Acta Biochim Biophys Sin, 2016, 48(5), 485–486 doi: 10.1093/abbs/gmw015 Advance Access Publication Date: 29 March 2016 New Phenomenon
New Phenomenon
Role of Neu-p11/luzindole in the regulation of insulin signaling pathways and insulin resistance Xiuping Li1, Shichang Cai2,*, Weidong Yin3, Xiaobo Hu3, Sujun Zhang4, Zhengming Li1, Xing Li2, and Moshe Laudon5 1
Department of Laboratory, Hunan University of Medicine, Huaihua 418000, China, 2Basic Medical Sciences, Hunan University of Medicine, Huaihua 418000, China, 3Institute of Cardiovascular Disease, Key Laboratory Arteriosclerology of Hunan Province, University of South China, Hengyang 412000, China, 4Department of Experimental Animal, University of South China, Hengyang 412000, China, and 5Neurim Pharmaceuticals Ltd., Tel-Aviv 69710, Israel *Correspondence address. Tel: +86-745-2382481; Fax: +86-745-2382082; E-mail: [email protected]
The prevalence of Type 2 diabetes (T2D) has been globally increased since the last decade. T2D is a condition of relative insulin insufficiency, in which hyperglycemia develops when the insulin secretory capacity of β-cells is no longer sufficient to meet the insulin requirement in the setting of insulin resistance [1]. Previous studies have shown that there is a close correlation between T2D and insulin receptor substrate-1 (IRS-1) levels, and that lack of expression or abnormal phosphorylation of IRS-1 can lead to insulin resistance [2]. Phosphoinositide 3-kinase (PI3K) plays a key role in insulin signaling and its activity has been shown to be blunted in tissues from T2D subjects. PI3K activation is critical for insulin-mediated metabolic effects such as increased glucose uptake and glycogen synthesis [3]. Glycogen synthase kinase-3β (GSK3β), a downstream target of insulin signaling, is activated by phosphorylated ( p) Akt [4]. Phosphorylation by p-Akt inhibits the activity of GSK3β [5]. GSK3β phosphorylation and inactivation are considered to be important mechanisms of cell survival [6]. Melatonin (Mel) is a circulating hormone that is predominantly released from the pineal gland [7]. Some studies suggest that Mel may potentially play a role in diabetes and its associated metabolic disturbances by regulating insulin secretion. Luzindole is a nonspecific Mel receptor antagonist that can block some Mel functions [8]. Although Mel has an extensive range of biological effects, studies have revealed that it is rapidly metabolized with a half-life of 20–30 min once it gets ingested in humans. Therefore, the effect of Mel cannot be studied from direct administration of this drug. Moreover, extraction and synthesis of Mel is complicated and a high dose of Mel is associated with side effects. Neu-P11 is a novel type of nonselective agonist of Mel. It has several characteristics including the ease with which it can be synthesized in vitro and administered effectively for a longer time with fewer side effects. It can also substitute for Mel to interact with its receptors and consequently has an extensive range of biological actions [9]. In this study, we investigated the relationship between Mel and its receptor agonist Neu-P11, and the relationship between insulin
signaling pathway and the alteration of p-IRS-1/PI3K/p-GSK3β signaling in insulin-resistant 3T3-L1 adipocytes. Insulin-resistant 3T3-L1 adipocytes were utilized in this study to investigate the alteration of p-IRS-1/PI3K/p-GSK3β signaling activity following Neu-P11 treatment. 3T3-L1 pre-adipocytes were purchased from the Institute of Basic Medicine, Chinese Academy of Medical Sciences (Beijing, China). Neu-P11 was a kind gift from Neurim Pharmaceuticals Ltd (Habarzel, Israel). A 5 mM stock solution was prepared by dissolving Neu-P11 in dimethylsulfoxide. Cells were differentiated in adipogenic cocktail medium containing 3-isobutyl-1-methylxanthine (Sigma, St Louis, USA), insulin, and dexamethasone, and visualized by oil red O staining. Insulin-resistant 3T3-L1 adipocytes were cultured in Dulbecco’s modified Eagle’s medium (Gibco, Gaithersburg, USA) with high glucose/high insulin for 24 h. Glucose uptake was measured in the differentiated cells by an enzymatic method to assess the adipocyte model. Cells were divided into six groups: (i) insulin-resistant group, (ii) Mel group (cells were treated with 10 nM Mel for 6 h), (iii) Neu-P11 group (cells were treated with 10 nM Neu-P11 for 6 h), (iv) Mel + luzindole group (cells were treated with 10 nM Mel + 100 nM luzindole for 6 h), (v) Neu-P11 + luzindole group (cells were treated with 10 nM Neu-P11 + 100 nM luzindole for 6 h), and (vi) Mel + Neu-P11. Then all six groups were treated with 50, 100, and 200 nM insulin for 6 h to evaluate the effects of these drugs on adipocytes glucose uptake. Subsequently, cells were cultured with or without Mel, Neu-P11, or luzindole for 6 h and followed by a final incubation with different doses of insulin for 6 h. The expression levels of p-IRS-1, PI3K, and p-GSK3β were analyzed by western blot analysis using anti-p-IRS antibody (Cell Signaling Technology, Beverly, USA), anti-PI3K antibody (Santa Cruz, Santa Cruz, USA), and anti-p-GSK3β antibody (Santa Cruz), respectively. Our results showed that p-IRS-1, PI3K, and p-GSK3β expression levels were decreased significantly in insulin-resistant adipocytes compared with normal adipocytes (P < 0.05; Fig. 1); however, the
© The Author 2016. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. 485
486
Neu-p11/luzindole and insulin resistance
Figure 1. The changes of protein expression in different groups Adipocytes were cultured with and without Mel, Neu-P11, or luzindole for 6 h. IS, insulin sensitivity; IR, insulin resistance; M, IR + Mel; N, IR + Neu-P11; M + L, IR + Mel + luzindole; N + L, IR + Neu-P11 + luzindole. Protein expressions were analyzed by western blot analysis. (A) PI3K-P85. (B) p-GSK3β. (C) p-IRS-1. *P < 0.05 vs. IR; #P < 0.05 vs. M; $P < 0.05 vs. N. n = 3.
expression levels of p-IRS-1, PI3K, and p-GSK3β were increased significantly when insulin-resistant adipocytes were treated with Mel or Neu-P11 (P < 0.05; Fig. 1). In contrast, the changes were not significant in adipocytes when treated with the Mel + luzindole or Neu-P11 + luzindole, compared with the insulin-resistant group (P > 0.05; Fig. 1). Representative western blots of three proteins in different groups were shown in Supplementary Fig. S1. Therefore, our results suggest that Mel and Neu-P11, when used singly, can improve the expression levels of p-IRS-1 and PI3K and p-GSK3β. Based on the results of our study, Neu-P11 may improve insulin resistance by influencing the activity of the insulin signaling pathway. The key elements of the insulin signaling pathway produced the expected changes following Neu-P11 treatment. Our studies have shown that the expression levels of p-IRS-1, PI3K, and p-GSK3 were decreased in the insulinresistant group when compared with the insulin-sensitive group. When treated with a combination of Mel and Neu-P11, the expression levels of p-IRS-1 and PI3K and p-GSK3 were enhanced, compared with the insulin-resistant group. The difference was statistically significant, suggesting that Mel and Neu-P11 can increase the expression of p-IRS-1 and PI3K and p-GSK3. When Mel and Neu-P11 were combined with luzindole separately, the expression levels of the p-IRS-1 and PI3K and p-GSK3 were significantly decreased. Mel and Neu-P11 can enhance the expressions of p-IRS-1 and PI3K and p-GSK3, and our previous study has shown that the function of Mel and Neu-P11 is blocked when luzindole is combined with the MTNR1B receptors [9]. It is unclear why luzindole has a suppressive effect on the insulin signaling pathway. Further studies are needed to clarify this. In addition, Neu-P11 is an agonist of Mel, and it has similar functions as Mel. It has been previously reported that Mel stimulates the production of some immune factors and enhances the function of the immune system [10]. Therefore, the relationship between Neu-P11 and immune function may deserve further exploration.
Supplementary Data Supplementary data is available at ABBS online.
Funding This work was supported by the grants from the Scientific Research Projects of the Department of Education in Hunan Province (No. 14C0909) and the Scientific Research Projects of Hunan University of Medicine (No. 2014KY02).
References 1. Roy T, Rury RH. Normal weight individuals who develop Type 2 diabetes: the personal fat threshold. Clin Sci 2015, 128: 405–410. 2. Morino K, Petersen KF, Dufour S, Befroy D, Frattini J, Shatzkes N, Neschen S, et al. Reduced mitochondrial density and increased IRS-1 serine phosphorylation in muscle of insulin-resistant offspring of type 2 diabetic parents. Clin Invest 2005, 115: 3587–3593. 3. Shepherd PR, Withers DJ, Siddle K. Phosphoinositide 3-kinase: the key switch mechanism in insulin signaling. Biochem J 1998, 333: 471–490. 4. Qu ZS, Li L, Sun XJ, Zhao YW, Zhang J, Geng Z, Fu JL, et al. Glycogen synthase kinase-3 regulates production of amyloid-peptides and Tau phosphorylation in diabetic rat brain. Scientific World J 2014, 2014: 878123. 5. Yadav HN, Singh M, Sharma PL. Involvement of GSK-3β in attenuation of the cardioprotective effect of ischemic preconditioning in diabetic rat heart. Mol Cell Biochem 2010, 343: 75–81. 6. Wang J, Yin H, Huang Y, Guo C, Xia C, Liu Q, Zhang L. Panax quinquefolius saponin of stem and leaf attenuates intermittent high glucose-induced oxidative stress injury in cultured human umbilical vein endothelial cells via PI3K/Akt/GSK-3 pathway. Evid Based Complement Alternat Med 2013, 2013: 1–7. 7. Espino J, Pariente JA, Rodriguez AB. Role of melatonin on diabetes-related metabolic disorders. World J Diabetes 2011, 2: 82–91. 8. Stumpf I, Mühlbauer E, Peschke E. Involvement of the cGMP pathway in mediating the insulin-inhibitory effect of melatonin in pancreatic β-cells. J Pineal Res 2008, 45: 31–327. 9. Li X, Cai S, Yin W, Hu X. Neu-p11 reduces clock/apelin expression in insulin-resistant mouse adipocyte model. Acta Biochim Biophys Sin 2013, 45: 798–800. 10. Calvo JR, González-Yanes C, Maldonado MD. Maldonado. The role of melatonin in the cells of the innate immunity: a review. J Pineal Res 2013, 55: 103–120.
New Phenomenon
Role of Neu-p11/luzindole in the regulation of insulin signaling pathways and insulin resistance Xiuping Li1, Shichang Cai2,*, Weidong Yin3, Xiaobo Hu3, Sujun Zhang4, Zhengming Li1, Xing Li2, and Moshe Laudon5 1
Department of Laboratory, Hunan University of Medicine, Huaihua 418000, China, 2Basic Medical Sciences, Hunan University of Medicine, Huaihua 418000, China, 3Institute of Cardiovascular Disease, Key Laboratory Arteriosclerology of Hunan Province, University of South China, Hengyang 412000, China, 4Department of Experimental Animal, University of South China, Hengyang 412000, China, and 5Neurim Pharmaceuticals Ltd., Tel-Aviv 69710, Israel *Correspondence address. Tel: +86-745-2382481; Fax: +86-745-2382082; E-mail: [email protected]
The prevalence of Type 2 diabetes (T2D) has been globally increased since the last decade. T2D is a condition of relative insulin insufficiency, in which hyperglycemia develops when the insulin secretory capacity of β-cells is no longer sufficient to meet the insulin requirement in the setting of insulin resistance [1]. Previous studies have shown that there is a close correlation between T2D and insulin receptor substrate-1 (IRS-1) levels, and that lack of expression or abnormal phosphorylation of IRS-1 can lead to insulin resistance [2]. Phosphoinositide 3-kinase (PI3K) plays a key role in insulin signaling and its activity has been shown to be blunted in tissues from T2D subjects. PI3K activation is critical for insulin-mediated metabolic effects such as increased glucose uptake and glycogen synthesis [3]. Glycogen synthase kinase-3β (GSK3β), a downstream target of insulin signaling, is activated by phosphorylated ( p) Akt [4]. Phosphorylation by p-Akt inhibits the activity of GSK3β [5]. GSK3β phosphorylation and inactivation are considered to be important mechanisms of cell survival [6]. Melatonin (Mel) is a circulating hormone that is predominantly released from the pineal gland [7]. Some studies suggest that Mel may potentially play a role in diabetes and its associated metabolic disturbances by regulating insulin secretion. Luzindole is a nonspecific Mel receptor antagonist that can block some Mel functions [8]. Although Mel has an extensive range of biological effects, studies have revealed that it is rapidly metabolized with a half-life of 20–30 min once it gets ingested in humans. Therefore, the effect of Mel cannot be studied from direct administration of this drug. Moreover, extraction and synthesis of Mel is complicated and a high dose of Mel is associated with side effects. Neu-P11 is a novel type of nonselective agonist of Mel. It has several characteristics including the ease with which it can be synthesized in vitro and administered effectively for a longer time with fewer side effects. It can also substitute for Mel to interact with its receptors and consequently has an extensive range of biological actions [9]. In this study, we investigated the relationship between Mel and its receptor agonist Neu-P11, and the relationship between insulin
signaling pathway and the alteration of p-IRS-1/PI3K/p-GSK3β signaling in insulin-resistant 3T3-L1 adipocytes. Insulin-resistant 3T3-L1 adipocytes were utilized in this study to investigate the alteration of p-IRS-1/PI3K/p-GSK3β signaling activity following Neu-P11 treatment. 3T3-L1 pre-adipocytes were purchased from the Institute of Basic Medicine, Chinese Academy of Medical Sciences (Beijing, China). Neu-P11 was a kind gift from Neurim Pharmaceuticals Ltd (Habarzel, Israel). A 5 mM stock solution was prepared by dissolving Neu-P11 in dimethylsulfoxide. Cells were differentiated in adipogenic cocktail medium containing 3-isobutyl-1-methylxanthine (Sigma, St Louis, USA), insulin, and dexamethasone, and visualized by oil red O staining. Insulin-resistant 3T3-L1 adipocytes were cultured in Dulbecco’s modified Eagle’s medium (Gibco, Gaithersburg, USA) with high glucose/high insulin for 24 h. Glucose uptake was measured in the differentiated cells by an enzymatic method to assess the adipocyte model. Cells were divided into six groups: (i) insulin-resistant group, (ii) Mel group (cells were treated with 10 nM Mel for 6 h), (iii) Neu-P11 group (cells were treated with 10 nM Neu-P11 for 6 h), (iv) Mel + luzindole group (cells were treated with 10 nM Mel + 100 nM luzindole for 6 h), (v) Neu-P11 + luzindole group (cells were treated with 10 nM Neu-P11 + 100 nM luzindole for 6 h), and (vi) Mel + Neu-P11. Then all six groups were treated with 50, 100, and 200 nM insulin for 6 h to evaluate the effects of these drugs on adipocytes glucose uptake. Subsequently, cells were cultured with or without Mel, Neu-P11, or luzindole for 6 h and followed by a final incubation with different doses of insulin for 6 h. The expression levels of p-IRS-1, PI3K, and p-GSK3β were analyzed by western blot analysis using anti-p-IRS antibody (Cell Signaling Technology, Beverly, USA), anti-PI3K antibody (Santa Cruz, Santa Cruz, USA), and anti-p-GSK3β antibody (Santa Cruz), respectively. Our results showed that p-IRS-1, PI3K, and p-GSK3β expression levels were decreased significantly in insulin-resistant adipocytes compared with normal adipocytes (P < 0.05; Fig. 1); however, the
© The Author 2016. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. 485
486
Neu-p11/luzindole and insulin resistance
Figure 1. The changes of protein expression in different groups Adipocytes were cultured with and without Mel, Neu-P11, or luzindole for 6 h. IS, insulin sensitivity; IR, insulin resistance; M, IR + Mel; N, IR + Neu-P11; M + L, IR + Mel + luzindole; N + L, IR + Neu-P11 + luzindole. Protein expressions were analyzed by western blot analysis. (A) PI3K-P85. (B) p-GSK3β. (C) p-IRS-1. *P < 0.05 vs. IR; #P < 0.05 vs. M; $P < 0.05 vs. N. n = 3.
expression levels of p-IRS-1, PI3K, and p-GSK3β were increased significantly when insulin-resistant adipocytes were treated with Mel or Neu-P11 (P < 0.05; Fig. 1). In contrast, the changes were not significant in adipocytes when treated with the Mel + luzindole or Neu-P11 + luzindole, compared with the insulin-resistant group (P > 0.05; Fig. 1). Representative western blots of three proteins in different groups were shown in Supplementary Fig. S1. Therefore, our results suggest that Mel and Neu-P11, when used singly, can improve the expression levels of p-IRS-1 and PI3K and p-GSK3β. Based on the results of our study, Neu-P11 may improve insulin resistance by influencing the activity of the insulin signaling pathway. The key elements of the insulin signaling pathway produced the expected changes following Neu-P11 treatment. Our studies have shown that the expression levels of p-IRS-1, PI3K, and p-GSK3 were decreased in the insulinresistant group when compared with the insulin-sensitive group. When treated with a combination of Mel and Neu-P11, the expression levels of p-IRS-1 and PI3K and p-GSK3 were enhanced, compared with the insulin-resistant group. The difference was statistically significant, suggesting that Mel and Neu-P11 can increase the expression of p-IRS-1 and PI3K and p-GSK3. When Mel and Neu-P11 were combined with luzindole separately, the expression levels of the p-IRS-1 and PI3K and p-GSK3 were significantly decreased. Mel and Neu-P11 can enhance the expressions of p-IRS-1 and PI3K and p-GSK3, and our previous study has shown that the function of Mel and Neu-P11 is blocked when luzindole is combined with the MTNR1B receptors [9]. It is unclear why luzindole has a suppressive effect on the insulin signaling pathway. Further studies are needed to clarify this. In addition, Neu-P11 is an agonist of Mel, and it has similar functions as Mel. It has been previously reported that Mel stimulates the production of some immune factors and enhances the function of the immune system [10]. Therefore, the relationship between Neu-P11 and immune function may deserve further exploration.
Supplementary Data Supplementary data is available at ABBS online.
Funding This work was supported by the grants from the Scientific Research Projects of the Department of Education in Hunan Province (No. 14C0909) and the Scientific Research Projects of Hunan University of Medicine (No. 2014KY02).
References 1. Roy T, Rury RH. Normal weight individuals who develop Type 2 diabetes: the personal fat threshold. Clin Sci 2015, 128: 405–410. 2. Morino K, Petersen KF, Dufour S, Befroy D, Frattini J, Shatzkes N, Neschen S, et al. Reduced mitochondrial density and increased IRS-1 serine phosphorylation in muscle of insulin-resistant offspring of type 2 diabetic parents. Clin Invest 2005, 115: 3587–3593. 3. Shepherd PR, Withers DJ, Siddle K. Phosphoinositide 3-kinase: the key switch mechanism in insulin signaling. Biochem J 1998, 333: 471–490. 4. Qu ZS, Li L, Sun XJ, Zhao YW, Zhang J, Geng Z, Fu JL, et al. Glycogen synthase kinase-3 regulates production of amyloid-peptides and Tau phosphorylation in diabetic rat brain. Scientific World J 2014, 2014: 878123. 5. Yadav HN, Singh M, Sharma PL. Involvement of GSK-3β in attenuation of the cardioprotective effect of ischemic preconditioning in diabetic rat heart. Mol Cell Biochem 2010, 343: 75–81. 6. Wang J, Yin H, Huang Y, Guo C, Xia C, Liu Q, Zhang L. Panax quinquefolius saponin of stem and leaf attenuates intermittent high glucose-induced oxidative stress injury in cultured human umbilical vein endothelial cells via PI3K/Akt/GSK-3 pathway. Evid Based Complement Alternat Med 2013, 2013: 1–7. 7. Espino J, Pariente JA, Rodriguez AB. Role of melatonin on diabetes-related metabolic disorders. World J Diabetes 2011, 2: 82–91. 8. Stumpf I, Mühlbauer E, Peschke E. Involvement of the cGMP pathway in mediating the insulin-inhibitory effect of melatonin in pancreatic β-cells. J Pineal Res 2008, 45: 31–327. 9. Li X, Cai S, Yin W, Hu X. Neu-p11 reduces clock/apelin expression in insulin-resistant mouse adipocyte model. Acta Biochim Biophys Sin 2013, 45: 798–800. 10. Calvo JR, González-Yanes C, Maldonado MD. Maldonado. The role of melatonin in the cells of the innate immunity: a review. J Pineal Res 2013, 55: 103–120.
