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diabetes research and clinical practice 96 (2012) 53–59
Contents available at Sciverse ScienceDirect
Diabetes Research and Clinical Practice journ al h ome pa ge : www .elsevier.co m/lo cate/diabres
Thymosin beta 4 ameliorates hyperglycemia and improves insulin resistance of KK Cg-Ay/J mouse Jian Zhu a, Li-Ping Su b, Lei Ye c, Kok-Onn Lee d, Jian-Hua Ma a,* a
Department of Endocrinology, Nanjing First Hospital, Nanjing Medical University, 210006 Nanjing, China Division of Bioengineering, National University of Singapore, Singapore c Department of Medicine, University of Minnesota, Minneapolis, MN, USA d Department of Medicine, National University of Singapore, Singapore b
article info
abstract
Article history:
Object: To evaluate the efficacy of thymosin beta 4 (Tb4) on hyperglycemia and insulin
Received 31 August 2011
sensitivity in a mouse model of type 2 diabetes mellitus (T2DM).
Received in revised form
Methods: KK mice were divided into the following groups: KK control group, with saline
7 November 2011
treatment; KK Tb4 group, with daily Tb4 100 ng/10 g body weight intraperitoneal injection for
Accepted 5 December 2011
12 weeks. Non-diabetic C57BL mice were used as normal control. OGTT, plasma insulin,
Published on line 3 January 2012
HbA1c, serum adiponectin, Tb4, cholesterol, and triglyceride were measured before and after Tb4 treatment. The phosphorylated AKT and total AKT protein levels of skeletal muscle
Keywords:
from all groups were determined.
Thymosin beta 4
Results: After Tb4 treatment, repeat OGTT showed a significant decrease in glucose profiles
Insulin resistance
in the KK Tb4 group compared with the KK control group. The KK-Tb4 group had reduced
Diabetes mellitus
mean HbA1c and triglyceride levels, and increased adiponectin compared with KK control group. C57BL mice showed normal glucose homeostasis. The phosphorylated AKT levels of skeletal muscle were significantly increased in KK Tb4 group compared with KK control group after glucose stimulation. C57BL mice showed no changes in phosphorylated AKT levels after Tb4 treatment. Conclusions: Tb4 improved glucose intolerance and ameliorated insulin resistance in KK mouse. Tb4 may be a potential alternative insulin sensitizer for treatment of T2DM. # 2011 Elsevier Ireland Ltd. All rights reserved.
1.
Introduction
Insulin resistance is a key feature of type 2 diabetes mellitus and is associated with an increased risk of cardiovascular disease (CVD) [1–3]. However, apart from metformin, other drugs that improve insulin resistance have had serious adverse effects and have either been withdrawn from the market, or subjected to serious warnings. Rosiglitazone, an insulin sensitizer which acts by binding to the PPAR receptors in fat cells and making the cells more responsive to insulin, has been found to be
associated with an increased risk of heart attack [4]. Pioglitazone has recently been thought to be associated with bladder cancer. Both these drugs are not recommended for long-term use because of the potential of causing long bone fractures. Thus, it is important to explore novel potential insulin sensitizers that not only improve the insulin resistance but also decrease the risk of cardiovascular event. Thymosin beta 4 (Tb4) is a major intracellular G-actinsequestering peptide. Recently, there is increasing evidence that Tb4 may play an important role in wound healing and
* Corresponding author. Tel.: +86 13905187504. E-mail address: [email protected] (J.-H. Ma). 0168-8227/$ – see front matter # 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.diabres.2011.12.009
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diabetes research and clinical practice 96 (2012) 53–59
tissue remodeling as well as being a potential treatment of myocardial infarction [5–8]. In addition, Tb4 can promote the migration of myoblasts to facilitate skeletal muscle regeneration [9]. Our previous study showed that skeletal myoblast transplantation by direct injection into the hindlimb muscles could attenuate hyperglycemia and hyperinsulinaemia in the KK Cg-Ay/J (KK) mouse [10]. Furthermore, previous study showed that Tb4 levels decreased significantly in corneas of diabetic patients compared with healthy subjects [11], and exogenous Tb4 administration would promote wound healing in diabetic and nondiabetic corneas [12,13]. The present study was designed to determine the role of Tb4 on glucose tolerance and hyperinsulinaemia in a KK mouse model of type 2 diabetes mellitus, and possible underlying mechanisms. This will be served as a stepping-stone to develop new strategy for treatment of T2DM using Tb4.
2.
Methods
2.1.
Animals and treatment
KK Cg- Ay/J (KK) mice were purchased from the Jackson Laboratory (Bar Harbor, ME, USA) and maintained at the Animal Holding Unit of the National University of Singapore (NUS). C57BL mice were obtained from the Animal Resources Centre, NUS. All mice were housed in individual cages at 25 8C with a 12 h light-dark cycle and had free access to food (Lab Diet 5K52; PMI Nutrition International, Brentwood, MO, USA) and water. KK mice at 12–14 weeks old would be screened fasting oral glucose tolerance test (OGTT) and used for experiments. KK mouse meeting the following criteria would be used: [1] fasting blood glucose >6.5 mmol/l and [2] blood glucose > 20 mmol/l at 30 min or 60 min, and >11 mmol/l at 2 h during GTT. KK mice which met the criteria and C57BL mice were each divided into two groups receiving: (1) saline, (2) 100 ng/10 g body weight of Tb4. Tb4 was i.p. injected once daily for 12 weeks.
2.2.
Oral glucose tolerance test
All mice in the study had OGTT after a 16-h overnight fasting. Each mouse was treated by oral gavage with 1 g/kg body weight of glucose diluted in distilled water (100 mg/ml). Blood samples from the tail vein were collected at 0 (before glucose injection), 30, 60 and 120 min after glucose challenge. The blood glucose concentration was determined by Accu-Chek Advantage glucometer (Roche, Germany).
2.3.
Metabolites measurement
Blood samples were obtained from cavernous sinus of mice with a capillary tube under anesthesia. Isolated plasma was stored at 80 8C until analysis. Plasma insulin concentrations were determined by an insulin ELISA kit (Millipore, Billerica, MA, USA). Tb4 was determined by thymosin b4 EIA kit (Bachem, Torrance, CA, USA). Plasma cholesterol and triglyceride concentrations were determined using standard assays (Infinity Cholesterol; Thermo Electron, Thebarton, SA, Australia and Accutrend GCT, Roche). Adiponectin was
determined by a mouse adiponectin ELISA Kit (Millipore, Billerica, MA, USA). HbA1c was measured by an analyzer (DCA 2000+; Bayer HealthCare, Elkhart, IN, USA).
2.4.
Inflammation assay
Plasma pro-inflammation cytokines including ICAM-1, INF-g, MCP-1, TNF-a, IL-1b and leptin were determined by an antibody mouse inflammation array kit (RayBiotech, USA) according to the manufacturer’s instruction.
2.5.
Western blot analysis
Total skeletal muscle protein was extracted using an extraction reagent (Phosphosafe, Merck, Darmstadt, Germany) and protein concentration was determined using Bradford reagent (Bio-Rad Laboratories, Hercules, CA, USA). Proteins (10 mg per sample) were separated on SDS-polyacrylamide gel and transferred to nitrocellulose membrane. The blots were incubated overnight with diluted 1:2000 anti-AKT, 1:1000 anti-phosphorylated (Ser 473) AKT (pAKT) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) respectively. Anti-rabbit IgG conjugated with horseradish peroxidase (dilution: 1:2000) was used as second antibody and the membrane was visualized by chemiluminescence (Thermo Scientific, Rockford, IL, USA) and exposed to X-ray film (Thermo Scientific). Optical density of the bands was quantified by Quantity One Image software (Bio-Rad, USA). The concentration of each protein sample was expressed as percentage of the C57BL saline group samples at fasting state.
2.6.
Statistical analysis
Data are presented as mean SEM. The areas under the glucose and insulin curves were calculated using trapezoidal integration. Comparisons between two groups were performed using the two-tailed unpaired Student’s t test. Comparisons among more than two groups were done by ANOVA using Bonferroni’s test. Statistical analysis was performed with SPSS 13.0 software. A p value of less than 0.05 was considered statistically significant.
3.
Results
3.1.
Plasma Tb4 concentration of KK mice
At baseline, the average plasma concentrations of Tb4 were 2.93 0.41 mg/ml in KK control group and 2.82 0.26 mg/ml in KK Tb4 group, respectively. Tb4 levels were significantly lower than that in C57BL control group (5.78 0.35 mg/ml) and C57BL Tb4 group (5.77 0.76 mg/ml). This was the initial observation that suggested to us to test the injection of Tb4 into KK mice to determine whether exogenous Tb4 supplementation can improve glucose tolerance and insulin sensitivity of KK mice. At 12 weeks after Tb4 treatment, blood samples were drew from KK or C57BL mice for measurement of Tb4 at 36–48 h after last time of Tb4 intraperitoneal injection. As shown in Fig. 1, plasma Tb4 concentrations increased slightly to 6.00 0. 0.45 mg/ml in C57BL Tb4 group and 2.94 0.23 mg/ml in KK
diabetes research and clinical practice 96 (2012) 53–59
3.2.
Fig. 1 – Plasma Tb4 concentration of KK mice was much lower than that of C57BL mice. Data are presented as the mean W SEM (n = 8 in KK control (CTL) group and KK Tb4 group, n = 6 in C57BL CTL group and C57BL Tb4 group, respectively). Significance: *p < 0.05 vs. C57BL CTL group at baseline, #p < 0.05 vs. C57BL CTL group after 12 weeks treatment.
Tb4 improved glucose tolerance of KK mouse
Results of OGTT demonstrated that KK mice developed hyperglycemia and glucose intolerance at 12–14 weeks of age, and glucose concentrations of the two KK groups were significantly higher than those in the C57BL groups ( p < 0.05) (Fig. 2A). KK control group animals remained hyperglycemic and glucose intolerant at 6 weeks after saline treatment (Fig. 2B). Blood glucose was reduced in the KK Tb4 group when compared with KK control group ( p < 0.05). Glycemic excursion remained normal in two C57BL groups. KK control animals still had severe hyperglycemia and glucose intolerance at 12 weeks after saline treatment (Fig. 2C). By contrast, KK Tb4 group animals showed significant reduction in blood glucose ( p < 0.01). Glycemic excursion in two C57BL groups remained normal and no difference was found between C57BL control group and C57BL Tb4 group.
3.3. Tb4 group, respectively. However, there were no statistic differences between Tb4 concentration at baseline and that after 12 weeks treatment both in C57BL and KK mice.
55
Plasma insulin during OGTT
KK mice developed hyperinsulinemia at 12–14 weeks of age. Plasma insulin concentrations of KK groups were significantly higher than those in the C57BL groups ( p < 0.05) (Fig. 2D).
Fig. 2 – Tb4 ameliorates hyperglycemia of KK mice. Glucose tolerance tests of the KK control (CTL) group (black line) (n = 8), KK Tb4 group (red line) (n = 8),C57BL CTL group (blue line) (n = 6), C57BL Tb4 group (pink line) (n = 6) at (A) baseline, (B) 6 weeks and (C) 12 weeks after treatment. (D–F) Plasma insulin of the KK CTL group (black line), KK Tb4 group (red line), C57BL CTL group (blue line), C57BL Tb4 group (pink line) during glucose tolerance tests at (D) baseline, (E) 6 weeks and (F) 12 weeks after treatment. Significance:*p < 0.05 vs. C57BL CTL group; #p < 0.05 vs. KK CTL group. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
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diabetes research and clinical practice 96 (2012) 53–59
KK mice remained hyperinsulinemia at 6 weeks after Tb4 treatment. There were no significant differences in plasma insulin levels between KK control group and KK Tb4 group (Fig. 2E). At 12 weeks after saline or Tb4 treatment, KK control group animals remained severely hyperinsulinemia (Fig. 2F). The KK Tb4 group showed slight reduction in plasma insulin levels (insulin AUC = 8.13 1.44 ng ml 1 h 1) when compared with the KK control group (insulin AUC = 6.99 0.86 ng ml 1 h 1). However, there were no statistically significant differences between two KK groups. Plasma insulin in C57BL groups remained normal compared with values before Tb4 treatment. No differences were found between C57BL control group and C57BL Tb4 group.
3.4.
Other plasma metabolites
The results of the fasting plasma metabolites are shown in Table 1. Fasting insulin and triglyceride in the two KK groups were significantly higher than those of C57BL groups at 12 to 14 weeks of age. At 6 weeks after Tb4 treatment, fasting insulin and triglyceride were still higher in the two KK groups than those in C57BL groups. At 12 weeks after saline treatment, the KK control group had further increased fasting insulin levels, while the KK Tb4 group remained unchanged as compared with that at 6 weeks after treatment. KK mice had significantly lower adiponectin compared to C57BL mice at 12 to 14 weeks of age. Adiponectin did not change in the KK control group at 12 weeks after saline treatment. Though adiponectin levels were increased in the KK Tb4 group, this was not significantly higher than that of the KK control group at 12 weeks after saline treatment. HbA1c was higher in the two KK groups compared to the C57BL group before treatment ( p < 0.05). At 12 weeks after
treatment, HbA1c in the KK control group further increased compared with the KK Tb4 group ( p < 0.05) and that of the C57BL group ( p < 0.05). However, HbA1c was significantly lower in the KK Tb4 group. Reduced blood HbA1c demonstrated that KK Tb4 group mice achieved significantly better glucose control after Tb4 treatment.
3.5.
Body weight change
The mean fasting body weight of the KK control group increased from 42.45 1.82 g at baseline to 47.86 2.48 g after 12 weeks. Body weight of KK Tb4 group increased to 49.55 2.32 g at 12 weeks after Tb4 treatment. Though there was a tendency that Tb4 treatment would increase body weight of KK mouse, no statistically significant difference in body weight was found between the two KK groups.
3.6.
Inflammation assay
At 12 weeks after Tb4 treatment, the pro-inflammation cytokines levels, including ICAM-1, INF-g, MCP-1, TNF-a, IL1b and leptin, in both KK groups were slightly higher than those in C57BL groups. However, there was no statistically significant difference in cytokine levels between the KK control group and the KK Tb4 group (data not shown).
3.7.
Protein level of insulin signal pathway
Western blot results suggested that no differences in levels of insulin receptor, phosphatidylinositol 3-kinase (PI3 K) and AKT in the two KK groups at baseline compared with C57BL groups at 12 weeks after Tb4 treatment. Glucose stimulation did not significantly change the levels of these proteins (data not shown). However, pAKT was significantly increased in the
Table 1 – Body weight and metabolitic parameters of the KK and C57BL mice. Animal groups
Baseline Weight (g) Adiponectin (mg/ml) Cholesterol (mmol/l) Triglyceride (mmol/l) Fasting insulin (ng/ml) HbA1c (%) 6 weeks after treatment Weight (g) Cholesterol (mmol/l) Triglyceride (mmol/l) Fasting insulin (ng/ml) 12 weeks after treatment Weight (g) Adiponectin (mg/ml) Cholesterol (mmol/l) Triglyceride (mmol/l) Fasting insulin (ng/ml) HbA1c (%) * #
p < 0.05 vs. C57BL control group. p < 0.05 vs. KK control group.
C57BL control (n = 6)
C57BL Tb4 (n = 6)
KK control (n = 8)
20.52 1.44 18.84 0.60 3.92 0.04 1.96 0.17 0.20 0.02 4.41 0.07
20.57 1.68 19.84 3.00 4.01 0.08 1.81 0.07 0.23 0.02 4.40 0.08
42.45 1.82* 10.97 1.84* 4.10 0.02 2.40 0.12* 2.85 0.42* 6.76 0.62*
43.78 1.81* 11.51 1.94* 4.38 0.21 2.32 0.13* 2.79 0.57* 6.80 0.34*
22.80 1.50 4.07 0.07 2.07 0.10 0.22 0.12
22.45 1.53 4.04 0.04 2.05 0.20 0.20 0.13
45.22 1.71* 4.18 0.05 2.50 0.12* 3.98 0.46*
46.69 1.86* 4.25 0.06 2.25 0.13* 4.14 0.46*
23.86 2.49 20.08 1.76 4.18 0.06 1.97 0.13 0.19 0.02 4.49 0.08
22.78 1.45 21.82 2.70 3.90 0.09 1.96 0.08 0.21 0.05 4.48 0.05
47.86 2.48* 10.93 1.72* 4.33 0.09 2.55 0.15* 5.14 0.95* 7.21 0.63*
49.55 2.32* 15.61 2.41* 4.34 0.16 2.29 0.18*,# 4.30 0.33*,# 5.42 0.26*,#
KK Tb4 (n = 8)
diabetes research and clinical practice 96 (2012) 53–59
57
significance. These results indicated that higher pAKT levels in KK Tb4 group mice were due to improved insulin sensitivity by Tb4 treatment.
4.
Fig. 3 – Tb4 improved AKT activity of skeletal muscle in response to glucose stimulation. (A) Western blot analyses of protein levels of pAKT and AKT at basal condition and 0.5 h after GTT at 12 weeks after treatment. (B) pAKT/AKT values are normalized to GAPDH protein expression levels and expressed relative to C57BL control (CTL) group at basal condition. White bar: basal condition. Black bar: 0.5 h after oral injection of glucose. Each lane represents a single mouse. Data are presented as the mean W SEM (n = 8 in KK group, n = 6 in C57BL group). Significance: *p < 0.05 vs. C57BL CTL group; #p < 0.05 vs. KK CTL group.
KK Tb4 group compared with KK control group after glucose stimulation (Fig. 3). These results suggest that Tb4 treatment increased AKT activity in response to glucose challenge, which might lead to increased uptake of glucose by skeletal muscles. The elevated pAKT in skeletal muscle may due to higher insulin concentration or more sensitivity to insulin or both, in KK mice, thus, we measured blood glucose levels and insulin levels in two KK groups at 30 minus after oral glucose stimulation. The results (Fig. 4) showed that blood glucose level in KK Tb4 group was lower than that in KK control group ( p < 0.05). Although average insulin level of KK Tb4 group (3.41 0.35 ng/ml) was lower than counterpart of KK control group (3.92 0.47 ng/ml), the difference did not reach statistic
Discussion
Insulin resistance in peripheral tissues is an essential element in the pathogenesis of T2DM. The KK mouse, an animal model of T2DM, is characterized by insulin resistance, hyperglycemia and hyperinsulinaemia [14]. In the present study, we demonstrated for the first time that Tb4 administration could effectively improve glucose tolerance and ameliorate hyperinsulinemia of KK mouse. We further showed that Tb4 treatment could decrease triglyceride levels and increase the AKT activity of skeletal muscle of KK mouse. Tb4 thus may be potentially useful in improving insulin resistance in patients with type 2 diabetes mellitus. The ultimate goal of T2DM treatment is to reduce mortality and the risk of microvascular and macrovascular complications [15]. The latter, mainly cardiovascular disease is the most frequent cause of increased mortality among T2DM patients [16–18]. Several studies have suggested a causal association between insulin resistance and cardiovascular disease [2,3,19,20]. Tb4, a small ubiquitous protein containing 43 amine acids, has structure/function activity via its actin-binding domain and numerous biological affects on cells [5]. In addition to cell motility, tissue development and maintenance, Tb4 can stimulate migration of cardiomyocytes and endothelial cells and promotes survival of cardiomyocytes [8,21]. Our present study demonstrated that the plasma Tb4 concentration was lower in KK mice compared to that of C57BL mice. Furthermore, we demonstrated that a 12-week Tb4 treatment could lead to significant reductions in hyperglycemia, triglycerides and HbA1c, as well as an increase in adiponectin in KK mouse. Those results indicated that Tb4 and related compounds might be a novel potential alternative anti-diabetic agent for treatment of T2DM. Chronic, low-grade inflammation has been implicated in the path-physiology of T2DM [22,23]. A number of studies have shown that many drugs such as statins and angiotensin
Fig. 4 – Tb4 treatment did not increase insulin secretion in KK mouse. Blood glucose (A) and plasma insulin levels (B) at 30 min after glucose stimulation. Data are presented as the mean W SEM for 8 mice. Significance: #p < 0.05 vs. KK control (CTL) group.
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diabetes research and clinical practice 96 (2012) 53–59
converting enzyme (ACE) inhibitors as well as angiotensin receptor blockers, which have apparent ‘‘anti-inflammatory’’ properties, could reduce the incidence and/or delay the onset of T2DM. High-dose aspirin has been shown to inhibit cyclooxygenase and IkappaB kinase-beta and reduce fasting plasma glucose concentration [15]. In addition, a study has suggested that recombinant IL-1-receptor antagonist, a specific inhibitors of inflammatory pathway, reduced inflammation and improved b-cell function and glycaemic control of patients with T2DM [24]. Tb4 has been shown to be able to exert anti-inflammatory function through suppressing NFkappaB pathway and attenuating neutrophil-associated inflammatory processes [25,26]. However, the concentrations of proinflammation cytokines such as ICAM-1, INF-g, MCP-1, TNF-a, IL-1b and leptin in KK groups were higher than C57BL groups at 12 weeks after Tb4 treatment in this study. Although there was tendency of decrease in those pro-inflammation cytokines in KK Tb4 group compared with KK control group, no statistically significant differences could been found. However, an increased adiponectin level was found in KK Tb4 group (about 50% increase as compared with KK control group). Adiponectin is solely secreted by adipocytes and acts as a hormone with insulin-sensitizing property. Findings from animal studies and metabolic studies in human suggest several mechanisms through which adiponectin may decrease the risk of T2DM, including suppression of hepatic gluconeogenesis, stimulation of fatty acid oxidation in the liver, stimulation of fatty acid oxidation and glucose uptake in skeletal muscle. All these together suggest that Tb4 may regulate blood adiponectin level to achieve anti-diabetic effect. Skeletal muscle, a long-living tissue, is responsible for about 75% of whole-body glucose metabolism. Insulin and exercise are important physiological stimulators for skeletal muscle glucose uptake. The mechanism of insulin-stimulated skeletal muscle glucose uptake has been well characterized and is dependent on insulin receptor substrate-1(IRS1) phosphorylation/PI3 K/AKT pathway which results in Glut-4 translocation [27]. Exercise-stimulated glucose uptake is insulin and PI3 K independent and links to AMP-activated protein kinase (AMPK) and p38 mitogen-activated protein kinase (MAPK) activation [28]. It has been well demonstrated that reduced glucose uptake in skeletal muscle plays an important role in development of T2DM [29]. In the present study, western blot results showed that Tb4 injection improved activity AKT in KK Tb4 group in response to glucose stimulation. The increased pAKT could potentially enhance its ability to activate various downstream effectors in the insulinmediated signaling pathway to transport glucose into skeletal muscle. Consistent with the change of pAKT, the blood glucose level at 30 min after glucose stimulation in KK Tb4 group was lower than KK control group. These results raise one question that whether Tb4 treatment increases insulin secretion from pancreatic b cells or improves insulin resistance of peripheral tissues. Thus, we also measured the insulin levels of mice from two KK groups at 30 min after oral glucose challenge. The results showed that average insulin level of KK control group was higher than KK Tb4 group, although the difference was not statistically significant. This suggested that increased insulin secretion was not the reason that Tb4 increased AKT activity. In addition, Tb4 has been shown to increase the pAKT on
cardiac cells by forming a functional complex with PINCH and integrin-linked kinase in vitro [21], which suggested Tb4 treatment in vivo might activate the AKT directly rather than improve insulin resistance of skeletal muscle. In this study, we sacrificed those mice at 36–48 h after last injection of Tb4, meanwhile, drew blood samples from KK or C57BL mice for measurement of Tb4. The results showed that there were no significant differences in Tb4 concentrations between two KK mice groups after treatment, as well as in two C57BL groups. It might be resulted from short half-life of Tb4. Mora et al. [30] had reported that a significant increase of Tb4 concentration in serum was starting 2 min after single intraperitoneal injection and lasting for 40 min. However, in some organs such as brain, kidney, liver, fat, the wet weight concentration of Tb4 increased significantly from the first 40 min to 8 h after injection in comparison to their baseline wet weight concentration. In addition, the peak wet weight concentration of Tb4 in muscle arrived at 6 h after Tb4 intraperitoneal injection and would last for 24 h, which indicated that Tb4 might take effect for long time in many organs. Although insulin independent glucose uptake pathway had not been investigated in this study, the role of Tb4 on AMPK and p38 MAPK activation was unknown. Together, these results still indicated that improvement of skeletal muscle insulin resistance may be a major mechanism by which Tb4 ameliorated hyperglycemia in KK mice. In conclusion, our preliminary data here indicates that Tb4 administration could improve glucose tolerance and ameliorate muscle insulin resistance. This peptide may be a novel potential alternative insulin sensitizer for treatment of T2DM and would be worthy of further investigation.
Conflict of interest The authors declare that they have no conflict of interest.
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Contents available at Sciverse ScienceDirect
Diabetes Research and Clinical Practice journ al h ome pa ge : www .elsevier.co m/lo cate/diabres
Thymosin beta 4 ameliorates hyperglycemia and improves insulin resistance of KK Cg-Ay/J mouse Jian Zhu a, Li-Ping Su b, Lei Ye c, Kok-Onn Lee d, Jian-Hua Ma a,* a
Department of Endocrinology, Nanjing First Hospital, Nanjing Medical University, 210006 Nanjing, China Division of Bioengineering, National University of Singapore, Singapore c Department of Medicine, University of Minnesota, Minneapolis, MN, USA d Department of Medicine, National University of Singapore, Singapore b
article info
abstract
Article history:
Object: To evaluate the efficacy of thymosin beta 4 (Tb4) on hyperglycemia and insulin
Received 31 August 2011
sensitivity in a mouse model of type 2 diabetes mellitus (T2DM).
Received in revised form
Methods: KK mice were divided into the following groups: KK control group, with saline
7 November 2011
treatment; KK Tb4 group, with daily Tb4 100 ng/10 g body weight intraperitoneal injection for
Accepted 5 December 2011
12 weeks. Non-diabetic C57BL mice were used as normal control. OGTT, plasma insulin,
Published on line 3 January 2012
HbA1c, serum adiponectin, Tb4, cholesterol, and triglyceride were measured before and after Tb4 treatment. The phosphorylated AKT and total AKT protein levels of skeletal muscle
Keywords:
from all groups were determined.
Thymosin beta 4
Results: After Tb4 treatment, repeat OGTT showed a significant decrease in glucose profiles
Insulin resistance
in the KK Tb4 group compared with the KK control group. The KK-Tb4 group had reduced
Diabetes mellitus
mean HbA1c and triglyceride levels, and increased adiponectin compared with KK control group. C57BL mice showed normal glucose homeostasis. The phosphorylated AKT levels of skeletal muscle were significantly increased in KK Tb4 group compared with KK control group after glucose stimulation. C57BL mice showed no changes in phosphorylated AKT levels after Tb4 treatment. Conclusions: Tb4 improved glucose intolerance and ameliorated insulin resistance in KK mouse. Tb4 may be a potential alternative insulin sensitizer for treatment of T2DM. # 2011 Elsevier Ireland Ltd. All rights reserved.
1.
Introduction
Insulin resistance is a key feature of type 2 diabetes mellitus and is associated with an increased risk of cardiovascular disease (CVD) [1–3]. However, apart from metformin, other drugs that improve insulin resistance have had serious adverse effects and have either been withdrawn from the market, or subjected to serious warnings. Rosiglitazone, an insulin sensitizer which acts by binding to the PPAR receptors in fat cells and making the cells more responsive to insulin, has been found to be
associated with an increased risk of heart attack [4]. Pioglitazone has recently been thought to be associated with bladder cancer. Both these drugs are not recommended for long-term use because of the potential of causing long bone fractures. Thus, it is important to explore novel potential insulin sensitizers that not only improve the insulin resistance but also decrease the risk of cardiovascular event. Thymosin beta 4 (Tb4) is a major intracellular G-actinsequestering peptide. Recently, there is increasing evidence that Tb4 may play an important role in wound healing and
* Corresponding author. Tel.: +86 13905187504. E-mail address: [email protected] (J.-H. Ma). 0168-8227/$ – see front matter # 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.diabres.2011.12.009
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diabetes research and clinical practice 96 (2012) 53–59
tissue remodeling as well as being a potential treatment of myocardial infarction [5–8]. In addition, Tb4 can promote the migration of myoblasts to facilitate skeletal muscle regeneration [9]. Our previous study showed that skeletal myoblast transplantation by direct injection into the hindlimb muscles could attenuate hyperglycemia and hyperinsulinaemia in the KK Cg-Ay/J (KK) mouse [10]. Furthermore, previous study showed that Tb4 levels decreased significantly in corneas of diabetic patients compared with healthy subjects [11], and exogenous Tb4 administration would promote wound healing in diabetic and nondiabetic corneas [12,13]. The present study was designed to determine the role of Tb4 on glucose tolerance and hyperinsulinaemia in a KK mouse model of type 2 diabetes mellitus, and possible underlying mechanisms. This will be served as a stepping-stone to develop new strategy for treatment of T2DM using Tb4.
2.
Methods
2.1.
Animals and treatment
KK Cg- Ay/J (KK) mice were purchased from the Jackson Laboratory (Bar Harbor, ME, USA) and maintained at the Animal Holding Unit of the National University of Singapore (NUS). C57BL mice were obtained from the Animal Resources Centre, NUS. All mice were housed in individual cages at 25 8C with a 12 h light-dark cycle and had free access to food (Lab Diet 5K52; PMI Nutrition International, Brentwood, MO, USA) and water. KK mice at 12–14 weeks old would be screened fasting oral glucose tolerance test (OGTT) and used for experiments. KK mouse meeting the following criteria would be used: [1] fasting blood glucose >6.5 mmol/l and [2] blood glucose > 20 mmol/l at 30 min or 60 min, and >11 mmol/l at 2 h during GTT. KK mice which met the criteria and C57BL mice were each divided into two groups receiving: (1) saline, (2) 100 ng/10 g body weight of Tb4. Tb4 was i.p. injected once daily for 12 weeks.
2.2.
Oral glucose tolerance test
All mice in the study had OGTT after a 16-h overnight fasting. Each mouse was treated by oral gavage with 1 g/kg body weight of glucose diluted in distilled water (100 mg/ml). Blood samples from the tail vein were collected at 0 (before glucose injection), 30, 60 and 120 min after glucose challenge. The blood glucose concentration was determined by Accu-Chek Advantage glucometer (Roche, Germany).
2.3.
Metabolites measurement
Blood samples were obtained from cavernous sinus of mice with a capillary tube under anesthesia. Isolated plasma was stored at 80 8C until analysis. Plasma insulin concentrations were determined by an insulin ELISA kit (Millipore, Billerica, MA, USA). Tb4 was determined by thymosin b4 EIA kit (Bachem, Torrance, CA, USA). Plasma cholesterol and triglyceride concentrations were determined using standard assays (Infinity Cholesterol; Thermo Electron, Thebarton, SA, Australia and Accutrend GCT, Roche). Adiponectin was
determined by a mouse adiponectin ELISA Kit (Millipore, Billerica, MA, USA). HbA1c was measured by an analyzer (DCA 2000+; Bayer HealthCare, Elkhart, IN, USA).
2.4.
Inflammation assay
Plasma pro-inflammation cytokines including ICAM-1, INF-g, MCP-1, TNF-a, IL-1b and leptin were determined by an antibody mouse inflammation array kit (RayBiotech, USA) according to the manufacturer’s instruction.
2.5.
Western blot analysis
Total skeletal muscle protein was extracted using an extraction reagent (Phosphosafe, Merck, Darmstadt, Germany) and protein concentration was determined using Bradford reagent (Bio-Rad Laboratories, Hercules, CA, USA). Proteins (10 mg per sample) were separated on SDS-polyacrylamide gel and transferred to nitrocellulose membrane. The blots were incubated overnight with diluted 1:2000 anti-AKT, 1:1000 anti-phosphorylated (Ser 473) AKT (pAKT) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) respectively. Anti-rabbit IgG conjugated with horseradish peroxidase (dilution: 1:2000) was used as second antibody and the membrane was visualized by chemiluminescence (Thermo Scientific, Rockford, IL, USA) and exposed to X-ray film (Thermo Scientific). Optical density of the bands was quantified by Quantity One Image software (Bio-Rad, USA). The concentration of each protein sample was expressed as percentage of the C57BL saline group samples at fasting state.
2.6.
Statistical analysis
Data are presented as mean SEM. The areas under the glucose and insulin curves were calculated using trapezoidal integration. Comparisons between two groups were performed using the two-tailed unpaired Student’s t test. Comparisons among more than two groups were done by ANOVA using Bonferroni’s test. Statistical analysis was performed with SPSS 13.0 software. A p value of less than 0.05 was considered statistically significant.
3.
Results
3.1.
Plasma Tb4 concentration of KK mice
At baseline, the average plasma concentrations of Tb4 were 2.93 0.41 mg/ml in KK control group and 2.82 0.26 mg/ml in KK Tb4 group, respectively. Tb4 levels were significantly lower than that in C57BL control group (5.78 0.35 mg/ml) and C57BL Tb4 group (5.77 0.76 mg/ml). This was the initial observation that suggested to us to test the injection of Tb4 into KK mice to determine whether exogenous Tb4 supplementation can improve glucose tolerance and insulin sensitivity of KK mice. At 12 weeks after Tb4 treatment, blood samples were drew from KK or C57BL mice for measurement of Tb4 at 36–48 h after last time of Tb4 intraperitoneal injection. As shown in Fig. 1, plasma Tb4 concentrations increased slightly to 6.00 0. 0.45 mg/ml in C57BL Tb4 group and 2.94 0.23 mg/ml in KK
diabetes research and clinical practice 96 (2012) 53–59
3.2.
Fig. 1 – Plasma Tb4 concentration of KK mice was much lower than that of C57BL mice. Data are presented as the mean W SEM (n = 8 in KK control (CTL) group and KK Tb4 group, n = 6 in C57BL CTL group and C57BL Tb4 group, respectively). Significance: *p < 0.05 vs. C57BL CTL group at baseline, #p < 0.05 vs. C57BL CTL group after 12 weeks treatment.
Tb4 improved glucose tolerance of KK mouse
Results of OGTT demonstrated that KK mice developed hyperglycemia and glucose intolerance at 12–14 weeks of age, and glucose concentrations of the two KK groups were significantly higher than those in the C57BL groups ( p < 0.05) (Fig. 2A). KK control group animals remained hyperglycemic and glucose intolerant at 6 weeks after saline treatment (Fig. 2B). Blood glucose was reduced in the KK Tb4 group when compared with KK control group ( p < 0.05). Glycemic excursion remained normal in two C57BL groups. KK control animals still had severe hyperglycemia and glucose intolerance at 12 weeks after saline treatment (Fig. 2C). By contrast, KK Tb4 group animals showed significant reduction in blood glucose ( p < 0.01). Glycemic excursion in two C57BL groups remained normal and no difference was found between C57BL control group and C57BL Tb4 group.
3.3. Tb4 group, respectively. However, there were no statistic differences between Tb4 concentration at baseline and that after 12 weeks treatment both in C57BL and KK mice.
55
Plasma insulin during OGTT
KK mice developed hyperinsulinemia at 12–14 weeks of age. Plasma insulin concentrations of KK groups were significantly higher than those in the C57BL groups ( p < 0.05) (Fig. 2D).
Fig. 2 – Tb4 ameliorates hyperglycemia of KK mice. Glucose tolerance tests of the KK control (CTL) group (black line) (n = 8), KK Tb4 group (red line) (n = 8),C57BL CTL group (blue line) (n = 6), C57BL Tb4 group (pink line) (n = 6) at (A) baseline, (B) 6 weeks and (C) 12 weeks after treatment. (D–F) Plasma insulin of the KK CTL group (black line), KK Tb4 group (red line), C57BL CTL group (blue line), C57BL Tb4 group (pink line) during glucose tolerance tests at (D) baseline, (E) 6 weeks and (F) 12 weeks after treatment. Significance:*p < 0.05 vs. C57BL CTL group; #p < 0.05 vs. KK CTL group. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
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diabetes research and clinical practice 96 (2012) 53–59
KK mice remained hyperinsulinemia at 6 weeks after Tb4 treatment. There were no significant differences in plasma insulin levels between KK control group and KK Tb4 group (Fig. 2E). At 12 weeks after saline or Tb4 treatment, KK control group animals remained severely hyperinsulinemia (Fig. 2F). The KK Tb4 group showed slight reduction in plasma insulin levels (insulin AUC = 8.13 1.44 ng ml 1 h 1) when compared with the KK control group (insulin AUC = 6.99 0.86 ng ml 1 h 1). However, there were no statistically significant differences between two KK groups. Plasma insulin in C57BL groups remained normal compared with values before Tb4 treatment. No differences were found between C57BL control group and C57BL Tb4 group.
3.4.
Other plasma metabolites
The results of the fasting plasma metabolites are shown in Table 1. Fasting insulin and triglyceride in the two KK groups were significantly higher than those of C57BL groups at 12 to 14 weeks of age. At 6 weeks after Tb4 treatment, fasting insulin and triglyceride were still higher in the two KK groups than those in C57BL groups. At 12 weeks after saline treatment, the KK control group had further increased fasting insulin levels, while the KK Tb4 group remained unchanged as compared with that at 6 weeks after treatment. KK mice had significantly lower adiponectin compared to C57BL mice at 12 to 14 weeks of age. Adiponectin did not change in the KK control group at 12 weeks after saline treatment. Though adiponectin levels were increased in the KK Tb4 group, this was not significantly higher than that of the KK control group at 12 weeks after saline treatment. HbA1c was higher in the two KK groups compared to the C57BL group before treatment ( p < 0.05). At 12 weeks after
treatment, HbA1c in the KK control group further increased compared with the KK Tb4 group ( p < 0.05) and that of the C57BL group ( p < 0.05). However, HbA1c was significantly lower in the KK Tb4 group. Reduced blood HbA1c demonstrated that KK Tb4 group mice achieved significantly better glucose control after Tb4 treatment.
3.5.
Body weight change
The mean fasting body weight of the KK control group increased from 42.45 1.82 g at baseline to 47.86 2.48 g after 12 weeks. Body weight of KK Tb4 group increased to 49.55 2.32 g at 12 weeks after Tb4 treatment. Though there was a tendency that Tb4 treatment would increase body weight of KK mouse, no statistically significant difference in body weight was found between the two KK groups.
3.6.
Inflammation assay
At 12 weeks after Tb4 treatment, the pro-inflammation cytokines levels, including ICAM-1, INF-g, MCP-1, TNF-a, IL1b and leptin, in both KK groups were slightly higher than those in C57BL groups. However, there was no statistically significant difference in cytokine levels between the KK control group and the KK Tb4 group (data not shown).
3.7.
Protein level of insulin signal pathway
Western blot results suggested that no differences in levels of insulin receptor, phosphatidylinositol 3-kinase (PI3 K) and AKT in the two KK groups at baseline compared with C57BL groups at 12 weeks after Tb4 treatment. Glucose stimulation did not significantly change the levels of these proteins (data not shown). However, pAKT was significantly increased in the
Table 1 – Body weight and metabolitic parameters of the KK and C57BL mice. Animal groups
Baseline Weight (g) Adiponectin (mg/ml) Cholesterol (mmol/l) Triglyceride (mmol/l) Fasting insulin (ng/ml) HbA1c (%) 6 weeks after treatment Weight (g) Cholesterol (mmol/l) Triglyceride (mmol/l) Fasting insulin (ng/ml) 12 weeks after treatment Weight (g) Adiponectin (mg/ml) Cholesterol (mmol/l) Triglyceride (mmol/l) Fasting insulin (ng/ml) HbA1c (%) * #
p < 0.05 vs. C57BL control group. p < 0.05 vs. KK control group.
C57BL control (n = 6)
C57BL Tb4 (n = 6)
KK control (n = 8)
20.52 1.44 18.84 0.60 3.92 0.04 1.96 0.17 0.20 0.02 4.41 0.07
20.57 1.68 19.84 3.00 4.01 0.08 1.81 0.07 0.23 0.02 4.40 0.08
42.45 1.82* 10.97 1.84* 4.10 0.02 2.40 0.12* 2.85 0.42* 6.76 0.62*
43.78 1.81* 11.51 1.94* 4.38 0.21 2.32 0.13* 2.79 0.57* 6.80 0.34*
22.80 1.50 4.07 0.07 2.07 0.10 0.22 0.12
22.45 1.53 4.04 0.04 2.05 0.20 0.20 0.13
45.22 1.71* 4.18 0.05 2.50 0.12* 3.98 0.46*
46.69 1.86* 4.25 0.06 2.25 0.13* 4.14 0.46*
23.86 2.49 20.08 1.76 4.18 0.06 1.97 0.13 0.19 0.02 4.49 0.08
22.78 1.45 21.82 2.70 3.90 0.09 1.96 0.08 0.21 0.05 4.48 0.05
47.86 2.48* 10.93 1.72* 4.33 0.09 2.55 0.15* 5.14 0.95* 7.21 0.63*
49.55 2.32* 15.61 2.41* 4.34 0.16 2.29 0.18*,# 4.30 0.33*,# 5.42 0.26*,#
KK Tb4 (n = 8)
diabetes research and clinical practice 96 (2012) 53–59
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significance. These results indicated that higher pAKT levels in KK Tb4 group mice were due to improved insulin sensitivity by Tb4 treatment.
4.
Fig. 3 – Tb4 improved AKT activity of skeletal muscle in response to glucose stimulation. (A) Western blot analyses of protein levels of pAKT and AKT at basal condition and 0.5 h after GTT at 12 weeks after treatment. (B) pAKT/AKT values are normalized to GAPDH protein expression levels and expressed relative to C57BL control (CTL) group at basal condition. White bar: basal condition. Black bar: 0.5 h after oral injection of glucose. Each lane represents a single mouse. Data are presented as the mean W SEM (n = 8 in KK group, n = 6 in C57BL group). Significance: *p < 0.05 vs. C57BL CTL group; #p < 0.05 vs. KK CTL group.
KK Tb4 group compared with KK control group after glucose stimulation (Fig. 3). These results suggest that Tb4 treatment increased AKT activity in response to glucose challenge, which might lead to increased uptake of glucose by skeletal muscles. The elevated pAKT in skeletal muscle may due to higher insulin concentration or more sensitivity to insulin or both, in KK mice, thus, we measured blood glucose levels and insulin levels in two KK groups at 30 minus after oral glucose stimulation. The results (Fig. 4) showed that blood glucose level in KK Tb4 group was lower than that in KK control group ( p < 0.05). Although average insulin level of KK Tb4 group (3.41 0.35 ng/ml) was lower than counterpart of KK control group (3.92 0.47 ng/ml), the difference did not reach statistic
Discussion
Insulin resistance in peripheral tissues is an essential element in the pathogenesis of T2DM. The KK mouse, an animal model of T2DM, is characterized by insulin resistance, hyperglycemia and hyperinsulinaemia [14]. In the present study, we demonstrated for the first time that Tb4 administration could effectively improve glucose tolerance and ameliorate hyperinsulinemia of KK mouse. We further showed that Tb4 treatment could decrease triglyceride levels and increase the AKT activity of skeletal muscle of KK mouse. Tb4 thus may be potentially useful in improving insulin resistance in patients with type 2 diabetes mellitus. The ultimate goal of T2DM treatment is to reduce mortality and the risk of microvascular and macrovascular complications [15]. The latter, mainly cardiovascular disease is the most frequent cause of increased mortality among T2DM patients [16–18]. Several studies have suggested a causal association between insulin resistance and cardiovascular disease [2,3,19,20]. Tb4, a small ubiquitous protein containing 43 amine acids, has structure/function activity via its actin-binding domain and numerous biological affects on cells [5]. In addition to cell motility, tissue development and maintenance, Tb4 can stimulate migration of cardiomyocytes and endothelial cells and promotes survival of cardiomyocytes [8,21]. Our present study demonstrated that the plasma Tb4 concentration was lower in KK mice compared to that of C57BL mice. Furthermore, we demonstrated that a 12-week Tb4 treatment could lead to significant reductions in hyperglycemia, triglycerides and HbA1c, as well as an increase in adiponectin in KK mouse. Those results indicated that Tb4 and related compounds might be a novel potential alternative anti-diabetic agent for treatment of T2DM. Chronic, low-grade inflammation has been implicated in the path-physiology of T2DM [22,23]. A number of studies have shown that many drugs such as statins and angiotensin
Fig. 4 – Tb4 treatment did not increase insulin secretion in KK mouse. Blood glucose (A) and plasma insulin levels (B) at 30 min after glucose stimulation. Data are presented as the mean W SEM for 8 mice. Significance: #p < 0.05 vs. KK control (CTL) group.
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diabetes research and clinical practice 96 (2012) 53–59
converting enzyme (ACE) inhibitors as well as angiotensin receptor blockers, which have apparent ‘‘anti-inflammatory’’ properties, could reduce the incidence and/or delay the onset of T2DM. High-dose aspirin has been shown to inhibit cyclooxygenase and IkappaB kinase-beta and reduce fasting plasma glucose concentration [15]. In addition, a study has suggested that recombinant IL-1-receptor antagonist, a specific inhibitors of inflammatory pathway, reduced inflammation and improved b-cell function and glycaemic control of patients with T2DM [24]. Tb4 has been shown to be able to exert anti-inflammatory function through suppressing NFkappaB pathway and attenuating neutrophil-associated inflammatory processes [25,26]. However, the concentrations of proinflammation cytokines such as ICAM-1, INF-g, MCP-1, TNF-a, IL-1b and leptin in KK groups were higher than C57BL groups at 12 weeks after Tb4 treatment in this study. Although there was tendency of decrease in those pro-inflammation cytokines in KK Tb4 group compared with KK control group, no statistically significant differences could been found. However, an increased adiponectin level was found in KK Tb4 group (about 50% increase as compared with KK control group). Adiponectin is solely secreted by adipocytes and acts as a hormone with insulin-sensitizing property. Findings from animal studies and metabolic studies in human suggest several mechanisms through which adiponectin may decrease the risk of T2DM, including suppression of hepatic gluconeogenesis, stimulation of fatty acid oxidation in the liver, stimulation of fatty acid oxidation and glucose uptake in skeletal muscle. All these together suggest that Tb4 may regulate blood adiponectin level to achieve anti-diabetic effect. Skeletal muscle, a long-living tissue, is responsible for about 75% of whole-body glucose metabolism. Insulin and exercise are important physiological stimulators for skeletal muscle glucose uptake. The mechanism of insulin-stimulated skeletal muscle glucose uptake has been well characterized and is dependent on insulin receptor substrate-1(IRS1) phosphorylation/PI3 K/AKT pathway which results in Glut-4 translocation [27]. Exercise-stimulated glucose uptake is insulin and PI3 K independent and links to AMP-activated protein kinase (AMPK) and p38 mitogen-activated protein kinase (MAPK) activation [28]. It has been well demonstrated that reduced glucose uptake in skeletal muscle plays an important role in development of T2DM [29]. In the present study, western blot results showed that Tb4 injection improved activity AKT in KK Tb4 group in response to glucose stimulation. The increased pAKT could potentially enhance its ability to activate various downstream effectors in the insulinmediated signaling pathway to transport glucose into skeletal muscle. Consistent with the change of pAKT, the blood glucose level at 30 min after glucose stimulation in KK Tb4 group was lower than KK control group. These results raise one question that whether Tb4 treatment increases insulin secretion from pancreatic b cells or improves insulin resistance of peripheral tissues. Thus, we also measured the insulin levels of mice from two KK groups at 30 min after oral glucose challenge. The results showed that average insulin level of KK control group was higher than KK Tb4 group, although the difference was not statistically significant. This suggested that increased insulin secretion was not the reason that Tb4 increased AKT activity. In addition, Tb4 has been shown to increase the pAKT on
cardiac cells by forming a functional complex with PINCH and integrin-linked kinase in vitro [21], which suggested Tb4 treatment in vivo might activate the AKT directly rather than improve insulin resistance of skeletal muscle. In this study, we sacrificed those mice at 36–48 h after last injection of Tb4, meanwhile, drew blood samples from KK or C57BL mice for measurement of Tb4. The results showed that there were no significant differences in Tb4 concentrations between two KK mice groups after treatment, as well as in two C57BL groups. It might be resulted from short half-life of Tb4. Mora et al. [30] had reported that a significant increase of Tb4 concentration in serum was starting 2 min after single intraperitoneal injection and lasting for 40 min. However, in some organs such as brain, kidney, liver, fat, the wet weight concentration of Tb4 increased significantly from the first 40 min to 8 h after injection in comparison to their baseline wet weight concentration. In addition, the peak wet weight concentration of Tb4 in muscle arrived at 6 h after Tb4 intraperitoneal injection and would last for 24 h, which indicated that Tb4 might take effect for long time in many organs. Although insulin independent glucose uptake pathway had not been investigated in this study, the role of Tb4 on AMPK and p38 MAPK activation was unknown. Together, these results still indicated that improvement of skeletal muscle insulin resistance may be a major mechanism by which Tb4 ameliorated hyperglycemia in KK mice. In conclusion, our preliminary data here indicates that Tb4 administration could improve glucose tolerance and ameliorate muscle insulin resistance. This peptide may be a novel potential alternative insulin sensitizer for treatment of T2DM and would be worthy of further investigation.
Conflict of interest The authors declare that they have no conflict of interest.
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