 cells do not precisely reflect the pathogenesis of malignant cells, we confirmed the key findings in a human neuroblastoma cell line, SK-N-SH. In conclusion, sunitinib directly inhibits catecholamine synthesis and secretion in pheochromocytoma PC-12 cells.
AJP Endocrinology and Metabolism 08/2012; 303(8):E1006-14. · 4.75 Impact Factor
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Naomi Takeichi,
Sanae Midorikawa,
Atsushi Watanabe,
Banyar Than Naing,
Hideki Tamura,
Toshiko Wakakuri-Kano,
Akira Ishizaki,
Hitoshi Sugihara,
Sumiko Nissato,
Yuria Saito, [......],
Hisato Hara,
Tatsuhiko Ikeda,
Kazuo Shimizu,
Shinichi Suzuki,
Hitoshi Shimano,
Masashi Kawamoto,
Takashi Shimada,
Tsuyoshi Watanabe,
Shinichi Oikawa,
Kazuhiro Takekoshi
[show abstract]
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ABSTRACT: Recently, TMEM127 was shown to be a new pheochromocytoma susceptibility gene; this is consistent with its function as a tumour suppressor gene (Journal of Clinical Endocrinology and Metabolism, 2009, 94, 2817). Most pheochromocytomas arise from the adrenal medulla, and in approximately half of the cases, the tumours are bilateral (Journal of Clinical Endocrinology and Metabolism, 2009, 94, 2817; Journal of the American Medical Association, 2004, 292, 943; Human Mutation, 2010, 31, 41; Science, 2009, 325, 1139). The aim of the present study was to determine whether TMEM127 mutations are involved in the pathogenesis of pheochromocytomas/paragangliomas in Japanese subjects.
For this study, 74 unrelated patients with pheochromocytoma/paraganglioma who tested negative for mutations and deletions in RET, VHL, SDHB and SDHD were recruited through a multi-institutional collaborative effort in Japan. The TMEM127 gene sequence was determined in their germline DNA, and tumour DNA was analysed for the loss of heterozygosity. In addition, their TMEM127 gene sequences were compared with sequences from 114 normal healthy, ethnically matched controls.
Among the 74 eligible patients, two unrelated patients (2·7%) with bilateral adrenal pheochromocytoma were found to have an identical germline TMEM127 mutation (c.116_119delTGTC, p.Ile41ArgfsX39) associated with 2q deletion loss of heterozygosity, which was also previously described in a Brazilian case (Journal of the American Medical Association, 2004, 292, 943). We also determined that none of the 114 normal healthy controls had this deletion mutation.
This is the first report showing that TMEM127 mutation plays a pathological role in pheochromocytoma in an Asian population. Although our surveillance is limited, the prevalence and the phenotype of this gene mutation appear to be similar to those reported in previous studies.
Clinical Endocrinology 04/2012; 77(5):707-14. · 3.17 Impact Factor
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Nihon Naika Gakkai Zasshi 04/2012; 101(4):949-58.
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[show abstract]
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ABSTRACT: Sunitinib is an oral multitargeted receptor tyrosine kinase inhibitor with antiangiogenic and antitumor activity that mainly targets vascular endothelial growth factor receptors (VEGFRs). Very recently, sunitinib has been shown to be an active agent for the treatment of malignant pheochromocytomas. However, it is unclear whether sunitinib acts only through an antiangiogenic mechanism or whether it may also directly target tumor cells. Sunitinib markedly induced apoptosis of PC12 cells in a dose-dependent and time-dependent manner. Furthermore, in support of these findings, we found that sunitinib induced a reduction in the expression of the antiapoptotic molecule Bcl-2 as well as dephosphorylation of the proapoptotic molecule BAD, which results in the activation of BAD in these cells. Consistent with these apoptotic effects, our results showed that sunitinib inhibited phosphorylation of Akt and mTOR and was followed by a reduction of S6K1, which is a well-known target of mTOR. Knockdown of VEGFR-2 attenuated the sunitinib-induced effects, including apoptosis and inhibition of signaling pathways such as the phosphorylation of Akt as well as mTOR, and Bcl-2, which confirmed that these effects could be mediated by VEGFR-2. In addition, silencing of S6K1 induced apoptosis accompanied by a decrease in the phosphorylation of BAD and Bcl-2, similar to that observed with sunitinib treatment. Thus, these results together suggest that sunitinib initially exerts its apoptotic effect through the inhibition of VEGFR-2, which, when followed by reduction of its downstream effectors, including Akt/mTOR/S6K1, may lead to inhibition of the antiapoptotic molecule Bcl-2 and activation of the proapoptotic molecule BAD in PC12 cells. However, PC12 cells do not precisely reflect the pathogenesis of malignant cells. Therefore, we confirmed the key findings by replicating these experiments in human neuroblastoma SK-N-SH cells.
AJP Endocrinology and Metabolism 08/2011; 302(6):E615-25. · 4.75 Impact Factor
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Nippon rinsho. Japanese journal of clinical medicine 03/2011; 69 Suppl 2:511-4.
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Yasushi Kawakami
Nippon rinsho. Japanese journal of clinical medicine 07/2010; 68 Suppl 7:443-6.
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ABSTRACT: Recently, mutations in nuclear genes encoding two mitochondrial complex II subunit proteins, Succinate dehydrogenase D (SDHD) and SDHB, have been found to be associated with the development of familial pheochromocytomas and paragangliomas (hereditary pheochromocytoma/paraganglioma syndrome: HPPS). Growing evidence suggests that the mutation of SDHB is highly associated with abdominal paraganglioma and the following distant metastasis (malignant paraganglioma). In the present study, we used multiplex ligation dependent probe amplification (MLPA) analysis to identify a large heterozygous SDHB gene deletion encompassing sequences corresponding to the promoter region, in addition to exon 1 and exon 2 malignant paraganglioma patient in whom previously characterized SDHB mutations were undetectable. This is the first Japanese case report of malignant paraganglioma, with a large SDHB deletions. Our present findings strongly support the notion that large deletions in the SDHB gene should be considered in patients lacking characterized SDHB mutations.
Endocrine Journal 04/2010; 57(4):351-6. · 2.03 Impact Factor
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ABSTRACT: Urotensin II (U-II), initially identified as a cyclic peptide from fish urophysis, acts both as a strong vasoconstrictor and vasodilator in the vasculature via its receptor, G-protein coupled receptor 14. In addition, U-II and its receptor are co-expressed in the adrenal medulla, as well as in human pheochromocytomas, suggesting that this peptide may have some function in chromaffin cells. However, the precise role of U-II in these cells is unknown. In the present study, we initially demonstrate that U-II and its receptors mRNA are co-expressed in the rat pheochromocytoma cell line PC12. Moreover, U-II has not effect on tyrosine hydroxylase (TH), the rate-limiting enzyme involved in the biosynthesis of catecholamine, in terms of enzyme activity or at the mRNA level. However, U-II does induce an increase in the phosphorylation of TH specifically at Ser31 without affecting phosphorylation at the two other sites (Ser19 and Ser40). U-II also markedly activates extracellular signal-regulated kinases (ERKs) and p38, but not Jun N-terminal kinase. Blockade of the epidermal growth factor (EGF) receptor by AG1478 significantly reduces activation of ERK, suggesting that EGF receptor transactivation could act upstream of the ERK pathway in PC12 cells. Furthermore, U-II significantly increases dopamine secretion from PC12 cells. Finally, we show that U-II induced significant DNA synthesis in a ERKs and P38 mitogen-activated protein kinase-dependent manner. The results obtained indicate that U-II may exert its effects as a neuromodulator in chromaffin cells.
Journal of Neuroendocrinology 12/2009; 22(2):83-91. · 3.14 Impact Factor
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Kazumasa Isobe,
Ling Fu,
Ichirou Tatsuno,
Hideto Takahashi,
Sumiko Nissato,
Hisato Hara,
Toru Yashiro,
Kazumi Suzukawa,
Kazuhiro Takekoshi,
Hitoshi Shimano, Yasushi Kawakami
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ABSTRACT: Recent studies have demonstrated that serum adiponectin and its receptors in adipose and muscle tissues are suppressed in diabetic or obese individuals. Patients with pheochromocytoma are frequently diabetic.
Using real-time PCR, we examined mRNA expressions of adiponectin (Adp) and adiponectin receptor 1 (AdpR1) and AdpR2 in pheochromocytoma tissues from 49 patients. We also measured levels of serum total and high molecular weight (HMW) adiponectin levels in 10 pheochromocytomas and 33 normal volunteers.
In pheochromocytoma tissue, AdpR1 mRNA expression was higher in adrenaline (A)-type tumors than in noradrenaline (NA)-type tumors. AdpR1 expression was significantly higher in A-type non-diabetics than in NA-type non-diabetics (p<0.05). AdpR1 mRNA expression was significantly associated with the tumor tissue adrenaline content (p<0.005) in linear regression analysis, which suggest that adrenaline positively regulates AdpR1 mRNA expression.Serum total and HMW Adp levels in patients with NA-type pheochromocytomas were approximately 3 times higher than those of healthy volunteers. After adrenalectomy, levels of adiponectin normalized.
Our results indicate that serum total and HMW Adp, and AdpR1 gene expressions in pheochromocytoma tissue, are associated with the level of catecholamine produced in the tumor. It is tempting to speculate that catecholamine induces adiponectin production and signaling.
Journal of atherosclerosis and thrombosis 09/2009; 16(4):442-7. · 2.69 Impact Factor
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Tomohito Saito,
Yukihito Saito,
Koichiro Matsumura,
Yu Tsubota,
Tomohiro Maniwa,
Hiroyuki Kaneda,
Ken-ichiro Minami,
Noriko Sakaida,
Yoshiko Uemura,
Gen Kawa,
Nae Yamamoto,
Yoshimitsu Fujii,
Kazumasa Isobe, Yasushi Kawakami,
Tadashi Matsuda,
Kazuhiro Takekoshi
[show abstract]
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ABSTRACT: Recently, nuclear genes encoding two mitochondrial complex II subunit proteins, SDHD and SDHB, have been found to be associated with the development of familial pheochromocytomas and paragangliomas (hereditary pheochromocytoma/paraganglioma syndrome: HPPS). Growing evidence suggests that the mutation of SDHB is highly associated with abdominal paraganglioma and the following distant metastasis (malignant paraganglioma). In the present study, we report the case of a novel SDHB mutation (L157X) in a Japanese patient with abdominal paraganglioma following malignant lung metastasis. In addition, we identified an asymptomatic carrier of the SDHB mutation in this family.
Endocrine Journal 04/2009; 56(3):451-8. · 2.03 Impact Factor
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Nihon Naika Gakkai Zasshi 11/2008; 97(10):2558-65.
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ABSTRACT: Recently, nuclear genes encoding two mitochondrial complex II subunit proteins, SDHD and SDHB, have been found to be associated with the development of familial pheochromocytomas and paragangliomas (hereditary pheochromocytoma/paraganglioma syndrome: HPPS). Growing evidence suggests that a mutation of SDHB is highly associated with abdominal (or thoracic) paraganglioma and the following distant metastasis (malignant paraganglioma). Previously, we identified a novel heterozygous G to A point mutation at the first base of intron 3 of the SDHB gene (IVS3+1G>A) in a malignant abdominal paraganglioma from a Japanese patient. In the present study, we report another case of SDHB mutation (R46Q) in a Japanese patient with both abdominal and thoracic paraganglioma following malignant metastasis. In addition, we identified an asymptomatic carrier of SDHB mutation in this family. Our report highlights the pathogenic role of the SDHB mutation (R46Q) in malignant paraganglioma. We also discuss the desired protocol that should be adopted to follow up an asymptomatic carrier of this mutation.
Endocrine Journal 06/2008; 55(2):299-303. · 2.03 Impact Factor
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Manabu Kusakabe,
Kazumi Suzukawa,
Toru Nanmoku,
Naoshi Obara,
Yasushi Okoshi,
Harumi Y Mukai,
Yuichi Hasegawa,
Hiroshi Kojima, Yasushi Kawakami,
Haruhiko Ninomiya,
Toshiro Nagasawa
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ABSTRACT: Acute promyelocytic leukemia (APL) is characterized by chromosomal rearrangements of 17q21, leading to fusion of the gene-encoding retinoic acid receptor alpha (RARA) with a number of alternative partner genes. Signal transducer and activator of transcription 5 beta (STAT5B) is one of the alternative partners. We report a rare case of APL with STAT5B-RARA fusion transcript and the normal chromosome 17 on G-banding. Administration of all trans-retinoic acid improved disseminated intravascular coagulation without decrease of the leukemia cells in his peripheral blood and bone marrow. The molecular mechanism of fusion between STAT5B and RARA by chromosomal rearrangement is discussed based on the data from genome database. Clinical characteristics of APL with STAT5B-RARA are also discussed.
European Journal Of Haematology 06/2008; 80(5):444-7. · 2.61 Impact Factor
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ABSTRACT: We investigated the effects of beta(3)-adrenoceptor agonist, 5-[(2R)-2-[[(2R)-2-(3-chlorophenyl)-2-hydroxyethyl]amino]propyl]-1,3-benzodioxole-2,2-dicarboxylate (CL-316,243) in obese diabetic KKAy mice. Two weeks' subcutaneous administration of CL-316,243 reduced serum levels of glucose, insulin, triglyceride, free fatty acid and tumor necrosis factor-alpha (TNF-alpha), and increased adiponectin. Adiponectin, adiponectin receptors and beta(3)-adrenoceptor mRNA expressions were reduced in epididymal white adipose tissue in KKAy mice, and CL-316,243 recovered these mRNA expressions. Meanwhile, CL-316,243 suppressed the overexpressed mRNA level of TNF-alpha in both epididymal white adipose tissue and brown adipose tissue. These data suggest that the normalization of adiponectin, adiponectin receptors and TNF-alpha may result in the amelioration of obesity-induced insulin resistance.
European Journal of Pharmacology 05/2008; 584(1):202-6. · 2.52 Impact Factor
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Yukitaka Katsura,
Kazumi Suzukawa,
Toru Nanmoku,
Noriko Nemoto,
Takayuki Machino,
Naoshi Obara,
Yasushi Okoshi,
Harumi Yamamoto Mukai,
Yuichi Hasegawa,
Hiroshi Kojima, Yasushi Kawakami,
Toshiro Nagasawa
Cancer Genetics and Cytogenetics 11/2007; 178(1):85-8. · 1.39 Impact Factor
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ABSTRACT: Adiponectin reportedly reduces insulin resistance. Exercise has also been shown to lessen insulin resistance, although it is not well known whether exercise increases levels of adiponectin and/or its receptors nor whether it effects are dependent on exercise intensity and/or period. We previously reported that blood adiponectin levels increased by 150% in animals that exercised at a rate of 30 m/min for 60 minutes, 2 days per week, and adiponectin receptor 1 (AdipoR1) mRNA levels in muscle increased up to 4 times in response to exercise at a rate of 25 m/min for 30 min, 5 days per week for 12 weeks.
In light of this information, we examined the effects of short-term exercise on adiponectin, and adiponectin receptor levels in rats, using ELISA and real-time PCR.
Our data showed that adiponectin mRNA levels in adipose tissue increased by 280% in rats exercised at a rate of 30 m/min for 60 minutes for 2 weeks and correlated with the exercise time periods. No effects of short-term exercise on adiponectin receptor 1 mRNA in muscle were observed.
Thus, long-term exercise may be required to regulate adiponectin receptor 1 mRNA expression in muscle and adiponectin mRNA expression in adipose tissue.
Journal of atherosclerosis and thrombosis 11/2007; 14(5):261-5. · 2.69 Impact Factor
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ABSTRACT: We have identified a novel fusion partner of MLL, namely the mastermind like 2 (MAML2 gene), in secondary acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) with inv(11)(q21q23). RT-PCR and sequencing revealed that exon 7 of MLL was fused to exon 2 of MAML2 in the AML and MDS cells. The inv(11)(q21q23) results in the creation of a chimeric RNA encoding a putative fusion protein containing 1,408 amino acids from the NH2-terminal part of MLL and 952 amino acids from the COOH-terminal part of MAML2. The NH2-terminal part of MAML2, a basic domain including a binding site of the intracellular domain of NOTCH, was deleted in MLL-MAML2. MLL-MAML2 in secondary AML/MDS and MECT1-MAML2 in mucoepithelioid carcinoma, benign Wartin's tumor, and clear cell hidradenoma consist of the same COOH-terminal part of MAML2. A luciferase assay revealed that MLL-MAML2 suppressed HES1 promoter activation by the NOTCH1 intracellular domain. MAML2 involving a chimeric gene might contribute to carcinogenesis in multiple neoplasms by the disruption of NOTCH signaling.
Genes Chromosomes and Cancer 10/2007; 46(9):813-9. · 3.31 Impact Factor
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ABSTRACT: Recently, the insulin-sensitizing adipokine adiponectin and the insulin resistance-inducing adipokine tumor necrosis factor-alpha (TNF-alpha) were reported to inhibit each other's production in adipocytes. We investigated the effects of two beta(3)-adrenoceptor agonists, 5-[(2R)-2-[[(2R)-2-(3-chlorophenyl)-2-hydroxyethyl]amino]propyl]-1,3-benzodioxole-2,2-dicarboxylate (CL-316,243) and (+/-)-(R(*),R(*))-[4-[2-[[2-(3-chlorophenyl)-2-hydroxyethyl]amino]propyl]phenoxy]acetic acid (BRL37344), on the gene expression of adiponectin, two adiponectin receptors, and TNF-alpha in adipose tissues of C57BL/6J mice. CL-316,243 and BRL37344 downregulated adiponectin, but upregulated adiponectin receptor 2 (not receptor 1) in epididymal or/and subcutaneous white adipose tissues and in brown adipose tissue. TNF-alpha expression was upregulated only in epididymal adipose tissue. To further explore these effects, we treated differentiated 3T3-L1 adipocytes with the non-selective beta-adrenoceptor agonist isoproterenol. As a result, adiponectin receptor 2 (but not receptor 1) gene expression and TNF-alpha protein expression increased, but gene expression and secretion of adiponectin decreased. The upregulation of adiponectin receptor 2 by isoproterenol is most likely via beta(2),beta(3)-adrenoceptors, adenylyl cyclases, and protein kinase A (PKA). However, the accompanying activation of AMP-activated protein kinase (AMPK) may inhibit this upregulation. Our results suggest that upregulation of TNF-alpha and downregulation of adiponectin by beta-adrenoceptor activation may contribute to the pathogenesis of catecholamine-induced insulin resistance, and that upregulation of adiponectin receptor 2 may be a feedback result of reduced adiponectin.
European Journal of Pharmacology 09/2007; 569(1-2):155-62. · 2.52 Impact Factor
