Anti-diabetes effect of chronic intermittent hypobaric hypoxia through improving liver insulin resistance in diabetic rats

    Anti-diabetes effect of chronic intermittent hypobaric hypoxia through improving liver insulin resistance in diabetic rats Yan-Ming Tian, Yan Liu, Sheng Wang, Yi Dong, Tong Su, Hui-Jie Ma, Yi Zhang PII: DOI: Reference:

S0024-3205(16)30103-5 doi: 10.1016/j.lfs.2016.02.053 LFS 14738

To appear in:

Life Sciences

Received date: Revised date: Accepted date:

12 September 2015 6 January 2016 12 February 2016

Please cite this article as: Tian Yan-Ming, Liu Yan, Wang Sheng, Dong Yi, Su Tong, Ma Hui-Jie, Zhang Yi, Anti-diabetes effect of chronic intermittent hypobaric hypoxia through improving liver insulin resistance in diabetic rats, Life Sciences (2016), doi: 10.1016/j.lfs.2016.02.053

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ACCEPTED MANUSCRIPT Anti-diabetes effect of chronic intermittent hypobaric hypoxia

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through improving liver insulin resistance in diabetic rats

Yan-Ming Tiana,c, Yan Liub, Sheng Wanga,c, Yi Donga, Tong Sua,

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Hui-Jie Maa,c*, Yi Zhanga,c*

Department of Physiology, Hebei Medical University, Shijiazhuang, 050017, P.R.

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Department of Endocrinology, the Third Hospital of Hebei Medical University,

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Shijiazhuang, 050051, P.R. China.

Hebei Collaborative Innovation Center for Cardio-cerebrovascular Disease,

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c

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China.

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Shijiazhuang, 050000, P.R. China.

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Running Title: Chronic intermittent hypobaric hypoxia against diabetes

* Corresponding author: Yi Zhang, Ph.D, Department of Physiology, Hebei Medical University, Zhong Shan Dong Rd., Shijiazhuang 050017, China. E-mail: [email protected], Tel: +86 311 8626 5663, fax: +86 311 8626 6811.

* Co-corresponding author: Hui-Jie MA, Ph.D, Department of Physiology, Hebei Medical University, Zhong Shan Dong Rd., Shijiazhuang 050017, China. E-mail: [email protected], Tel: +86 311 8626 1164, fax: +86 311 8626 6811.

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ACCEPTED MANUSCRIPT ABSTRACT Aim: Cumulating evidence demonstrated that chronic intermittent hypobaric

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hypoxia (CIHH) had beneficial effects on the body. The present study was to

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investigate the anti-diabetes effect of CIHH in type-2 diabetic rats for the first time. Main methods: Sprague–Dawley rats were randomly divided into 4 groups: control group (CON), diabetes mellitus group (DM, induced by high-fat diet combined with

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low-dose streptozotocin), CIHH treatment group (CIHH, simulated 5000-m altitude, 6

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h per day for 28 days), and diabetes mellitus plus CIHH treatment group (DM+CIHH). Histopathology of liver, systolic arterial blood pressure (SAP), blood biochemicals,

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glucose and insulin tolerance were determined. The expression of proteins associated

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with insulin signaling pathway as well as hypoxia induced factors were assayed.

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Key findings: Diabetic rats showed impaired glucose tolerance, dyslipidemia, hepatic steatosis and hepatic insulin resistance in addition to increased SAP. However,

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SAP, serum triglyceride and cholesterol were decreased, and hepatic steatosis and insulin resistance were improved in DM+CIHH rats. Furthermore, the protein expression of glucokinase (GCK), insulin receptor substrates (IRS-1 and IRS-2), and HIF1α were increased, while the expression of phosphoenolpyruvate carboxykinase (PEPCK), was markedly reduced in DM+CIHH rats. Significance: We conclude that CIHH treatment has anti-diabetes effects through ameliorating insulin resistance via hepatic HIF-insulin signaling pathway in type-2 diabetic rats. Keywords: CIHH, insulin resistance, Type 2 diabetes mellitus, IRS, HIF

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ACCEPTED MANUSCRIPT INTRODUCTION Diabetes mellitus, a common metabolic disease with rapidly increasing in

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prevalence in modern society, has become one of the most challenging public health

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problems in the world [1]. Type 2 diabetes mellitus (T2DM) is the major diabetes

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mellitus in human beings (>90% of diabetes mellitus) and a risk factor for cardiovascular diseases [2]. In addition, T2DM results in a lot of chronic complications including retinopathy, nephropathy, peripheral neuropathy and

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autonomic neuropathy [3]. Numerous researches showed that insulin resistance,

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defined as a reduced sensitivity of target tissues to insulin, played a vital role in the pathogenesis of T2DM [4]. In hepatic insulin resistance, the decreased hepatic

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glycogen synthesis and increased hepatic glucose production result in hyperglycemia

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and compensatory hyperinsulinemia [5], as well as dyslipidemia and hepatic steatosis, which further aggravates insulin resistance [6].

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It has been found since 70's that populations living in high altitude have lower blood glucose levels or even lower incidence of type 2 diabetes mellitus than plain

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inhabitant [7, 8]. High altitude hypoxia adaptation has been found to improve glucose tolerance in subjects with metabolic syndrome [9, 10] and T2DM patients [11]. Chronic intermittent hypobaric hypoxia (CIHH) has been proved to have beneficial effects on the body such as cardio-protection and anti-hypertension [12]. Our previous study

displayed

that

CIHH

protected

cardiovascular

system

against

ischemia/reperfusion injury through multiple mechanisms or pathways [13-18]. It was also proved that CIHH regulated immune function and protected rat against collagen-induced arthritis [19]. Recently, we found that CIHH improved the abnormal metabolism, such as decreasing of fasting blood glucose and insulin resistance [20], as well as ameliorating nonalcoholic fatty liver disease (NAFLD) in fructose-induced

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ACCEPTED MANUSCRIPT metabolic syndrome rats [21]. So we hypothesized that CIHH treatment had anti-diabetes effects through

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improving insulin resistance. The aim of present study was to investigate the

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anti-diabetes effects of CIHH and the mechanism in T2DM rats.

MATERIALS AND METHODS

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Animals and CIHH treatment

Adult male Sprague–Dawley rats (body weight 250–300 g, provided by the Animal

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Center of Hebei Medical University) were randomly divided into four groups: diabetes mellitus group (DM), chronic intermittent hypobaric hypoxia treatment group

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(CIHH), diabetes mellitus plus CIHH treatment group (DM+CIHH) and control group

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(CON). DM and DM+CIHH rats were treated by high-fat diet for 4 weeks and intraperitoneal (i.p.) injection of streptozotocin (STZ) to induce T2DM. CIHH rats

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were exposed to hypobaric hypoxia simulating 5000-m altitude (PB = 404mmHg, Po2 = 84mmHg) for 4 weeks, 6 h/day in a hypobaric chamber. DM+CIHH rats were

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treated with 4-week CIHH after T2DM formation. All animals were housed in a temperature-controlled room (22 ± 1°C) with a 12 h/12 h light/dark cycle and had free access to water and food. Systolic arterial blood pressure (SAP) were measured in conscious rats by a tail-cuff pressure meter (LE5001, Panlab) weekly, and body weight, amounts of food consumption, water intake and urinary volume of rats in 24 hours were collected each week. All the experiments were conducted in compliance with the Guide for the Care and Use of Laboratory Animals (National Research Council, 1996). Induction of T2DM model The diabetic rat model was developed according to a modified method previously

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ACCEPTED MANUSCRIPT described [22]. Briefly, Type 2 diabetes was induced by feeding rats with a high-fat diet (20% lard, 20% sucrose, 10% yolk powder, 2.0% cholate, and 48% chow;

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20.12J/g) for 4 weeks followed by a single i.p. injection of low dose STZ (30 mg/kg,

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dissolved in citrate buffer, pH 4.2). Successful development of hyperglycemia was

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checked 1 week after STZ injection (5 weeks after the onset of high-fat diet feeding). The CON and CIHH rats were fed normal chow diet (13.77J/g) and injected with vehicle. Fasting blood glucose (FBG) was assessed in STZ-injected rats using a

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One-Touch Ultra blood glucose meter (ACCU-CHEK, Germany). When FBG in rats

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≥ 11.1 mmol/L, the diabetes model was thought successful and used in experiment. Determination of blood biochemicals

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Blood samples were collected from the angular vein of rats after 12 h fasting at the

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end of the ninth week of experiment (4 weeks after the onset of CIHH) and centrifuged at 3500 rpm for 10 minutes to get serum for assay. Fasting blood glucose,

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serum triglyceride (TG) and cholesterol (CHO) were measured by enzyme coupled colorimetric method with commercial kits (BioSino, China).

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Glucose and insulin tolerance test At the end of the ninth week of experiment (4 weeks after onset of CIHH treatment), animals were fasted overnight and then intraperitoneal injected with glucose (50% solution; 2g/kg body weight), blood samples were taken at 0- (before injection), 30-, 60-, and 120-min after glucose injection. Plasma glucose was determined as described above. The area under the blood glucose curve (AUC) of glucose tolerance tests (GTTs) were calculated according to equation: AUC = (blood glucose at 0-min + blood glucose at 30-min) × 0.25 + (blood glucose at 30-min + blood glucose at 60-min) × 0.25 + (blood glucose at 60-min + blood glucose at 120-min) × 0.5.

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ACCEPTED MANUSCRIPT Three days after GTTs, insulin tolerance tests (ITTs) were performed by insulin intraperitoneal injection (1 U/kg body weight) in rats after a 4-h fasting, followed by

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blood collection at 0 (before injection), 30- and 60-min after insulin injection. Blood

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glucose was determined as described above.

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Serum insulin values were determined by radioimmunoassay, and homeostatic model assessment-insulin resistance (HOMA-IR) scores were calculated from the glucose and insulin values by the following equations: HOMA-IR index = FBG

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(mmol/L) × Fasting insulin (FINS, units/L)/22.5 to assess changes in insulin

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resistance during the experimental period [23]. Histology Examination

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At the end of the experiments (three days after ITTs), rats were fasted overnight

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and euthanized by a sodium pentobarbital overdose (66 mg/kg, intraperitoneal). The hepatic tissues were quickly removed and immerged in 4% paraformaldehyde for 48 h

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and then dehydrated in gradient ethanol step by step. After embedding in wax, tissues were sectioned at 5 μm thickness using microtome (Leica, Germany) and stained with

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hematoxylin-eosin (HE) and examined by a light microscope digital camera (Nikon Instruments, Japan). Western blot analysis To clarify the molecular mechanisms of CIHH treatment, the expression of proteins related to glucose metabolism and insulin signaling in the liver were examined. The hepatic tissues were homogenized in a lysis buffer and protein concentration was determined by the Bradford assay (TIANGEN, China). Protein samples (50 µg) were separated by SDS–PAGE, transferred to a PVDF membrane (Millipore Corporation, USA) that was blocked for 1 h with 5% (w/v) non-fat milk in tris-buffered saline, and incubated with antibodies against HIF1α (1:1000, Wanleibio, China), IRS-2 (1:1000,

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ACCEPTED MANUSCRIPT Cell Signaling Technology, USA), HIF2α (1:1000, abcam, UK), IRS-1 (1:1000, Affinity, USA), PEPCK (1:1000, Sangon Biotech, China), and GCK(1:1000, AVIVA

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SYSTEMS BIOLOGY, USA) overnight at 4 ℃. The same membrane was stripped

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and re-blotted with an anti-β-actin antibody (1:5000, Affinity, USA) for normalization.

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Blots were developed by the chemiluminescent detection method. The protein blots was quantified by densitometry using NIH image software and normalized to β-actin. Data analyses

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All data were expressed as means ± S.E.M.. Results were assessed using one-way

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ANOVA followed by SNK test for multiple comparisons. t test was used for comparison between two groups. A value of p < 0.05 was considered statistically

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significant.

RESULTS

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Effect of CIHH on general condition of animals Compared with CON and CIHH rats, DM and DM+CIHH rats displayed

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polyphagia, polyuria and weight loss (P<0.05-0.01, Fig. 1). The success rate of achieving the T2DM model in the present study was 75% (at first, sixteen rats were involved in DM and DM+CIHH groups, and twelve T2DM rats were left and used in the experiment). CIHH treatment caused no amelioration of diabetic symptom in DM rats, indicating CIHH treatment could not improve diabetic symptom apparently. Effect of CIHH on blood glucose and lipids The serum glucose was increased in DM and DM+CIHH rats compared with CON and CIHH rats (P<0.01). There was no difference in serum glucose between CON and CIHH rats, and between DM and DM+CIHH rats, respectively (P>0.05). The serum TG and CHO were increased in DM rats compared with CON rats (P<0.01), and were

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ACCEPTED MANUSCRIPT decreased in DM+CIHH rats compared with DM rats (P<0.01, Fig. 2). There was no statistical difference in TG and CHO between CON and CIHH rats (P>0.05). The

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results suggested that hyperlipidemia in diabetic rats can be improved by CIHH

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treatment.

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Effect of CIHH on glucose tolerance and insulin sensitivity

During 120 min of glucose tolerance tests, the blood glucose concentrations were increased in DM rats compared with CON rats, and were decreased in DM+CIHH rats

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compared with DM rats (P<0.01). No difference of blood glucose was found between

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CON and CIHH rats (P>0.05, Fig 3a). The calculated AUC values for glucose response were consistent with the glucose tolerance test (Fig. 3b).

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The results of insulin tolerance tests (ITTs) showed that the blood glucose levels

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were significantly higher in DM rats at every minute after insulin treatment, and reduced blood glucose levels were observed in DM rats treated with CIHH at

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comparable time point, indicating CIHH treatment might cause improved insulin resistance in DM animals. In addition, there was no difference found in glucose levels

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at all time point in healthy animals with or without CIHH treatment, indicating in this animal model, CIHH might not cause further improvement in insulin sensitivity in healthy animals (Fig. 4). Compared with CON rats, DM rats showed significantly increased HOMA-IR (P<0.01), and CIHH treatment led to significantly decreased serum insulin levels and HOMA-IR in DM rats (P<0.05-0.01, Fig. 2). Therefore, CIHH treatment improved insulin resistance in diabetic rats. Effect of CIHH on systolic artery blood pressure Significantly increased blood pressure was found in both DM and DM +CIHH rats when compared to both CON and CIHH animals (P<0.01). Intriguingly, increase of

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ACCEPTED MANUSCRIPT blood pressure could be largely improved by CIHH treatment (P<0.01, Fig. 5). There was no significant difference in arterial blood pressure in rats between CIHH and

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pressure in diabetic rats but had no effect on healthy animals.

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CON group (P>0.05). Data indicated that CIHH might improve increased blood

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Histological Analysis

Significant hepatic micro-/macro-steatosis were found in DM rats, combined with hepatocellular ballooning degeneration (Fig. 6). And the pathological changes were

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substantially alleviated in DM+CIHH rats, which suggested a protective effect of

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CIHH on hepatic steatosis in diabetic rats.

Effect of CIHH on liver protein expression

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PEPCK, a key enzyme of gluconeogenesis, was significantly higher expressed at

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protein level in liver from DM rats when compared to CON rats, but was significantly lower in DM+CIHH rats than that in DM rats (P<0.05). The hepatic expression of

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PEPCK was also higher in CIHH rats than that in CON rats (P<0.05, Fig. 7). The protein expression of glucokinase (GCK), a key enzyme of glycolysis, was

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lower in DM but higher in CIHH rats compared to CON rats (P<0.05), and was higher in DM+CIHH rats than that in DM rats (P<0.05, Fig. 7). The protein levels of IRS-1 and IRS-2, the important elements in hepatic insulin signal pathway, were decreased in DM rats compared to CON rats (P<0.05). Compared with DM rats, both IRS-1 and IRS-2 were higher in DM+CIHH rats (P<0.05). And there was no difference in IRS-1 and IRS-2 expression between CON and CIHH rats (P>0.05, Fig. 8). The protein levels of both HIF1α and HIF2α were decreased in DM rats compared to CON rats (P<0.05-0.01). Compared to DM rats, HIF1α was higher (P<0.05) while HIF2α was unchanged in DM+CIHH rats (P>0.05). And the expression of HIF1α was

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ACCEPTED MANUSCRIPT higher while HIF2α was lower in CIHH rats than that in CON rats (P<0.05, Fig. 9).

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DISCUSSION

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In the present study, the diabetic rats displayed classical symptoms of diabetes

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including polyphagia, polydipsia and low body weight with hyperglycemia, dyslipidemia, hepatic steatosis, high blood pressure and insulin resistance. The manifestation of diabetic rats was obviously improved after four-week CIHH

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treatment. In addition, glucose tolerance and insulin sensitivity were increased in

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CIHH-treated diabetes rats. The results demonstrated that CIHH antagonized diabetes through improving insulin resistance and abnormal glycometabolism in type-2

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diabetic rats.

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It is well known that hepatic insulin resistance is a key factor for the development of T2DM. Under physiological condition, insulin stimulates the combination of

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insulin receptor with insulin receptor substrate (IRS) and activates AKT, GSK3β and mTOR signalling pathway, consequently suppresses hepatic gluconeogenesis and

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promotes glycogen synthesis and lipogenesis [24]. As an important pathogenesis of T2DM, hepatic insulin resistance can induce hyperglycemia by increase of hepatic glucose production and induce hepatic steatosis by lipometabolism disturbance, which further results in systemic insulin resistance and abnormal high blood glucose. Although numerous genetic and physiological factors interact to produce and aggravate insulin resistance, human studies implicate dysregulated signaling by the insulin receptor substrate proteins IRS1 and IRS2 as a common underlying mechanism [25], and studies revealed that many insulin responses, including the regulation of carbohydrate and lipid metabolism, required IRS1 and IRS2 [26]. Consistent with the previous report that CIHH improved the insulin resistance in

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ACCEPTED MANUSCRIPT fructose-induced metabolic syndrome rats [20], this study supported that the hepatic insulin resistance occurred with the decrease of both IRS-1 and IRS-2 proteins in DM

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rats, and CIHH treatment effectively prevented the decrease of IRS-1 and IRS-2

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protein in DM rats. It suggested that CIHH improved hepatic insulin resistance

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through antagonizing the down-regulation of hepatic IRS-1 and IRS-2 in DM rats. In physiological condition, insulin inhibits gluconeogenesis through suppressing PEPCK via AKT signaling. It was reported that hyperglycemia and insulin resistance

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in T2DM patients were closely related to the enhancement of gluconeogenesis [27].

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Glucokinase (GCK), a rate-limiting enzyme in glycolysis, plays an important role in the glucose metabolism. There were reports that GCK was decreased in diabetic

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animals and patients [28, 29], and pharmacological activation of GCK could decrease

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blood glucose in diabetic animals and patients [30]. This indicates that depressed glycolysis is related to insulin resistance. Recent research showed that both PEPCK

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and GCK were regulated by IRS [31]. In order to further investigate the effect of CIHH on the regulation of glucose metabolism, we measured the protein levels of

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PEPCK and GCK. The results displayed that the expression of PEPCK was up-regulated while GCK was down-regulated in DM rats, and the up-regulation of PEPCK and down-regulation of GCK were effectively antagonized in CIHH-treated DM rats. It suggested that gluconeogenesis was enhanced and glycolysis was reduced in diabetic rats, and CIHH treatment had antagonistic effects on them through enhancing GCK and inhibiting PEPCK. Hypoxia-inducible factors (HIFs), as the major transcription factors on hypoxia stimulation, are heterodimers consisting of an O2-regulated HIF-α subunit and a constitutively expressed HIF-β subunit. A great number of researches indicate that HIF contributes to the regulation of diverse function and is involved in the diseases

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ACCEPTED MANUSCRIPT such as tumor and metabolism syndrome [32]. The disturbance of HIF signaling was related to hypoinsulinism [33], insulin resistance [34], dysfunction of lipocytes and

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inflammation [35] in diabetes. An impaired glucose tolerance was reported in HIF1α

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knock-out mouse [36]. The effect of HIF2α may depend on the intensity of activation.

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There were reports that the over-activation of HIF2α resulted in severe hepatic steatosis [37, 38], but low level activation of HIF-2α would enhance the hepatic insulin signaling via IRS-2 [39, 40]. There was also a report that insulin resistance

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was aggravated in Hif-2α heterozygous-null mice [41]. All of these studies suggest

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that there is a cross-talk between hypoxia and insulin signaling. And it is known that HIF1α is activated by CIHH [42]. Our results showed that hepatic HIF1α and HIF2α were down-regulated in the DM rats, and CIHH effectively antagonized the

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decreasing of HIF1α, but not HIF2α in DM rats. It suggested that HIF1α was involved in the anti-diabetes effect of CIHH.

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CONCLUSION

In conclusion, this study demonstrated for the first time that CIHH had

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anti-diabetes effect, improving abnormal glucose and lipid metabolism, alleviating hepatic steatosis, and decreasing high blood pressure in rats, which might be achieved through ameliorating insulin resistance via hepatic HIF-insulin signaling pathway. CIHH may be a potential novel therapeutics to treat diabetes.

Acknowledgement This work was supported by National Basic Research Program of China (2006CB504100, 2012CB518200).

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hypoxia and insulin signaling through Phd3 regulates hepatic glucose and lipid metabolism and ameliorates diabetes. Nature medicine. 2013;19:1325-30. [40] Wei K, Piecewicz SM, McGinnis LM, Taniguchi CM, Wiegand SJ, Anderson K, et al. A liver Nature medicine. 2013;19:1331-7.

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Hif-2alpha-Irs2 pathway sensitizes hepatic insulin signaling and is modulated by Vegf inhibition.

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[41] Choe SS, Shin KC, Ka S, Lee YK, Chun JS, Kim JB. Macrophage HIF-2alpha ameliorates adipose tissue inflammation and insulin resistance in obesity. Diabetes. 2014;63:3359-71. [42] Sanchis-Gomar F, Vina J, Lippi G. Intermittent hypobaric hypoxia applicability in myocardial

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infarction prevention and recovery. Journal of cellular and molecular medicine. 2012;16:1150-4.

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ACCEPTED MANUSCRIPT Figure Legend Fig. 1. Effect of CIHH on food intake (A), water intake (B), urine volume (C), and

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body weight (D) in rats. The arrows indicate the onset of high-fat feeding, the timing

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of STZ injection and the onset of CIHH treatment. CON: control group, CIHH: CIHH

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group, DM: DM group, DM+CIHH: DM+CIHH group, All data were expressed as

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mean ± SEM, n=6 for each group, * P<0.05 **P<0.01 vs. CON.

Fig. 2. Effect of CIHH on blood biochemicals and insulin resistance index in rats.

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FBG: Fasting blood glucose, TG: Triglyceride, CHO: Cholesterol, INS: Insulin, HOMA-IR: Insulin resistance index, CON: control group, CIHH: CIHH group, DM:

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DM group, DM+CIHH: DM+CIHH group, All data were expressed as mean ± SEM,

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n=6 for each group, *P<0.05 **P<0.01 vs CON, #P<0.05 ##P<0.01 vs DM.

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Fig. 3. Effect of CIHH on glucose tolerance (A) and area under the curve (B) in rats. AUC: area under the curve, CON: control group, CIHH: CIHH group, DM: DM

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group, DM+CIHH: DM+CIHH group, All data were expressed as mean ± SEM, n=6 for each group, **P<0.01 vs CON, ## P<0.01 vs DM.

Fig. 4. Effect of CIHH on insulin tolerance (A) and the percentage of basal blood glucose (B) in rats. CON: control group, CIHH: CIHH group, DM: DM group, DM+CIHH: DM+CIHH group, All data were expressed as mean ± SEM, n = 6 for each group, **P<0.01 vs CON, ## P<0.01 vs. DM.

Fig. 5. Effect of CIHH on artery blood pressure in rats. The arrows indicate the onset of high-fat feeding, the timing of STZ injection and the onset of CIHH treatment.

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ACCEPTED MANUSCRIPT CON: control group; CIHH: CIHH group; DM: DM group; DM+CIHH: DM+CIHH group. All data were expressed as mean ± SEM. n = 6 for each group, **P<0.01 vs

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CON, ## P<0.01 vs DM.

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Fig. 6. Histological analysis of liver by HE staining (magnification, 400×). The arrow shows the fat vacuoles in hepatocytes, CON: control group, CIHH: CIHH group, DM:

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DM group, DM+CIHH: DM+CIHH group.

Fig. 7. Expression of glycometabolism related protein in rats. CON: control group,

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CIHH: CIHH group, DM: DM group, DM+CIHH: DM+CIHH group, All data were

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#P<0.05 vs DM.

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expressed as mean ± SEM. n = 4 for each group, *P<0.05 **P<0.01 vs CON,

Fig. 8. Expression of IRS protein in rats. CON: control group, CIHH: CIHH group,

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DM: DM group, DM+CIHH: DM+CIHH group, All data were expressed as mean ± SEM. n = 4 for each group, *P<0.05 **P<0.01 vs CON, #P<0.05 vs DM.

Fig. 9. Expression of HIF protein in rats. CON: control group, CIHH: CIHH group, DM: DM group, DM+CIHH: DM+CIHH group, All data were expressed as mean ± SEM. n = 4 for each group, *P<0.05 **P<0.01 vs CON, #P<0.05 vs DM.

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