Lose Weight and Gain Muscle

Anti-inflammatory and Insulin-Sensitizing Effects of Resveratrol

The number of obese individuals worldwide has reached 2.1 billion, leading to an explosion of obesity-related health problems associated with increased morbidity and mortality. Obese individuals develop resistance to the cellular actions of insulin, characterized by an impaired ability of insulin to regulate metabolic pathway. Insulin action is initiated through the binding to and the activation of its cell-surface receptor through a series of intramolecular transphosphorylation reactions. Once activated, the insulin receptor phosphorylates several substrates on their tyrosine residues; among such substrates are members of the insulin receptor substrate family (IRS-1, -2, -3, -4). The insulin signal is further spread through a phosphorylation network involving intracellular proteins that propagate the various metabolic actions of insulin. Insulin is essential for the maintenance of carbohydrate and lipid homeostasis. A large fraction of glucose absorbed from the small intestine is immediately taken up by hepatocytes, which convert it into glycogen. However, when the liver is saturated with glycogen (roughly 5% of liver mass), any additional glucose taken up by hepatocytes is shunted into pathways leading to synthesis of fatty acids, which will be esterified into triglyceride (TG) to be exported to adipose tissue as very low-density lipoproteins (VLDLs). Insulin inhibits lipolysis in adipose tissue by inhibiting hormone-sensitive lipase (HSL), the enzyme regulating free fatty acid (FFA) release from adipose tissue. Insulin also regulates glucose homeostasis at many sites, reducing hepatic glucose production (HGP) (via decreased glucose biosynthesis; gluconeogenesis and glycogen breakdown; glycogenolysis) and increasing the rate of glucose uptake, primarily into skeletal muscle and adipose tissue.

Insulin resistance, which can be considered the result of a signaling defect, occurs when normal circulating concentrations of the hormone are insufficient to regulate key metabolic pathways in adipose tissue, skeletal muscles, and/or liver. One of the approaches used to assess insulin sensitivity involves measuring the ability of insulin to phosphorylate intracellular substrates such as IRS-1, IRS-2, or Akt (also known as protein kinase B) since defects in the activation of these molecules are known to lead to the inability of insulin pathway. Increasingly, insulin resistance has been recognized as the integral feature of the so-called metabolic syndrome, which includes glucose intolerance, insulin resistance, obesity, hypertriglyceridemia, low HDL cholesterol, hypertension, and accelerated atherosclerosis. Particularly in this study, we researched on obesity related insulin resistance. Obesity is characterized by a chronic inflammatory response, abnormal elevations in cytokines production and the activation of signaling pathways involved in the inflammatory process. The manifestations of pro-inflammation state include the increased circulation of pro-inflammation molecules and surrogate markers of inflammation. So there may be some connections between inflammation and insulin resistance. Growing evidence suggests that in animal models of both genetic (e.g. the Fatty Zucker rat) and acquired (e.g. diet-induced obesity or DIO) obesity, the pathogenesis of insulin resistance in key metabolic tissues, such as liver, adipose tissue, aorta and skeletal muscle, involves activation of IκB kinase (IKK) and subsequently of nuclear factor-kB (NF-kB), a key transcriptional mediator of cellular inflammation. The altered production of proinflammatory molecules by adipose tissue has been implicated in the metabolic complications of obesity. Compared with adipose tissue of lean individuals, adipose tissue of the obese expresses increased amounts of proinflammatory proteins such as tumor necrosis factor-alpha (TNF-a), interleukin-6 (IL-6), inducible nitric oxide synthase (iNOS), tumor growth factor beta-1 (TGF-β1), C-reactive protein (CRP), soluble intracellular cell adhesion molecule-1 (ICAM-1) and monocyte chemotactic protein-1 (MCP-1) (18–25), and procoagulant proteins such as plasminogen activator inhibitor type-1 (PAI-1), tissue factor, and factor VII (26–28). Obese mice deficient in TNF-a and iNOS are more sensitive to insulin than are obese wild-type mice (21, 29). Proinflammatory molecules have direct effects on cellular metabolism. For example, TNF-a directly decreases insulin sensitivity and increases lipolysis in adipocytes (30, 31). IL-6 leads to hypertriglyceridemia in vivo by stimulating lipolysis and hepatic triglyceride secretion (32). Therefore, insulin resistance induced by obesity deeply related to inflammation.

 A number of large-scale epidemiological studies have suggested that a prolonged and moderate consumption of red wine by the Southern French and other Mediterranean populations, despite a high-fat diet, little exercise, and widespread smoking, is associated with a very low incidence of cardiovascular diseases, mainly coronary heart disease (the so-called French paradox) [1]. Although possible compounds of ‘French paradox’ have yet to be identified, the principal candidates are a number of polyphenolic compounds such as stilbenes and flavonoids (for review, see, e.g., ref. [2]) and resveratrol received attention as a potential compound. Resveratrol; 3,4,5-trihydroxystilbene (RESV) is a natural phytoalexin (phytoestrogen) synthesized in response to injury or fungal attack, found in grape skin, and notably, in red wine in its two isomers, trans and cis (ref. [3]; see Fig. 1).

 Recent studies suggest that resveratrol, by activating silent mating type information regulation 2 homolog 1 (sirtuin 1; SIRT1), safely mimics the effects of dietary restriction in laboratory animals.4  Sirtuin-related enzymes, were originally defined as a family of NAD-dependent enzymes that deacetylate lysine residues on various proteins. Certain sirtuins also have ADP-ribosyltransferase activity. The mammalian sirtuins, SIRT1-7, are implicated in a variety of cellular functions, ranging from gene silencing, control of the cell cycle and apoptosis, to energy homeostasis (11). Resveratrol has since been shown to extend the lifespan of evolutionarily distant species including S. cerevisiae, C. elegans and D. melanogaster in a SIRT1-dependent manner6–9. A recent study found that resveratrol improves health and extends maximum lifespan by 59% in a vertebrate fish10. In mammalian cells, resveratrol produces SIRT1-dependent effects that are consistent with improved cellular function and organismal health11–15. Many studies have demonstrated that resveratrol has a wide range of pharmacological properties, which have been comprehensively reviewed by Bhat et al. (1). Resveratrol has been suggested to be cardio-protective via various mechanisms such as its antioxidant activity (2), inhibition of platelet aggregation (3), induction of nitric oxide (NO) production (4), and modulation of the synthesis of hepatic apolipoprotein and lipids (5). Resveratrol is also reported to have chemo-preventive activity; Jang et al. (6) have suggested that it inhibits all three phases of tumor development: initiation, promotion, and progression. Considerable evidence demonstrates anti-inflammatory properties of resveratrol, including inhibition of reactive oxygen species (ROS) in neutrophils (7), monocytes (8), and macrophages (9). The release of various cytokines from macrophages and lymphocytes, such as IL-6 (13, 14), IFNγ, IL-2, TNF-α, and IL-12 (15), has been shown to be inhibited by resveratrol. In stimulated macrophages the expression of iNOS and the release of nitric oxide are reduced by resveratrol (14, 16, 17). A reduction of cyclooxygenase (COX)-1 (18) and COX-2 (19) expression and activity by resveratrol is also apparent. Others have shown an inhibition of matrix metalloproteinase-9 (MMP-9) expression by resveratrol (20, 21). Recently the effect of resveratrol on NF-κB has been of particular interest. It has been suggested that resveratrol acts on NF-κB by the inhibition of IKK, which results in the prevention of translocation of NF-κB into the nucleus (28) or through its sirtuin-like activity, which deacetylates NF-κB (29, 30). Also, there are studies regarding the effects of resveratrol on insulin resistance. In the high fat diet induced obese mice, Baur JA. et. reported prolonged intake of resveratrol improves insulin sensitivity and lowers blood glucose levels ( and Lagouge M. et. reported that -> ) and another report showed that treatment of resveratrol with high dose showed to reduce body weight and improve mitochondrial functions. 16,17

 Although resveratrol has various beneficial effects, there is no study about the effects of resveratrol with low dose for short periods on insulin signaling in target tissue related to inflammation in insulin resistance mice model. Therefore the purposes of this study are to evaluate the effects of resveratrol on insulin resistance induced by inflammation in adipocytes and insulin signaling in high fat diet induced obese mice models.

 Materials and methods

 trans-Resveratrol and carboxyl methyl cellulose (CMC) was purchased from Sigma (M.O., U.S.A.). Antibodies and their sources were as follow : anti-phospho-insulin receptor / insulin-like growth factor-1 receptor (p-IR/IGF1R) (Y1162 and Y1163; polyclonal Ab) from Invitrogen (C.A., U.S.A.); anti-insulin receptor β (IRβ) (polyclonal Ab) from BD Biosciences (C.A., U.S.A.); anti-phospho-Akt (S473; polyclonal Ab), anti-phospho-Akt (T308; polyclonal Ab) from Cell Signaling Technology (M.A., U.S.A.); anti-β actin (monoclonal Ab) from Sigma (M.O., U.S.A.); anti-Akt (polyclonal Ab), anti-inducible nitric oxide synthase (iNOS) (polyclonal Ab), anti-IkBα (C21) (polyclonal Ab), anti-phospho-IkBα (S32; monoclonal Ab) and anti-rabbit immunoglobulin G (IgG) or anti-mouse immunoglobulin G (IgG) horseradish peroxidase (HRP)-linked secondary antibodies from Santa Cruz Biotechnology (C.A., U.S.A.). The sources of reagents and media using in cell experiments were as follow : Dulbecco’s Modified Eagle Medium (DMEM) from Hyclone (U.T., U.S.A.); fetal bovine serum (FBS), bovine serum (BS), trypsin and penicillin-streptomycin (p/s) from GIBCO (N.Y., U.S.A.); lipopolysaccharide (LPS), 3-isobutyl-1-methylxanthine (IBMX), dexamethasone, and insulin from Sigma (M.O., U.S.A.). All other reagents and chemicals were obtained from the usual commercial sources.

Male C57BL/6N mice (5 weeks of age) were supplied from Orient Co. (Songham, Korea). The animals were housed in 24 ± 1 ℃ at 50% humidity with a 12-h light/12-h dark cycle. All animals procedures were carried out in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of Jeju National University (ACUCC; approval No. 20090018).

 The mice divided into 3 groups of twelve mice each. Experimental groups were as follow : the mice were maintained on a normal diet (5L79; Orient Co. Songham, Republic of Korea ); ‘N’ group, the mice were fed high fat and high sucrose diet for 20 weeks ( D12331; Research Diets, Inc., N.J., U.S.A.); ‘H’ group and the mice maintained on high fat and high sucrose diet for 18 weeks and then treated with resveratrol for 2 weeks; H/R group. Resveratrol suspended in 0.5% CMC and treated at a dose of 30 ㎎/㎏/day per oral.

Body weights were measured on a weekly. At 20 weeks, after the fasting for 16hrs, blood was collected from tail vein and measured the blood glucose levels by One touch® Horizon (LifeScan, Inc. a Johnson & Johnson company, C.A., U.S.A.).

At 20 weeks, after the end of the experiment, the mice were fasted for 16 hours and eight mice in each group injected human insulin (LILLY KOREA INC.; Seoul, Korea) intraperitoneally at a dose of 5 U/㎏ for studying insulin signaling in liver. The mice were euthanized by cervical dislocation and the liver were excised. Liver were quickly placed in liquid nitrogen and stored in -80 ℃.

Approximately 100 ㎎ of frozen liver were placed in 1.2 ㎖ lysis buffer (20 mM Tris-Hcl, pH 7.4, with 5 mM EDTA pH 8.0, 10 mM Na2P4O7, 100 mM NaF, 2 mM Na3VO4, 1% NP-40, 13.2 ㎍/㎖ aprotinin, 13.2 ㎍/㎖ leupeptin, 1 mM PMSF) and homogenated for 90 seconds at 11,000 rpm in a tissue homogenizer. The homogenates were centrifuged two times. First, homogenates were cleared by centrifugation at 800 g for 15 minutes at 4 ℃ and secondly centrifuged at 10,000 g for 20 minutes at 4 ℃. The supernatants were stored at -80 ℃ until used. The protein concentration was measured using Bradford method (4).

The liver from mice which were not injected insulin were fixed in 10% formaldehyde. The fixed tissues embedded in paraffin and sectioned (4.0 ㎛). The sections were with hematoxylin–eosin and examined under light microscope. To determine the grades of fatty change in liver, the slides were blindly scored for overall pathology on a scale of 0-4; 0 or 1 being healthy tissue typically seen in normal mice and 4 being the worst.

A murine macrophage cell line, RAW264.7, were provided by Korean Cell Line Bank (KCLB, Seoul, Korea) and cultured in DMEM supplemented with 10% FBS and 1% p/s at 37 ℃ in a 5% CO2 humidified air atmosphere.

To establish of the inflammation in adipocytes, inflammatory media was obtained from RAW264.7 cells treated with LPS; LPS treated conditioned media (L/CM). RAW264.7 cells were stimulated by 1 ㎍/㎖ LPS and after 24 hours, the media was collected. We prepared also control media which is culture media with RAW264.7 for 24 hours (CM). L/CM and CM treated to mature adipocytes after they were filtered.

 3T3-L1 preadipocytes were provided by American Type Culture Collection (ATCC, M.D., U.S.A.) and cultured in DMEM supplemented with 10% BS and 1% p/s at 37 ℃ in a 5% CO2 humidified air atmosphere. At the day of the post-confluent (day 0), media switched to differentiation initiation media (DIM) consisted of DMEM with 10% FBS, 1% p/s, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 0.5 μM dexamethasone, and 10 ㎍/㎖ insulin and incubated for 48 hours. At ‘day 2′, DIM was switched to differentiation progression media (DPM) that was contained 10% FBS, 1% p/s and 10 ㎍/㎖ insulin in DMEM and incubated for 48 hours. At ‘day 4′ and ‘day 6′, cells were cultured in DMEM contained with 10% FBS and 1% p/s and then maintained until completely differentiated. In this study, utilized mature 3T3-L1 adipocytes cultured for 8–12 days after completion of the differentiation process.

The mature adipocytes were incubated L/CM with or without resveratrol at the concentrations of 10 μM, 25 μM and 50 μM for 24 hours. After treatment of human insulin at a dose of 10 ㎍/㎖ for 30 minutes, proteins were isolated from cell lysates and determined by using BCA protein assay (DC protein assay; Bio-Rad, C.A., U.S.A.)

 Aliquots of 60 ㎍ proteins for liver or 70 ㎍ proteins for mature 3T3-L1 adipocytes were resolved using 10% (v/v) SDS-PAGE and transferred onto PVDF membrane (Immobilon P; Millipore, M.A., U.S.A.) for 100 min at 110 V. Then incubated in blocking buffer TTBS (20mM Tris-Hcl pH 7.6, with 137mM NaCl, 0.05% Tween-20) supplemented with 5% (w/v) skimmed milk for 1h, washed in TTBS for 4 ⅹ 10min and incubated with the following antibodies: anti-p-IR / IGF1R (Y1162 and Y1163) (1:1000 dilution), anti-IRβ (1:1000 dilution), anti-Akt (1:1000 dilution), anti-p-Akt (S473) (1:1000 dilution), anti-p-Akt (T308) (1:1000 dilution), anti-iNOS (1:1000 dilution), anti-IkBα (1:1000 dilution), anti-p-IkBα (1:1000 dilution) and anti-β actin (1:5000 dilution) diluted in TTBS overnight at 4 ℃. Following 5 ⅹ 10 min washes in TTBS the membranes were incubated with anti-rabbit IgG or anti-mouse IgG HRP-linked secondary antibodies at 1:10,000 or 1:5000 in TTBS for 40min. Reactive bands were revealed using enhanced chemiluminesence (ECL) reagents (Intron Biotechnology, M.O., U.S.A.). The expressions of proteins were analyzed by Image J 1.42 software (National Institute of Mental Health (NIH) ; M.D., U.S.A.).

All results were represented as the mean ± S.E.. Comparison among more than two groups was carried out using one way analysis of variance (ANOVA) (SPSS program, ver. 12.0.0; SPSS InC., I.L., U.S.A.). P values <0.05 were considered significant.

Results

Effects of resveratrol on inflammatory factors in adipocytes

Western blot analysis of the cytoplasmic extracts was performed to study the effects of resveratrol on inflammation induced iNOS expression and IkBα degradation in adipocytes. As shown in Figure 1 and 2, compared with the adipocytes incubated in L/CM, resveratrol dose dependently (10, 25, 50 uM) decreased iNOS expression and the phosphorylation levels of IkBα were reduced at a dose of 50 uM by preventing IkBα degradation.

Effects of resveratrol on insulin pathway in adipocytes

To confirm the effects of resveratrol on insulin pathway in adipocytes placed in inflammatory status, western blot analysis was performed to determine the levels of p-Akt (S473) in adipocytes. As shown in Figure 3, L/CM significantly reduced the phosphorylation levels of Akt (S473). However, co-incubation with resveratrol prevented the inflammation induced down regulation of p-Akt (S473). These results showed that L/CM contained with inflammatory factors released from RAW264.7 cells emerged insulin resistance in adipocytes and resveratrol had ability to improve it.

Effects of resveratrol on body weight changes

After 2weeks of the study, body weights of ‘H’ and ‘H/R’ groups were continuously greater than body weights of ‘N’ group (34.9 ± 0.56 g, p < 0.001) until the end of the study. At 20 weeks, there was no difference between body weighs of ‘H’ (51.3 ± 1.6 g,) and ‘H/R’ (49.3 ± 2.8 g) (Figure 4).

Effects of resveratrol on blood glucose levels

The effect of resveratrol on blood glucose levels was observed at the end of the study. Compared with ‘N’ group (111 ± 3 mg/dl), the blood glucose level in ‘H’ group (190 ± 8 mg/dl, p < 0.001) was significantly increased. The treatment with resveratrol (‘H/R’ group) reduced blood glucose level (168.4 ± 6.9 mg/dl, p < 0.05 vs. ‘H’) (Figure 5).

Effects of resveratrol on insulin pathway in liver

Western blot analysis of the cytoplasmic extracts was performed to study the effects of resveratrol on insulin signaling in liver. The phosphorylation levels of IR and Akt (S473 and T308) were decreased in ‘H’ group. Although the phosphrylation levels of IR were not improved, the phosphorylation levels of Akt (S473) were greatly recovered in resveratrol treated group (Figure 6, 7 and 8).

Effects of resveratrol on iNOS expression in liver

Western blot analysis of the cytoplasmic extracts was performed to study the effects of resveratrol on iNOS expression in liver. Although there were no significant differences between ‘H’ and ‘H/R’ groups, the iNOS expression decreased in ‘H/R’ group compared with ‘H’ group. (Figure 9).

Effects of resveratrol on fatty changes in liver

In the histopathological study, high calorie diets significantly induced severe fatty changes in liver. Treatment of resveratrol attenuated fat accumulation and fatty changes in liver (Figure 10).

Discussion

Resveratrol improved insulin signaling and attenuated inflammation in 3T3-L1 adipocystes incubated with inflammatory media.

IκBα was involved in major inflammatory pathway, Nf-kB pathway, and formed the complex with NF-κB, restricting it to a cytoplasmic location. However, when incoming inflammatory signals lead to activation of IKKβ, IkBa was phosphorylated by IKKβ and dissociates from NF-κB. Then IkBa undergoes degradation and the released NF-κB translocates to the nucleus, where it binds to its cognate DNA response elements, leading to transactivation of inflammatory pathway genes; TNF-a, iNOS, MCP-1 etc.. As previous reports, TNFα, IL-6, IL-1β, and possibly other cytokines and macrophage-secreted factors exert paracrine effects to activate inflammatory pathways within insulin target cells. This leads to activation of Jun N-terminal kinase (JNK), IKKβ, and other serine kinases. In an insulin resistant state, JNK1 (33, 34) and IKK (28, 35) signaling is upregulated in insulin-resistant skeletal muscle, fat, and other tissues in humans and rodents. This is important, as these serine kinases activate transcription factor targets, including activator protein 1 (AP1) (c-Jun/Fos) and nuclear factor-κB (NF-κB), which then stimulate transcription of an overlapping set of inflammatory pathway genes (36). These serine kinases can also phosphorylate insulin receptor substrate (IRS) proteins, insulin receptors, and possibly other insulin signaling molecules. These serine phosphorylation events interfere with normal insulin action, creating a state of cellular insulin resistance. Accordingly, knockout (KO) or inhibition of JNK1 or IKKβ prevents insulin resistance in cell and mouse models (33, 37–40). Although inflammation leading to activation of the JNK/SAPK and IKK–NF-kB pathways has been implicated in the pathogenesis of obesity-related insulin resistance, iNOS is a pivotal downstream effector of insulin resistance in many pathologic states, including obesity. iNOS inhibitor reduced inflammation and hyperglycemia induced by LPS (61) and inhibition of iNOS by gene disruption reversed the decreased IRS-1 expression and thereby improved IRS-1–mediated insulin signaling in obese and diabetic (leptin-deficient) mice.64 Also, induction of iNOS and/or high levels of nitric oxide also can induce IKK–NF-kB and JNK/SAPK with induction of ER stress.79–81 On the basis of these reports, iNOS has been proposed as an important component of mechanisms that lead to insulin resistance, in which it functions as both a downstream effecter and an upstream enhancer of inflammation.

The present study shows resveratrol treatment reduced iNOS expression and IkBa degradation synchronized with the effect of improvement of phosphorylation of Akt in 3T3-L1 adipocytes incubated with inflammatory media (L/CM). These results suggest that resveratrol could improve insulin resistance by reducing inflammatory factors in adipocytes.

In obese mice models, resveratrol lowered the fasting blood glucose levels and recovered Akt phosphorylation in liver.

In obese mice models, resveratrol lowered the fasting blood glucose levels and recovered Akt phosphorylation in liver. To determine the in vivo effects of resveratrol, we set up high calorie diets induce obese mice which was commonly used as a model of established insulin resistance related to obesity. In the present study, high calorie diets significantly increased body weight and elevated blood glucose levels. Fatty changes in liver were severely progressed in mice with high calorie diets. However treatment of resveratrol attenuated the grade of fatty changes and increased phosphorylation of Akt in liver even though there was no effect on phosphorylation of insulin receptor.

Circulating glucose levels reflect a balance between glucose production by the liver and glucose uptake by the muscle.(10) The liver plays a key role in obesity-induced hyperglycemia and affects multiple pathways.5,48 As the glucose regulating mechanism, insulin promotes the synthesis of glycogen, while repressing glucose release in the liver. This effect is enhanced and coordinated through multiple genes that control glycolysis, fatty acid synthesis, gluconeogenesis (synthesis of glucose from proteins), and glycogenolysis (breakdown of glycogen).10 Obesity is the most common factor related to fatty change in liver; NAFLD (non-alcoholic fatty liver disease) and the ectopic accumulation of fat in the liver has strongly associated with hepatic and adipose tissue insulin resistance (28 –30) as well as reduced whole-body insulin sensitivity (28, 29). As previously study, knockout of insulin receptor in liver resulted in fasting and postprandial hyperglycemia, peripheral insulin resistance and hepatic steatosis, but knockout of insulin receptor in muscle alone or in combination with adipocyte showed normal blood glucose levels. (72) Andrew H. et. reported that hepatic insulin receptors loss in genetic and acquired obesity, and in our study, expression of insulin receptor in liver was reduced in mice with high calorie diets. Therefore insulin resistance in whole-body was deeply correlates with hepatic insulin resistance. Even though anti-inflammatory effects of resveratrol in liver were not shown significantly, but it would be possible mechanism influenced to insulin signaling.

In conclusion, resveratrol improves insulin resistance in liver linking to systemic insulin resistance by attenuating fatty change in liver.

Suggestion and future study

Our observations can be summarized as follows. 1) Resveratrol improved insulin resistance induced by inflammation in adipocytes by reducing inflammatory factors. 2) Resveratrol restored phosphorylation of Akt and attenuated the fatty change in liver. This may be one mechanism underlying the effects of resveratrol on insulin resistance which occurs in obese mice induced by high calorie diets. The mechanisms of resveratrol on insulin resistance in other insulin target tissues; muscle, adipose tissue and aorta in diet induced obesity will be elucidated in further studies.

About the Author

Trace amounts of O2 affect NO and N2O production during denitrifying enzyme activity (DEA) assays [An article from: Soil Biology and Biochemistry] $8.95 This digital document is a journal article from Soil Biology and Biochemistry, published by Elsevier in 2004. The article is delivered in HTML format and is available in your Amazon.com Media Library immediately after purchase. You can view it with any web browser.Description: We evaluated the potential of trace amounts of O”2 inadvertently introduced into anaerobic incubations to initiate the C”2…

The responses of lymphocytes from Asian and Caucasian diabetic patients and non-diabetics to hydrogen peroxide and sodium nitrite in the Comet assay [An ... Toxicology and Environmental Mutagenesis] $10.95 This digital document is a journal article from Mut.Res.-Genetic Toxicology and Environmental Mutagenesis, published by Elsevier in 2006. The article is delivered in HTML format and is available in your Amazon.com Media Library immediately after purchase. You can view it with any web browser.Description: Numerous factors may influence the incidence of diabetes in the population. The production of …

---

 Mail this post

Technorati Tags: nitric oxide assay, nitric oxide assay protocol