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Beneficial effects of oligopeptides from marine salmon skin in a rat model of type 2 diabetes
Nutrition 26 (2010) 1014–1020
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Basic nutritional investigation
Beneficial effects of oligopeptides from marine salmon skin in a rat model of type 2 diabetes Cui-Feng Zhu M.D. a, b, Hong-Bing Peng M. Med. a, Gui-Qin Liu B. Med. c, Fan Zhang M. Med. b, Yong Li Ph.D. a, * a b c
Department of Nutrition and Food Hygiene, School of Public Health, Peking University, Beijing, PR China Shenzhen Hospital, Peking University, Shenzhen PR China Shenzhen Traditional Chinese Medical Hospital of Futian District, Shenzhen, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 25 February 2009 Accepted 6 January 2010
Objective: This study aimed at investigating whether treatment with oligopeptides from marine salmon skin (OMSS) could modulate type 2 diabetes mellitus (T2DM)-related hyperglycemia and b-cell apoptosis in rats induced by high fat diet and low doses of streptozotocin and its therapeutic mechanisms. Methods: Groups of T2DM rats were treated with OMSS or bovine serum albumin (3.0 g/kg/d) for 4 wk and their blood samples, together with those of normal control rats, were collected before and 4 wk after treatment. The levels of fasting blood glucose (FBG) and insulin, serum superoxide dismutase (SOD), malondialdehyde (MDA), and glutathione (GSH), tumor necrosis factor-alpha (TNFa), and interferon-gamma (IFNg) in rats were determined. The islet cell apoptosis and Fas/ FasL expression were detected by TUNEL and immunohistochemistry. Results: In comparison with control rats, higher levels of FBG and frequency of apoptotic islet cells were detected in the bovine serum albumin group of diabetic rats, accompanied by higher levels of Fas expression in the pancreatic islets, serum TNFa, IFNg, and MDA, but lower levels of SOD and GSH. However, the levels of FBG and frequency of apoptotic islet cells were significantly reduced in OMSS-treated rats. Lower levels of Fas expression were observed in the pancreatic islets of OMSS-treated rats. Significantly reduced levels of serum TNFa, IFNg, and MDA, but increased levels of SOD and GSH, were detected in OMSS-treated rats. Conclusions: Treatment with OMSS significantly reduced FBG in diabetic rats. This antidiabetic activity may be mediated by down-regulating T2DM-related oxidative stress and inflammation, protecting the pancreatic b-cells from apoptosis. Ó 2010 Elsevier Inc. All rights reserved.
Keywords: Oligopeptides Type 2 diabetes Apoptosis Rat Salmon
Introduction Diabetes mellitus is a major threat to public health, as the incidence of diabetes is rapidly increasing in the world. Diabetes mellitus currently affects more than 194 million individuals worldwide [1]. It is estimated that the number of patients with diabetes will increase to 333 million in 2025 [2]. Among the major types of diabetes, type 2 diabetes mellitus (T2DM) is highly prevalent, accounting for about 90% of the total diabetic patients [3]. Although many therapeutic reagents, together with diet and exercise programs, can effectively correct hyperglycemia in T2DM patients, these therapeutic strategies fail to effectively inhibit the pathogenic process of T2DM and prevent * Corresponding author. Tel.: 86 10 82801177; fax: 86 10 82801177. E-mail address: [email protected] (Y. Li). 0899-9007/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.nut.2010.01.011
T2DM-related complications. Therefore, discovery and development of new therapeutic reagents will be of great significance. Both insulin resistance and b-cell dysfunction are important characters of T2DM [4]. During the pathogenic process of T2DM, many factors contribute to the development of insulin resistance and b-cell dysfunction and they include genetic, environmental, and individual behavioral factors, such as overweight, less exercise, hypertension, and stress. The b-cell dysfunction is crucial for T2DM development as individuals can develop T2DM in the absence of insulin resistance, while individuals with insulin resistance alone may fail to develop T2DM [4,5]. Indeed, many patients with T2DM show impaired b-cell function [6]. Furthermore, patients with T2DM have already lost 50% of b-cell function when they were diagnosed and suffer with further deterioration of b-cell function on an average of 7% yearly, highlighting the importance of b-cell defects in the pathogenesis
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of T2DM [7]. Hence, therapeutic strategies for the preservation of b-cell function may be crucial for the balance of glucose metabolism in T2DM patients. Numerous documents have demonstrated that oxidative stress-related production of reactive nitrogen species (RNS) and reactive oxygen species (ROS) is a risk factor for the cytotoxicity of insulin-producing b-cells in the pancreatic islets in the pathogenesis of T2DM [8]. In addition, chronic inflammation and associated proinflammatory cytokines, such as tumor necrosis factor-alpha (TNFa) and interferon-gamma (IFNg), as well as the death factors of Fas and FasL, are also dangerous events that can trigger b-cell apoptosis in the pancreatic islets, leading to b-cell dysfunction [9–11]. Indeed, b-cell apoptosis is responsible for the loss of b-cell mass in animal models of T2DM [12]. Modulating proinflammatory parameters have been shown to be beneficial for T2DM patients [12]. Thus, therapeutic approaches designed to reduce the sensitivity of b-cells to apoptosis triggers and to down-regulate inflammation may effectively protect the b-cells from apoptosis-related b-cell dysfunction, beneficial for T2DM patients. Medical nutrition therapy has been shown to improve in glucose homeostasis and be beneficial for T2DM patients, reducing T2DM-related complications [12]. Previous study has shown that treatment with dietary cod (morrhua) proteins improved the insulin sensitivity in insulin-resistant individuals and reduced insulin-resistance-related metabolic disorders, contributing to the prevention of T2DM [13]. Our previous findings demonstrated that treatment with oligopeptides from marine salmon (Oncorhynchus keta) skin (OMSS) inhibited inflammation by reducing the production of proinflammatory cytokines in mice [14]. Accordingly, we hypothesized that treatment with OMSS could modulate hyperglycemia in diabetic individuals by down-regulating proinflammatory cytokine production and reducing oxidative stress, leading to the preservation of the insulin-producing b-cells from apoptosis. A high fat diet can induce hyperlipidemia and associated oxidative stress, which usually results in lipid peroxidation and the production of malondialdehyde (MDA) [10]. Furthermore, the metabolism of oxidative stress-related RNS and ROS usually exhausts antioxidant superoxidize dismutase (SOD) and glutathione (GSH) [15]. Therefore, the levels of serum MDA, SOD, and GSH will be indicative of oxidative stress in vivo. Streptozotocin (STZ) is a glucosamine-nitrosourea compound and can be selectively toxic to the pancreatic b-cells through the GLUT2 transporter by oxidative stress-related DNA damage in the sensitive cells [16]. While treatment with a high dose of STZ usually induces aggressive b-cell death and insulin deficiency, treatment with multiple low doses of it commonly causes inflammation and partial loss of b-cells as well as b-cell-specific autoimmunity in animals [17]. In this study, we fed male Sprague-Dawley (SD) rats with a high fat diet for the induction of insulin resistance and oxidative stress and then injected them with three low doses of STZ for the partial damage of b-cells to generate a rat model of T2DM. Subsequently, we tested whether treatment with OMSS could modulate hyperglycemia in T2DM rats and determined whether OMSS treatment could alter T2DMrelated apoptosis of b-cells and the levels of oxidative stress and inflammation in T2DM rats.
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Animals were housed under specific pathogen-free conditions in a controlled humidity, temperature, and light/dark (12:12 h) -switching facility. The experimental protocols were approved by the Animal Experimentation Committee of Shenzhen Hospital, Beijing University.
Preparation and identification of OMSS OMSS was prepared from wild Chum Salmon (Oncorhynchus kern) that were caught from the East China Sea (with an average body weight of 1.47 kg) and provided by the CF Haishi Biotechnology (Beijing, China). Briefly, fishes were cleaned and the meat was minced, followed by defatting [18]. The materials were homogenized in distilled water and digested with the mixed proteases (3000 U/g protein) at 40 C for 3 h. The resultant hydrolysate was centrifuged at 26 000 g and the supernatants were subsequently filtered through ceramic membrane (200 mm) for the purification of it. OMSS powder was generated by ultrafiltration. The generated OMSS was analyzed by high-performance liquid chromatography (with a cutoff of 10 000 Da) to remove undigested proteins, desalination, cryoconcentration under vacuum at 70 C, decolorization with the medicinal charcoal (Hangmu Corporation, Hangzhou, China), and lyophilization (Waters Corp., Milford, MA, USA) using a Phenomenex C18 column (10 250 mm), acetonitrile 0.05 mol/L phosphate buffer (pH 3.2, 10:90) with a flow rate of 2.0 mL/ min. The analysis was monitored at 260 nm using a Water 486 tunable UV detector. Subsequently, the OMSS was characterized by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS, LDI-1700; Linear Scientific Inc., Reno, NV, USA). The amino acid composition of OMSS was further analyzed using an H835-50 automatic amino acid analyzer (Hitachi, Tokyo, Japan), and the amount of free amino acids was measured by high-performance liquid chromatography. The OMSS contained oligopeptides with molecular weights of 130–3000 Da (S1), accounting for >95% of total proteins with the purity of 90.6%.
Diet preparation and diabetes induction Control and experimental diets were prepared in our laboratory. The high fat diet contained 58%, 26%, and 16% calories from fats, carbohydrates, and proteins, respectively, and a total of 5.6 kcal/g. The control diet contained 29%, 39%, and 32% calories from fats, carbohydrates, and proteins, respectively, and a total of 2.8 kcal/g. Groups of SD rats were randomly divided and fed with control diet as normal control or with a high fat diet with free access of water for the induction of diabetes. After feeding for 4 wk, 10 rats from control and the high fat diet groups were sacrificed and their aorta blood samples were collected for the measurement of fasting blood glucose (FBG) and insulin. After confirming insulin resistance, the rats were injected intraperitoneally with 1% STZ (30 mg/kg in body weight) in citrate buffer (pH 4.0), while the rats in the control group received vehicle every 4 d for three injections. The levels of glucose in their blood were monitored every other day using a portable glucometer (Accu-Chek Active; Roche Diagnostics Ltd., Mannheim, Germany). Individual animals with two consecutive blood glucose levels 16.7 mmol/L were considered diabetic, while those animals with a level of blood glucose <16.7 mmol/L were excluded from the study.
Treatment and sampling After identifying diabetes, groups of diabetic rats (n ¼ 10 per group) were treated with 3 g/kg OMSS (OMSS group) or bovine serum albumin (BSA) in water (BSA group) daily by oral gavage for 4 wk. At the end of the experiment, the animals were fasted overnight and sacrificed. Their blood was collected and centrifuged at 1734 g for 5 min. Their sera were collected and stored at 80 C for further analysis. Their pancreatic tissues were dissected out for histological examination.
Immunohistochemical analysis The pancreatic tissues were fixed in a 4% paraformaldehyde solution, dehydrated with ethanol, and embedded in paraffin. The tissue sections at 4 mm were prepared and stained with hematoxylin and eosin or antibodies against human Fas or Annexin-V or isotype controls (Santa Cruz Biotechnology, CA, USA). The number of positive staining cells was determined by counting five randomly selected islets per rat in high-power fields (400).
Materials and methods Determination of serum TNFa and IFNg Animals Male SD rats at 6 to 8 mo of age and around 180–220 g in body weight were purchased from the Experimental Animal Center, Guanxi Medical University.
Serum concentrations of TNFa and IFNg were determined by ELISA using the ELISA kits (BioSource International, Camarillo, CA, USA), according to the manufacturer’s instructions.
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Table 1 Effect of OMSS on fasting blood glucose and serum insulin Group
FBG (mmol/L)
Fasting insulin (mIU/mL)
Control Vehicle OMSS
5.73 0.38 18.22 5.82* 13.57 2.56*,y
7.735 0.439 8.176 0.869 6.995 1.609
Data are expressed as mean SD of each group (n ¼ 10). * P < 0.05 versus control group. y P < 0.05 versus vehicle treatment of diabetic group.
Malondialdehyde, superoxidize dismutase, and glutathione assays The levels of serum MDA were evaluated by the Thiobarbituric Acid Reactive Substances (TBARS) assay using the spectrophotometer [19]. The contents of serum GSH were determined by the spectrophotometric assay, as described previously [20]. The serum SOD levels were determined based on the inhibition of SOD on the reduction of nitroblue tetrazolium by superoxide to form formazan using the xanthine and xanthine oxidase [21]. Hence, the SOD activity is inversely proportional to the amount of the formazane formed, measured at 560 nm. The absorbance was measured in an ELX800 spectrophotometer (Bio-Tek Instruments Inc., CA, NY, USA). The MDA, GSH, and SOD determination kits were purchased from Nanjing Jiancheng Biochemical Company (Nanjing, China) using standard MDA, GSH, or SOD as the positive control, respectively. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick end-labeling (TUNEL) assay Apoptotic cells in the pancreatic islets were measured by the TUNEL assays using the in situ apoptosis detection kit (Roche), according to the manufacturer’s instruction. The apoptotic cells were examined for at least 25 islets per rat and imaged under a light microscope with a magnification of 400. The percentage of apoptotic cells was calculated as the ratio of number of apoptotic cells/total number of nuclei counted. Statistical analysis Data are expressed as mean standard deviation. The difference among groups was tested by one-way ANOVA, followed by Scheffe’s modified F-test for multiple comparisons using the SPSS 13.0. A value of P < 0.05 was considered statistically significant.
Results Effect of OMSS treatment on FBG and serum insulin in diabetic SD rats Feeding with a high fat diet usually induces obesity and insulin resistance in sensitive animals and this, together with a low dose of STZ treatments, commonly causes severe diabetes. Following feeding with a high fat diet and treatment with STZ, their FBG and serum insulin levels were measured in Table 1. While control rats fed with a regular diet showed normal levels of FBG, the rats fed with a high fat diet and injected with STZ developed diabetes with high levels of FBG. Interestingly, the average levels of FBG in the OMSS group were significantly lower than that of the BSA group (P < 0.05). Furthermore, analysis of serum insulin revealed that there was no significant difference among these groups of rats. These data indicated that treatment with OMSS reduced FBG levels and improved hyperglycemia in diabetic rats but did not affect insulin expression and secretion in fasted diabetic rats. Effect of OMSS treatment on the pancreatic islets in diabetic rats Next, the effect of OMSS treatment on the pancreatic islets was histologically examined. While intact islets with healthy islet cells were observed in control rats, many shrunken and small islets with impaired cell membranes were detected in the
Fig. 1. Histological analysis of the pancreatic islets. Groups of diabetic SD rats were treated with OMSS or BSA for 4 wk and their pancreatic islets, together with those of normal control rats, were histologically examined by H&E staining. Data are representative of single islet from each group of rats. (A) Control rats; (B) BSA group of rats; and (C) OMSS group of rats.
BSA group of rats (Fig. 1). In contrast, all islets in the OMSS group of rats remained intact without obviously pathogenic signs. Hence, OMSS treatment preserved the islet cells from T2DMrelated damage. STZ is a potent toxicant and causes strong oxidative stress, leading to cytotoxicity, particularly for highly sensitive b-cells in the pancreatic islets. While a high dose of STZ usually causes extensive b-cell necrosis, multiple low doses of it commonly induce b-cell apoptosis. We further examined the effect of OMSS treatment on the pancreatic islet cell apoptosis in diabetic rats by the TUNEL assays (Fig. 2). While there were rare apoptotic islet cells in the pancreatic islets of control rats, many pancreatic islet cells already underwent apoptosis in BSA-treated diabetic rats that did not receive OMSS treatment. In contrast, a few apoptotic islet cells were observed in the OMSS group of rats. Quantitative analysis revealed that the percentage of apoptotic islet cells in the BSA group of rats was 30-fold higher than that in control rats (Table 2). However, a significantly reduced frequency of
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Table 2 The frequency of apoptotic islet cells Group
Positive (%)
Control Vehicle OMSS
0.870 0.321 28.321 2.145* 10.896 5.019*,y
Data are expressed as meanSD of each group (n ¼ 10). * P < 0.01 versus control group. y P < 0.05 versus vehicle-treated diabetic group.
and Annexin-V formation in the pancreatic islets by immunohistochemical analysis. There is no detectable FasL expression in the pancreatic islets from these groups of rats (data not shown). As shown in Figure 3, higher levels of Fas expression were detected in the pancreatic islets of the BSA group of rats, accompanied by stronger staining of anti-Annexin V, as compared with that in the control rats. In contrast, the levels of Fas expression and antiAnnexin V staining in the pancreatic islets of OMSS-treated rats were significantly lower than that in BSA-treated rats (Table 3). The lower levels of anti-Annexin V staining were consistent with lower frequency of apoptotic islet cells. Therefore, OMSS treatment down-regulated the expression of Fas in the b-cells of pancreatic islets, reducing the sensitivity of b-cells to apoptosis induction. Effects of OMSS on antioxidant defense system in diabetic rats
Fig. 2. Apoptotic islet cells in the rat pancreases. After treatment with OMSS or BSA for 4 wk, the effect of OMSS treatment on T2DM-related islet cell apoptosis was characterized by the TUNEL assay using DAB as the substrate. Data are representative of each group of rats (n ¼ 10) and the arrow indicates the apoptotic islet cells. (A) Control rats; (B) BSA group of rats; and (C) OMSS group of rats.
Oxidative stress induced by STZ treatment usually promotes the production of ROS, which can induce lipid peroxidation and MDA production as well as exhaustion of antioxidant SOD and GSH. We further determined the impact of OMSS treatment on the levels of serum MDA, SOD, and GSH in diabetic rats. As shown in Table 4, while low levels of serum MDA were detected in control rats, the levels of MDA in the BSA group were near six-fold higher than that in control rats, reflecting the oxidative stress-related high levels of lipid peroxidation in the BSA group of rats. In contrast, the levels of serum MDA were significantly reduced in the OMSS-treated rats, indicating that OMSS inhibited oxidative stress-related lipid peroxidation. Analysis of serum antioxidant SOD and GSH revealed that the levels of SOD and GSH in the BSA group of rats were significantly lower than that in the control rats (Table 4). However, higher levels of serum SOD and GSH were observed in the OMSS-treated rats and their serum SOD and GSH levels were significantly elevated, near to that of the control rats, as compared with that in the BSA group of rats. Together, these data demonstrated that OMSS treatment preserved antioxidant SOD and GSH and inhibited the oxidative stress-related lipid peroxidation in diabetic rats. Effect of OMSS treatment on diabetes-associated inflammation
apoptotic islet cells was observed in the OMSS-treated rats, as compared with that of BSA-treated rats. Apparently, OMSS treatment protected the islet cells from T2DM-associated islet cell apoptosis. Effects of OMSS on the expression of Fas/FasL and Annexin V Oxidative stress induced by STZ treatment can usually upregulate the expression of Fas in the b-cells of pancreatic islets, leading to high sensitivity of b-cells to apoptotic triggers. It is possible that OMSS treatment may modulate the expression of Fas and FasL in the pancreatic islet cells, reducing their sensitivity to apoptosis. We further examined the expression of Fas, FasL,
Finally, we tested whether OMSS treatment could modulate systemic inflammation in diabetic rats as inflammation and related proinflammatory cytokines are associated with the pathogenesis of T2DM induced by high fat feeding. We found that high levels of serum proinflammatory cytokines, such as TNFa and IFNg, were detected in the BSA group of rats, which was significantly higher than that in the control rats. In contrast, the levels of serum TNFa and IFNg in the OMSS group of rats were significantly reduced by 40.08% or 35.38%, respectively, as compared with that in the BSA group of rats (P < 0.05, Table 5). Therefore, the significantly reduced levels of proinflammatory cytokines in the OMSStreated rats demonstrated that OMSS treatment reduced diabetes-associated inflammation in diabetic rats.
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Fig. 3. Immunohistochemistry analysis of Fas and Annexin-V in the pancreatic islets. The effect of OMSS treatment on T2DM-related Fas expression and Annexin-V formation was determined by immunohistochemistry analysis using specific antibodies. Data are representative of each group of rats (n ¼ 10). A, B, and C, Fas staining; D, E, and F, Annexin-V staining. (A, D) Control rats; (B, E) BSA group of rats; (C, F) OMSS group of rats.
Discussion During the pathogenesis of T2DM, insulin resistance, b-cell dysfunction, and apoptosis are characteristic. High fat diet feeding usually induces insulin resistance and treatment with multiple low doses of STZ commonly promotes b-cell apoptosis in animals. In the present study, we employed a rat model of T2DM by feeding SD rats with a high fat diet and injecting them with multiple low doses of STZ for examining the effect of OMSS treatment on T2DM-related hyperglycemia, oxidative stress, Table 3 The frequency of Fasþ or Annexin-Vþ islet cells Group
Fasþ (%)
Annexin-Vþ (%)
Control Vehicle OMSS
2.35 0.19% 85.70 3.34* 62.93 2.87*,y
2.47 2.01 85.6 2.1* 76.2 3.4*
Data are expressed as mean SD of each group (n ¼ 10). * P < 0.01 versus control group. y P < 0.05 versus vehicle-treated diabetic group.
chronic inflammation, and associated b-cell apoptosis as this model has been shown to represent the pathogenesis of T2DM [22]. We found that all rats that had been injected with STZ developed diabetes within 1 wk post-STZ injection. Interestingly, OMSS treatment corrected hyperglycemia and significantly reduced FBG in diabetic rats. However, this treatment failed to modulate significantly the levels of serum insulin, as there was no significant difference in the levels of fasting serum insulin among groups of rats, regardless of whether they were treated with OMSS or control BSA. This was not surprising as 15% of b-cell mass can maintain a normal insulin level by high levels of expression and secretion of insulin from the remaining b-cells in the pancreatic islets. Importantly, the significantly reduced levels of FBG indicated that OMSS treatment improved the glucose metabolism and may aid in design of new therapy for T2DM in humans. Following the T2DM progression, hyperglycemia, oxidative stress, and inflammation can induce b-cell apoptosis, particularly after injecting with multiple low doses of STZ. Accordingly, we further examined the effect of OMSS treatment on islet cell
C.-F. Zhu et al. / Nutrition 26 (2010) 1014–1020 Table 4 Effect of OMSS on serum MDA, SOD, and GSH levels Group
MDA (nmol/mL)
SOD (U/mL)
GSH (mg/L)
Control Vehicle OMSS
8.90 2.69 51.82 16.91* 22.23 10.47y,x
107.88 8.30 74.93 2.20* 92.53 6.82x
474.75 47.41 335.75 66.32* 442.87 91.09z
Data are expressed as mean SD of each group (n ¼ 10). * P < 0.01 or yP < 0.05 versus control group. z P < 0.01 or xP < 0.05 versus vehicle-treated diabetic group.
apoptosis in diabetic rats. We found that few apoptotic islet cells were detected in control rats, while a higher frequency of apoptotic islet cells were observed, accompanied by significantly higher levels of anti-Annexin V staining in the BSA group of rats. However, the frequency of apoptotic islet cells in the OMSS-treated rats was significantly reduced. The significantly reduced frequency of apoptotic islet cells was accompanied by remarkable lower levels of anti-Annexin V staining, further indicating that OMSS treatment preserved the islet cells from T2DM-related apoptosis in our experimental model. There are many factors that can trigger islet cell apoptosis during the pathogenic process of T2DM. Hyperglycemia and chronic inflammation can up-regulate the expression of Fas and FasL in the pancreatic islets. High levels of Fas expression can increase the sensitivity of b-cells to apoptosis induction, while high levels of FasL expression can trigger apoptotic signal cascade and lead to b-cell apoptosis in the islets. We characterized the expression of Fas and FasL in the pancreatic islets of these groups of rats. We found that although little FasL expression was detected in the pancreatic islets of these groups of rats, significantly higher levels of Fas did display in the pancreatic islets of BSA group of rats. Surprisingly, significantly lower levels of Fas expression were observed in the pancreatic islets of the OMSS-treated rats. These data suggest that high levels of Fas expression in the pancreatic islets may, at least partially, contribute to the high frequency of islet cells undergoing apoptosis in diabetic rats, while OMSS may protect the islet cells from apoptosis by down-regulating the expression of Fas. However, the mechanisms by which OMSS treatment down-regulates the expression of Fas, inhibiting islet cell apoptosis in diabetic rats, remain to be further determined. Chronic inflammation and high levels of proinflammatory cytokines, such as TNFa and IFNg and others, are crucial for the pathogenesis of T2DM. TNFa has been widely accepted as a crucial factor, responsible for the development of impaired glucose metabolism and insulin resistance in obese and diabetic subjects [23,24]. TNFa can also trigger b-cell apoptosis through its receptor and downstream signaling events. Indeed, high levels of TNFa were detected in subjects with insulin resistance and T2DM [22]. IFNg, a Th1 proinflammatory cytokine, is important for defense against viral and intracellular pathogens and organ-specific autoimmune diseases [25]. IFNg can up-regulate the expression of MHC molecules and activate macrophages and dendritic cells, promoting antigen-presenting
Table 5 Effect of OMSS on serum TNFa and IFNg levels Group
TNFa (pg/mL)
IFNg (pg/mL)
Control Vehicle OMSS
39.450 8.243 109.850 10.973* 65.817 1.892y
47.220 11.133 126.922 21.711* 82.100 11.747y
Data are expressed as mean SD of each group (n ¼ 10). * P < 0.01 versus control group. y P < 0.05 versus vehicle-treated diabetic group.
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activity and inflammatory cascade. High levels of IFNg have been associated with the development of T2DM and latent autoimmune diabetes in adults [25]. We measured the levels of serum TNFa and IFNg and found that significantly higher levels of serum TNFa and IFNg were detected in the BSA group of rats, reflecting a high degree of inflammation. In contrast, the levels of serum TNFa and IFNg were significantly reduced in the OMSStreated rats. These data indicated that treatment with OMSS inhibited T2DM-related inflammation in diabetic rats. Given that TNFa and IFNg are important for T2DM-related inflammation and associated b-cell apoptosis, the significantly reduced levels of serum TNFa and IFNg may also contribute to low frequency of apoptotic islet cells in the OMSS-treated rats. These data extended our previous findings that OMSS treatment inhibited chronic inflammation in animals [14]. Oxidative stress is critical for the development of insulin resistance and b-cell damage in the pathogenic process of T2DM. Oxidative stress usually promotes the production of ROS and RNS, which can change in the mitochondrial membrane potential, and the release of cytochrome c, triggering cell apoptosis [26]. We determined the effect of OMSS treatment on systemic levels of oxidative stress. We found significantly higher levels of MDA, an indicative of lipid peroxidation, and lower levels of SOD and GSH in the BSA group of rats. However, following treatment with OMSS, the levels of serum MDA were significantly reduced while the levels of antioxidant SOD and GSH were dramatically elevated in the OMSS-treated rats, as compared with that in BSA group of rats. These data indicated that OMSS treatment inhibited hyperglycemia-related oxidative stress in diabetic rats. Notably, high levels of ROS and RNS have been demonstrated to be risk factors for the b-cell apoptosis in animal models and treatment with scavenger for nitric oxide has been shown to inhibit b-cell apoptosis [27,28]. Therefore, the reduced levels of serum MDA and elevated levels of antioxidant SOD and GSH by OMSS treatment may also protect the pancreatic b-cells from T2DM-related apoptosis. In summary, our data, to the best of our knowledge, demonstrate for the first time that OMSS treatment significantly reduced FBG and T2DM-related islet cell apoptosis in diabetic rats. Furthermore, OMSS treatment inhibited inflammation significantly by decreasing the levels of serum proinflammatory TNFa and IFNg and down-regulating the expression of Fas. In addition, OMSS treatment reduced oxidative stress-related formation of MDA but elevated the levels of serum antioxidant SOD and GSH. Therefore, it is possible that the effect of OMSS treatment on T2DM may be mediated by inhibiting inflammation, oxidative stress, and the Fas expression, protecting the b-cells in the pancreatic islets from apoptosis. Although further investigations are necessary for determination of mechanisms underlying the action of OMSS treatment in the diabetes progression, potentially, our findings may provide a basis for further design of new therapy for T2DM. Acknowledgments This study was supported by grants from the National Key Technologies R&D Program of China (No. 2006BAD27B01) and the Science & Technology Committee of Futian District State, Shenzhen City, Guangdong Province of China (No. 20080214142). Supplementary data Supplementary data associated with this article can be found, in the online version, at 10.1016/j.nut.2010.01.011.
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Basic nutritional investigation
Beneficial effects of oligopeptides from marine salmon skin in a rat model of type 2 diabetes Cui-Feng Zhu M.D. a, b, Hong-Bing Peng M. Med. a, Gui-Qin Liu B. Med. c, Fan Zhang M. Med. b, Yong Li Ph.D. a, * a b c
Department of Nutrition and Food Hygiene, School of Public Health, Peking University, Beijing, PR China Shenzhen Hospital, Peking University, Shenzhen PR China Shenzhen Traditional Chinese Medical Hospital of Futian District, Shenzhen, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 25 February 2009 Accepted 6 January 2010
Objective: This study aimed at investigating whether treatment with oligopeptides from marine salmon skin (OMSS) could modulate type 2 diabetes mellitus (T2DM)-related hyperglycemia and b-cell apoptosis in rats induced by high fat diet and low doses of streptozotocin and its therapeutic mechanisms. Methods: Groups of T2DM rats were treated with OMSS or bovine serum albumin (3.0 g/kg/d) for 4 wk and their blood samples, together with those of normal control rats, were collected before and 4 wk after treatment. The levels of fasting blood glucose (FBG) and insulin, serum superoxide dismutase (SOD), malondialdehyde (MDA), and glutathione (GSH), tumor necrosis factor-alpha (TNFa), and interferon-gamma (IFNg) in rats were determined. The islet cell apoptosis and Fas/ FasL expression were detected by TUNEL and immunohistochemistry. Results: In comparison with control rats, higher levels of FBG and frequency of apoptotic islet cells were detected in the bovine serum albumin group of diabetic rats, accompanied by higher levels of Fas expression in the pancreatic islets, serum TNFa, IFNg, and MDA, but lower levels of SOD and GSH. However, the levels of FBG and frequency of apoptotic islet cells were significantly reduced in OMSS-treated rats. Lower levels of Fas expression were observed in the pancreatic islets of OMSS-treated rats. Significantly reduced levels of serum TNFa, IFNg, and MDA, but increased levels of SOD and GSH, were detected in OMSS-treated rats. Conclusions: Treatment with OMSS significantly reduced FBG in diabetic rats. This antidiabetic activity may be mediated by down-regulating T2DM-related oxidative stress and inflammation, protecting the pancreatic b-cells from apoptosis. Ó 2010 Elsevier Inc. All rights reserved.
Keywords: Oligopeptides Type 2 diabetes Apoptosis Rat Salmon
Introduction Diabetes mellitus is a major threat to public health, as the incidence of diabetes is rapidly increasing in the world. Diabetes mellitus currently affects more than 194 million individuals worldwide [1]. It is estimated that the number of patients with diabetes will increase to 333 million in 2025 [2]. Among the major types of diabetes, type 2 diabetes mellitus (T2DM) is highly prevalent, accounting for about 90% of the total diabetic patients [3]. Although many therapeutic reagents, together with diet and exercise programs, can effectively correct hyperglycemia in T2DM patients, these therapeutic strategies fail to effectively inhibit the pathogenic process of T2DM and prevent * Corresponding author. Tel.: 86 10 82801177; fax: 86 10 82801177. E-mail address: [email protected] (Y. Li). 0899-9007/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.nut.2010.01.011
T2DM-related complications. Therefore, discovery and development of new therapeutic reagents will be of great significance. Both insulin resistance and b-cell dysfunction are important characters of T2DM [4]. During the pathogenic process of T2DM, many factors contribute to the development of insulin resistance and b-cell dysfunction and they include genetic, environmental, and individual behavioral factors, such as overweight, less exercise, hypertension, and stress. The b-cell dysfunction is crucial for T2DM development as individuals can develop T2DM in the absence of insulin resistance, while individuals with insulin resistance alone may fail to develop T2DM [4,5]. Indeed, many patients with T2DM show impaired b-cell function [6]. Furthermore, patients with T2DM have already lost 50% of b-cell function when they were diagnosed and suffer with further deterioration of b-cell function on an average of 7% yearly, highlighting the importance of b-cell defects in the pathogenesis
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of T2DM [7]. Hence, therapeutic strategies for the preservation of b-cell function may be crucial for the balance of glucose metabolism in T2DM patients. Numerous documents have demonstrated that oxidative stress-related production of reactive nitrogen species (RNS) and reactive oxygen species (ROS) is a risk factor for the cytotoxicity of insulin-producing b-cells in the pancreatic islets in the pathogenesis of T2DM [8]. In addition, chronic inflammation and associated proinflammatory cytokines, such as tumor necrosis factor-alpha (TNFa) and interferon-gamma (IFNg), as well as the death factors of Fas and FasL, are also dangerous events that can trigger b-cell apoptosis in the pancreatic islets, leading to b-cell dysfunction [9–11]. Indeed, b-cell apoptosis is responsible for the loss of b-cell mass in animal models of T2DM [12]. Modulating proinflammatory parameters have been shown to be beneficial for T2DM patients [12]. Thus, therapeutic approaches designed to reduce the sensitivity of b-cells to apoptosis triggers and to down-regulate inflammation may effectively protect the b-cells from apoptosis-related b-cell dysfunction, beneficial for T2DM patients. Medical nutrition therapy has been shown to improve in glucose homeostasis and be beneficial for T2DM patients, reducing T2DM-related complications [12]. Previous study has shown that treatment with dietary cod (morrhua) proteins improved the insulin sensitivity in insulin-resistant individuals and reduced insulin-resistance-related metabolic disorders, contributing to the prevention of T2DM [13]. Our previous findings demonstrated that treatment with oligopeptides from marine salmon (Oncorhynchus keta) skin (OMSS) inhibited inflammation by reducing the production of proinflammatory cytokines in mice [14]. Accordingly, we hypothesized that treatment with OMSS could modulate hyperglycemia in diabetic individuals by down-regulating proinflammatory cytokine production and reducing oxidative stress, leading to the preservation of the insulin-producing b-cells from apoptosis. A high fat diet can induce hyperlipidemia and associated oxidative stress, which usually results in lipid peroxidation and the production of malondialdehyde (MDA) [10]. Furthermore, the metabolism of oxidative stress-related RNS and ROS usually exhausts antioxidant superoxidize dismutase (SOD) and glutathione (GSH) [15]. Therefore, the levels of serum MDA, SOD, and GSH will be indicative of oxidative stress in vivo. Streptozotocin (STZ) is a glucosamine-nitrosourea compound and can be selectively toxic to the pancreatic b-cells through the GLUT2 transporter by oxidative stress-related DNA damage in the sensitive cells [16]. While treatment with a high dose of STZ usually induces aggressive b-cell death and insulin deficiency, treatment with multiple low doses of it commonly causes inflammation and partial loss of b-cells as well as b-cell-specific autoimmunity in animals [17]. In this study, we fed male Sprague-Dawley (SD) rats with a high fat diet for the induction of insulin resistance and oxidative stress and then injected them with three low doses of STZ for the partial damage of b-cells to generate a rat model of T2DM. Subsequently, we tested whether treatment with OMSS could modulate hyperglycemia in T2DM rats and determined whether OMSS treatment could alter T2DMrelated apoptosis of b-cells and the levels of oxidative stress and inflammation in T2DM rats.
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Animals were housed under specific pathogen-free conditions in a controlled humidity, temperature, and light/dark (12:12 h) -switching facility. The experimental protocols were approved by the Animal Experimentation Committee of Shenzhen Hospital, Beijing University.
Preparation and identification of OMSS OMSS was prepared from wild Chum Salmon (Oncorhynchus kern) that were caught from the East China Sea (with an average body weight of 1.47 kg) and provided by the CF Haishi Biotechnology (Beijing, China). Briefly, fishes were cleaned and the meat was minced, followed by defatting [18]. The materials were homogenized in distilled water and digested with the mixed proteases (3000 U/g protein) at 40 C for 3 h. The resultant hydrolysate was centrifuged at 26 000 g and the supernatants were subsequently filtered through ceramic membrane (200 mm) for the purification of it. OMSS powder was generated by ultrafiltration. The generated OMSS was analyzed by high-performance liquid chromatography (with a cutoff of 10 000 Da) to remove undigested proteins, desalination, cryoconcentration under vacuum at 70 C, decolorization with the medicinal charcoal (Hangmu Corporation, Hangzhou, China), and lyophilization (Waters Corp., Milford, MA, USA) using a Phenomenex C18 column (10 250 mm), acetonitrile 0.05 mol/L phosphate buffer (pH 3.2, 10:90) with a flow rate of 2.0 mL/ min. The analysis was monitored at 260 nm using a Water 486 tunable UV detector. Subsequently, the OMSS was characterized by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS, LDI-1700; Linear Scientific Inc., Reno, NV, USA). The amino acid composition of OMSS was further analyzed using an H835-50 automatic amino acid analyzer (Hitachi, Tokyo, Japan), and the amount of free amino acids was measured by high-performance liquid chromatography. The OMSS contained oligopeptides with molecular weights of 130–3000 Da (S1), accounting for >95% of total proteins with the purity of 90.6%.
Diet preparation and diabetes induction Control and experimental diets were prepared in our laboratory. The high fat diet contained 58%, 26%, and 16% calories from fats, carbohydrates, and proteins, respectively, and a total of 5.6 kcal/g. The control diet contained 29%, 39%, and 32% calories from fats, carbohydrates, and proteins, respectively, and a total of 2.8 kcal/g. Groups of SD rats were randomly divided and fed with control diet as normal control or with a high fat diet with free access of water for the induction of diabetes. After feeding for 4 wk, 10 rats from control and the high fat diet groups were sacrificed and their aorta blood samples were collected for the measurement of fasting blood glucose (FBG) and insulin. After confirming insulin resistance, the rats were injected intraperitoneally with 1% STZ (30 mg/kg in body weight) in citrate buffer (pH 4.0), while the rats in the control group received vehicle every 4 d for three injections. The levels of glucose in their blood were monitored every other day using a portable glucometer (Accu-Chek Active; Roche Diagnostics Ltd., Mannheim, Germany). Individual animals with two consecutive blood glucose levels 16.7 mmol/L were considered diabetic, while those animals with a level of blood glucose <16.7 mmol/L were excluded from the study.
Treatment and sampling After identifying diabetes, groups of diabetic rats (n ¼ 10 per group) were treated with 3 g/kg OMSS (OMSS group) or bovine serum albumin (BSA) in water (BSA group) daily by oral gavage for 4 wk. At the end of the experiment, the animals were fasted overnight and sacrificed. Their blood was collected and centrifuged at 1734 g for 5 min. Their sera were collected and stored at 80 C for further analysis. Their pancreatic tissues were dissected out for histological examination.
Immunohistochemical analysis The pancreatic tissues were fixed in a 4% paraformaldehyde solution, dehydrated with ethanol, and embedded in paraffin. The tissue sections at 4 mm were prepared and stained with hematoxylin and eosin or antibodies against human Fas or Annexin-V or isotype controls (Santa Cruz Biotechnology, CA, USA). The number of positive staining cells was determined by counting five randomly selected islets per rat in high-power fields (400).
Materials and methods Determination of serum TNFa and IFNg Animals Male SD rats at 6 to 8 mo of age and around 180–220 g in body weight were purchased from the Experimental Animal Center, Guanxi Medical University.
Serum concentrations of TNFa and IFNg were determined by ELISA using the ELISA kits (BioSource International, Camarillo, CA, USA), according to the manufacturer’s instructions.
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Table 1 Effect of OMSS on fasting blood glucose and serum insulin Group
FBG (mmol/L)
Fasting insulin (mIU/mL)
Control Vehicle OMSS
5.73 0.38 18.22 5.82* 13.57 2.56*,y
7.735 0.439 8.176 0.869 6.995 1.609
Data are expressed as mean SD of each group (n ¼ 10). * P < 0.05 versus control group. y P < 0.05 versus vehicle treatment of diabetic group.
Malondialdehyde, superoxidize dismutase, and glutathione assays The levels of serum MDA were evaluated by the Thiobarbituric Acid Reactive Substances (TBARS) assay using the spectrophotometer [19]. The contents of serum GSH were determined by the spectrophotometric assay, as described previously [20]. The serum SOD levels were determined based on the inhibition of SOD on the reduction of nitroblue tetrazolium by superoxide to form formazan using the xanthine and xanthine oxidase [21]. Hence, the SOD activity is inversely proportional to the amount of the formazane formed, measured at 560 nm. The absorbance was measured in an ELX800 spectrophotometer (Bio-Tek Instruments Inc., CA, NY, USA). The MDA, GSH, and SOD determination kits were purchased from Nanjing Jiancheng Biochemical Company (Nanjing, China) using standard MDA, GSH, or SOD as the positive control, respectively. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick end-labeling (TUNEL) assay Apoptotic cells in the pancreatic islets were measured by the TUNEL assays using the in situ apoptosis detection kit (Roche), according to the manufacturer’s instruction. The apoptotic cells were examined for at least 25 islets per rat and imaged under a light microscope with a magnification of 400. The percentage of apoptotic cells was calculated as the ratio of number of apoptotic cells/total number of nuclei counted. Statistical analysis Data are expressed as mean standard deviation. The difference among groups was tested by one-way ANOVA, followed by Scheffe’s modified F-test for multiple comparisons using the SPSS 13.0. A value of P < 0.05 was considered statistically significant.
Results Effect of OMSS treatment on FBG and serum insulin in diabetic SD rats Feeding with a high fat diet usually induces obesity and insulin resistance in sensitive animals and this, together with a low dose of STZ treatments, commonly causes severe diabetes. Following feeding with a high fat diet and treatment with STZ, their FBG and serum insulin levels were measured in Table 1. While control rats fed with a regular diet showed normal levels of FBG, the rats fed with a high fat diet and injected with STZ developed diabetes with high levels of FBG. Interestingly, the average levels of FBG in the OMSS group were significantly lower than that of the BSA group (P < 0.05). Furthermore, analysis of serum insulin revealed that there was no significant difference among these groups of rats. These data indicated that treatment with OMSS reduced FBG levels and improved hyperglycemia in diabetic rats but did not affect insulin expression and secretion in fasted diabetic rats. Effect of OMSS treatment on the pancreatic islets in diabetic rats Next, the effect of OMSS treatment on the pancreatic islets was histologically examined. While intact islets with healthy islet cells were observed in control rats, many shrunken and small islets with impaired cell membranes were detected in the
Fig. 1. Histological analysis of the pancreatic islets. Groups of diabetic SD rats were treated with OMSS or BSA for 4 wk and their pancreatic islets, together with those of normal control rats, were histologically examined by H&E staining. Data are representative of single islet from each group of rats. (A) Control rats; (B) BSA group of rats; and (C) OMSS group of rats.
BSA group of rats (Fig. 1). In contrast, all islets in the OMSS group of rats remained intact without obviously pathogenic signs. Hence, OMSS treatment preserved the islet cells from T2DMrelated damage. STZ is a potent toxicant and causes strong oxidative stress, leading to cytotoxicity, particularly for highly sensitive b-cells in the pancreatic islets. While a high dose of STZ usually causes extensive b-cell necrosis, multiple low doses of it commonly induce b-cell apoptosis. We further examined the effect of OMSS treatment on the pancreatic islet cell apoptosis in diabetic rats by the TUNEL assays (Fig. 2). While there were rare apoptotic islet cells in the pancreatic islets of control rats, many pancreatic islet cells already underwent apoptosis in BSA-treated diabetic rats that did not receive OMSS treatment. In contrast, a few apoptotic islet cells were observed in the OMSS group of rats. Quantitative analysis revealed that the percentage of apoptotic islet cells in the BSA group of rats was 30-fold higher than that in control rats (Table 2). However, a significantly reduced frequency of
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Table 2 The frequency of apoptotic islet cells Group
Positive (%)
Control Vehicle OMSS
0.870 0.321 28.321 2.145* 10.896 5.019*,y
Data are expressed as meanSD of each group (n ¼ 10). * P < 0.01 versus control group. y P < 0.05 versus vehicle-treated diabetic group.
and Annexin-V formation in the pancreatic islets by immunohistochemical analysis. There is no detectable FasL expression in the pancreatic islets from these groups of rats (data not shown). As shown in Figure 3, higher levels of Fas expression were detected in the pancreatic islets of the BSA group of rats, accompanied by stronger staining of anti-Annexin V, as compared with that in the control rats. In contrast, the levels of Fas expression and antiAnnexin V staining in the pancreatic islets of OMSS-treated rats were significantly lower than that in BSA-treated rats (Table 3). The lower levels of anti-Annexin V staining were consistent with lower frequency of apoptotic islet cells. Therefore, OMSS treatment down-regulated the expression of Fas in the b-cells of pancreatic islets, reducing the sensitivity of b-cells to apoptosis induction. Effects of OMSS on antioxidant defense system in diabetic rats
Fig. 2. Apoptotic islet cells in the rat pancreases. After treatment with OMSS or BSA for 4 wk, the effect of OMSS treatment on T2DM-related islet cell apoptosis was characterized by the TUNEL assay using DAB as the substrate. Data are representative of each group of rats (n ¼ 10) and the arrow indicates the apoptotic islet cells. (A) Control rats; (B) BSA group of rats; and (C) OMSS group of rats.
Oxidative stress induced by STZ treatment usually promotes the production of ROS, which can induce lipid peroxidation and MDA production as well as exhaustion of antioxidant SOD and GSH. We further determined the impact of OMSS treatment on the levels of serum MDA, SOD, and GSH in diabetic rats. As shown in Table 4, while low levels of serum MDA were detected in control rats, the levels of MDA in the BSA group were near six-fold higher than that in control rats, reflecting the oxidative stress-related high levels of lipid peroxidation in the BSA group of rats. In contrast, the levels of serum MDA were significantly reduced in the OMSS-treated rats, indicating that OMSS inhibited oxidative stress-related lipid peroxidation. Analysis of serum antioxidant SOD and GSH revealed that the levels of SOD and GSH in the BSA group of rats were significantly lower than that in the control rats (Table 4). However, higher levels of serum SOD and GSH were observed in the OMSS-treated rats and their serum SOD and GSH levels were significantly elevated, near to that of the control rats, as compared with that in the BSA group of rats. Together, these data demonstrated that OMSS treatment preserved antioxidant SOD and GSH and inhibited the oxidative stress-related lipid peroxidation in diabetic rats. Effect of OMSS treatment on diabetes-associated inflammation
apoptotic islet cells was observed in the OMSS-treated rats, as compared with that of BSA-treated rats. Apparently, OMSS treatment protected the islet cells from T2DM-associated islet cell apoptosis. Effects of OMSS on the expression of Fas/FasL and Annexin V Oxidative stress induced by STZ treatment can usually upregulate the expression of Fas in the b-cells of pancreatic islets, leading to high sensitivity of b-cells to apoptotic triggers. It is possible that OMSS treatment may modulate the expression of Fas and FasL in the pancreatic islet cells, reducing their sensitivity to apoptosis. We further examined the expression of Fas, FasL,
Finally, we tested whether OMSS treatment could modulate systemic inflammation in diabetic rats as inflammation and related proinflammatory cytokines are associated with the pathogenesis of T2DM induced by high fat feeding. We found that high levels of serum proinflammatory cytokines, such as TNFa and IFNg, were detected in the BSA group of rats, which was significantly higher than that in the control rats. In contrast, the levels of serum TNFa and IFNg in the OMSS group of rats were significantly reduced by 40.08% or 35.38%, respectively, as compared with that in the BSA group of rats (P < 0.05, Table 5). Therefore, the significantly reduced levels of proinflammatory cytokines in the OMSStreated rats demonstrated that OMSS treatment reduced diabetes-associated inflammation in diabetic rats.
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Fig. 3. Immunohistochemistry analysis of Fas and Annexin-V in the pancreatic islets. The effect of OMSS treatment on T2DM-related Fas expression and Annexin-V formation was determined by immunohistochemistry analysis using specific antibodies. Data are representative of each group of rats (n ¼ 10). A, B, and C, Fas staining; D, E, and F, Annexin-V staining. (A, D) Control rats; (B, E) BSA group of rats; (C, F) OMSS group of rats.
Discussion During the pathogenesis of T2DM, insulin resistance, b-cell dysfunction, and apoptosis are characteristic. High fat diet feeding usually induces insulin resistance and treatment with multiple low doses of STZ commonly promotes b-cell apoptosis in animals. In the present study, we employed a rat model of T2DM by feeding SD rats with a high fat diet and injecting them with multiple low doses of STZ for examining the effect of OMSS treatment on T2DM-related hyperglycemia, oxidative stress, Table 3 The frequency of Fasþ or Annexin-Vþ islet cells Group
Fasþ (%)
Annexin-Vþ (%)
Control Vehicle OMSS
2.35 0.19% 85.70 3.34* 62.93 2.87*,y
2.47 2.01 85.6 2.1* 76.2 3.4*
Data are expressed as mean SD of each group (n ¼ 10). * P < 0.01 versus control group. y P < 0.05 versus vehicle-treated diabetic group.
chronic inflammation, and associated b-cell apoptosis as this model has been shown to represent the pathogenesis of T2DM [22]. We found that all rats that had been injected with STZ developed diabetes within 1 wk post-STZ injection. Interestingly, OMSS treatment corrected hyperglycemia and significantly reduced FBG in diabetic rats. However, this treatment failed to modulate significantly the levels of serum insulin, as there was no significant difference in the levels of fasting serum insulin among groups of rats, regardless of whether they were treated with OMSS or control BSA. This was not surprising as 15% of b-cell mass can maintain a normal insulin level by high levels of expression and secretion of insulin from the remaining b-cells in the pancreatic islets. Importantly, the significantly reduced levels of FBG indicated that OMSS treatment improved the glucose metabolism and may aid in design of new therapy for T2DM in humans. Following the T2DM progression, hyperglycemia, oxidative stress, and inflammation can induce b-cell apoptosis, particularly after injecting with multiple low doses of STZ. Accordingly, we further examined the effect of OMSS treatment on islet cell
C.-F. Zhu et al. / Nutrition 26 (2010) 1014–1020 Table 4 Effect of OMSS on serum MDA, SOD, and GSH levels Group
MDA (nmol/mL)
SOD (U/mL)
GSH (mg/L)
Control Vehicle OMSS
8.90 2.69 51.82 16.91* 22.23 10.47y,x
107.88 8.30 74.93 2.20* 92.53 6.82x
474.75 47.41 335.75 66.32* 442.87 91.09z
Data are expressed as mean SD of each group (n ¼ 10). * P < 0.01 or yP < 0.05 versus control group. z P < 0.01 or xP < 0.05 versus vehicle-treated diabetic group.
apoptosis in diabetic rats. We found that few apoptotic islet cells were detected in control rats, while a higher frequency of apoptotic islet cells were observed, accompanied by significantly higher levels of anti-Annexin V staining in the BSA group of rats. However, the frequency of apoptotic islet cells in the OMSS-treated rats was significantly reduced. The significantly reduced frequency of apoptotic islet cells was accompanied by remarkable lower levels of anti-Annexin V staining, further indicating that OMSS treatment preserved the islet cells from T2DM-related apoptosis in our experimental model. There are many factors that can trigger islet cell apoptosis during the pathogenic process of T2DM. Hyperglycemia and chronic inflammation can up-regulate the expression of Fas and FasL in the pancreatic islets. High levels of Fas expression can increase the sensitivity of b-cells to apoptosis induction, while high levels of FasL expression can trigger apoptotic signal cascade and lead to b-cell apoptosis in the islets. We characterized the expression of Fas and FasL in the pancreatic islets of these groups of rats. We found that although little FasL expression was detected in the pancreatic islets of these groups of rats, significantly higher levels of Fas did display in the pancreatic islets of BSA group of rats. Surprisingly, significantly lower levels of Fas expression were observed in the pancreatic islets of the OMSS-treated rats. These data suggest that high levels of Fas expression in the pancreatic islets may, at least partially, contribute to the high frequency of islet cells undergoing apoptosis in diabetic rats, while OMSS may protect the islet cells from apoptosis by down-regulating the expression of Fas. However, the mechanisms by which OMSS treatment down-regulates the expression of Fas, inhibiting islet cell apoptosis in diabetic rats, remain to be further determined. Chronic inflammation and high levels of proinflammatory cytokines, such as TNFa and IFNg and others, are crucial for the pathogenesis of T2DM. TNFa has been widely accepted as a crucial factor, responsible for the development of impaired glucose metabolism and insulin resistance in obese and diabetic subjects [23,24]. TNFa can also trigger b-cell apoptosis through its receptor and downstream signaling events. Indeed, high levels of TNFa were detected in subjects with insulin resistance and T2DM [22]. IFNg, a Th1 proinflammatory cytokine, is important for defense against viral and intracellular pathogens and organ-specific autoimmune diseases [25]. IFNg can up-regulate the expression of MHC molecules and activate macrophages and dendritic cells, promoting antigen-presenting
Table 5 Effect of OMSS on serum TNFa and IFNg levels Group
TNFa (pg/mL)
IFNg (pg/mL)
Control Vehicle OMSS
39.450 8.243 109.850 10.973* 65.817 1.892y
47.220 11.133 126.922 21.711* 82.100 11.747y
Data are expressed as mean SD of each group (n ¼ 10). * P < 0.01 versus control group. y P < 0.05 versus vehicle-treated diabetic group.
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activity and inflammatory cascade. High levels of IFNg have been associated with the development of T2DM and latent autoimmune diabetes in adults [25]. We measured the levels of serum TNFa and IFNg and found that significantly higher levels of serum TNFa and IFNg were detected in the BSA group of rats, reflecting a high degree of inflammation. In contrast, the levels of serum TNFa and IFNg were significantly reduced in the OMSStreated rats. These data indicated that treatment with OMSS inhibited T2DM-related inflammation in diabetic rats. Given that TNFa and IFNg are important for T2DM-related inflammation and associated b-cell apoptosis, the significantly reduced levels of serum TNFa and IFNg may also contribute to low frequency of apoptotic islet cells in the OMSS-treated rats. These data extended our previous findings that OMSS treatment inhibited chronic inflammation in animals [14]. Oxidative stress is critical for the development of insulin resistance and b-cell damage in the pathogenic process of T2DM. Oxidative stress usually promotes the production of ROS and RNS, which can change in the mitochondrial membrane potential, and the release of cytochrome c, triggering cell apoptosis [26]. We determined the effect of OMSS treatment on systemic levels of oxidative stress. We found significantly higher levels of MDA, an indicative of lipid peroxidation, and lower levels of SOD and GSH in the BSA group of rats. However, following treatment with OMSS, the levels of serum MDA were significantly reduced while the levels of antioxidant SOD and GSH were dramatically elevated in the OMSS-treated rats, as compared with that in BSA group of rats. These data indicated that OMSS treatment inhibited hyperglycemia-related oxidative stress in diabetic rats. Notably, high levels of ROS and RNS have been demonstrated to be risk factors for the b-cell apoptosis in animal models and treatment with scavenger for nitric oxide has been shown to inhibit b-cell apoptosis [27,28]. Therefore, the reduced levels of serum MDA and elevated levels of antioxidant SOD and GSH by OMSS treatment may also protect the pancreatic b-cells from T2DM-related apoptosis. In summary, our data, to the best of our knowledge, demonstrate for the first time that OMSS treatment significantly reduced FBG and T2DM-related islet cell apoptosis in diabetic rats. Furthermore, OMSS treatment inhibited inflammation significantly by decreasing the levels of serum proinflammatory TNFa and IFNg and down-regulating the expression of Fas. In addition, OMSS treatment reduced oxidative stress-related formation of MDA but elevated the levels of serum antioxidant SOD and GSH. Therefore, it is possible that the effect of OMSS treatment on T2DM may be mediated by inhibiting inflammation, oxidative stress, and the Fas expression, protecting the b-cells in the pancreatic islets from apoptosis. Although further investigations are necessary for determination of mechanisms underlying the action of OMSS treatment in the diabetes progression, potentially, our findings may provide a basis for further design of new therapy for T2DM. Acknowledgments This study was supported by grants from the National Key Technologies R&D Program of China (No. 2006BAD27B01) and the Science & Technology Committee of Futian District State, Shenzhen City, Guangdong Province of China (No. 20080214142). Supplementary data Supplementary data associated with this article can be found, in the online version, at 10.1016/j.nut.2010.01.011.
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