Protective Effect of Aspirin in Relation to IGF-1 in Streptozotocin Induced Type-II Diabetic Rats

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Article Information:


Group: 2009
Subgroup: Volume 7, Issue 1, Winter
Date: March 2009
Type: Original Article
Start Page: 20
End Page: 25

Authors:

  • S Martha
  • Dept. of Pharmacology and Clinical Pharmacy, University College of Pharmaceutical Sciences, Kakatiya University, Warangal, AP, India
  • UK Veldandi
  • Dept. of Pharmacology and Clinical Pharmacy, University College of Pharmaceutical Sciences, Kakatiya University, Warangal, AP, India
  • R Devarakonda Krishna
  • Clinical Research Division, Covedien Inc., Hazelwood, Missouri, USA
  • N Pantam
  • Department of General Medicine, Mahatma Gandhi Memorial Hospital, 506 002, Warangal, AP, India
  • S Thungathurthi
  • Department of General Medicine, Mahatma Gandhi Memorial Hospital, Warangal, AP, India
  • N Reddy Yellu
  • Dept. of Pharmacology and Clinical Pharmacy, University College of Pharmaceutical Sciences, Kakatiya University, Warangal, AP, India

      Correspondence:

      Affiliation: Department of General Medicine, Mahatma Gandhi Memorial Hospital, 506 002
      City, Province: Warangal, AP
      Country: India
      Tel:
      Fax:
      E-mail: pantamnarayana@gmail.com

Abstract:


background:The present study aimed at investigating the protective effects of aspirin in relation to insulin like growth factor-1 (IGF-1) in streptozotocin (STZ) induced type-2 diabetic rats.
Materials and Methods: Rat pups were divided into four groups; on the 5th day of their age, group-I pups received citrate buffer solution and served as the normal group; group-II, treated on-ly with streptozotocin (80 mg/kg, i.p), served as the diabetic group; groups-III & IV, treated with aspirin (10 mg/kg/day, p.o) for one month (5-35 days) and two months (5-65 days) after streptozo-tocin, served as the treated groups. On the 35th and 65th days, blood samples were collected from all animals and fasting blood sugar, fasting insu-lin, IGF-1, insulin resistance and insulin sensi-tivity levels were estimated.
Results: Blood samples taken on the 35th & 65th days, from pups treated with streptozotocin per se and in combination with aspirin for one and two months showed significantly increased body weight, fasting blood glucose and insulin resis-tance levels and significantly lowered fasting in-sulin and insulin sensitivity levels, when com-pared to the normal control pups respectively. Pups treated with aspirin for one month had significantly raised IGF-1 levels, whereas those with two month treatments had significantly lo-wered IGF-1 levels when compared to the nor-mal pups (p <0.0001).
Conclusions: The study indicates that aspirin pretreatment seems to protect the pancreas from damage caused by STZ and maintains glucose levels in diabetic rats, while increasing insulin sensitivity and reducing insulin resistance, which may indicate an involvement of an insulin like pathway, particularly IGF-1.

Keywords: Insulin like growth factor-I; Strepto-zotocin; Aspirin; Type-2 diabetes

Manuscript Body:


Introduction

Type-2 diabetes mellitus is a metabolic disorder, characterized by hyperglycemia and insufficiency of secretion or action of endo-genous insulin.1 One of the major characteris-tics of type 2 diabetes mellitus, is insulin re-sistance; if the insulin resistance results from oxidative damage, then it could be predicted that chronic oxidative stress would lead to hyperinsulinaemia if plasma glucose is clamped at normal level by infusing the re-quired insulin. Increased oxidative stress, de-fined as a persistent imbalance between the production of highly reactive molecular spe-cies (chiefly oxygen and nitrogen) and anti-oxidant defenses, is a widely accepted under-lying factor in the development and progres-sion of diabetes and its complications.2 Hyperglycemia has also been known to pro-mote lipid peroxidation of low density lipo-protein (LDL) by a superoxide-dependent pathway to generate free radicals,3 which can be generated in glucose oxidation, believed to be the main source of free radicals that are not degraded by catalase or glutathione pe-roxidase, and, in the presence of transitional metals, can lead to production of extremely reactive hydroxyl radicals.4 Aspirin, a deriva-tive of salicylic acid, used as a NSAID, anti thrombotic, antioxidant and antidiabetic drug, provides a new approach in type 2 diabetes. Salicylates inhibit serine/ threonine caused insulin resistance and IKK-β activity and re-store insulin sensitivity, both in-vitro and in-vivo. Salicylate alters the phosphorylation patterns of IRS proteins, resulting in the de-crease of serine phosphorylation, increased tyrosine phosphorylation, and improved insu-lin action.5 Asprins principal mechanism for its pharmacological action is inhibition of arachidonate cyclooxygenase (COX),6 which is of two types viz. COX-I and COX-II; COX-I is a constitutive enzyme expressed in most tissues including blood platelets and is involved in cell-cell signaling and tissue ho-meostasis. COX-II is induced in inflammato-ry cells when they are activated and is be-lieved to be the enzyme that produces the prostanoid mediators of inflammation. Aspi-rin and also most of the non-steroidal anti in-flammatory drugs (NSAIDS) used currently, are inhibitors of both isoenzymes (COX-I & COX-II), though the degree of inhibition for each varies.7 Many of the antioxidants have the capability of decreasing blood sugar le-vels. Free radicals play a major role in di-abetes and cardiovascular disease; aspirin with its antioxidant properties is considered to be beneficial in such disorders.8 The insu-lin-like growth factor 1 (IGF1), insulin-like growth factor binding protein 3 (IGFBP3), insulin receptor substrate 1 (IRS1), insulin receptor substrate 2 (IRS2), and the vitamin D receptor (VDR) genes have been proposed as being directly or indirectly involved in in-sulin-related pathways. Polymorphisms of these genes have been identified, some of which have been shown to have effects on in-sulin resistance and/or colon cancer risk.9 In this study, we investigate the importance of the role played by aspirin in preventing type-II diabetes mellitus in relation to IGF-1 in rats, considering that aspirin has antioxidant, antidyslipidimic and antidiabetic properties to ascertain whether it plays a role in prevent-ing non-insulin diabetes mellitus.

Materials and methods

Materials Aspirin was donated by Natco Pharma Li-mited, Hyderabad, India. Diphenyl picryl hy-drazyl and streptozotocin were purchased from Sigma, St. Louis, USA, glucose kits from Excel diagnostics limited, Hyderabad, and Ethanol (analytical grade) from E. Merck Limited, Mumbai, India. The rat insulin Elisa kit was obtained from Mercodia AB., Swe-den, and Octeia Rat/Mouse IGF-1 kits were bought from Immunodiagnostic System Ltd., UK.  Animals Four pregnant female Wistar rats, weighing between 300-350 g, obtained from Mahaveer Enterprises, Hyderabad, were housed indivi-dually in acrylic cages in standard environ-mental conditions (20-250C), and fed with standard rodent diet and water ad libitum. The rats were delivered within 1-2 days. Ex-periments on animals were conducted in ac-cordance with internationally accepted prin-ciples for laboratory animal use. The experi-ment was conducted following approval by the related ethical committee. Induction of diabetes At 8 a.m., 5 day-old rat pups received a single 80 mg/kg intraperitoneal injection of streptozotocin (Sigma, St. Louis, MO) in 0.1 M sodium citrate buffer, pH 4.5. Control nondiabetic animals remained in a fasting state, receiving only citrate buffer. After 5-6 weeks, animals with blood glucose levels above 150 mg/dL were considered diabetic.10  Study design The 5 day-old rat pups (neonates) were di-vided into four groups as follows; Group I-14 pups, group II-8 pups, group III-7 pups, and group IV-9 pups. Group I pups received ci-trate buffer solution and served as the normal control group; group II were treated only with streptozotocin (80 mg/kg, i.p), and served as the diabetic controls, group-III treated with aspirin (aspirin dissolved in small volume of ethanol and mixed with milk) 10 mg/kg/day, p.o, for one month (5-35 days) after streptozotocin, served as the treated group for one month, and group IV, treated with aspirin (aspirin dissolved in small volume of ethanol and finally mixed with milk) (10 mg/kg/day, p.o) for two months (5-65 days) after streptozotocin, served as the treated group for two months. On days 35 and 65, blood samples were col-lected from all animals and fasting blood sugar levels, fasting insulin levels fasting IGF-1 levels, insulin resistance and insulin sensitivity levels were estimated for one month and two months. Insulin resistance was assessed using the previously validated homeostasis model assessment for insulin re-sistance, calculated from the fasting insulin and fasting glucose concentrations according to the formula:11 HOMA-IR=FI in mU/L or µU/mL × FPG in mg/dL / 405 Similarly, insulin sensitivity was assessed using the previously validated homeostasis model assessment for insulin sensitivity, cal-culated from the fasting insulin and glucose concentrations according to the formula:11 HOMA-S =1/HOMA-IR. Statistical analysis All variables are expressed as means±SD. Group differences of continuous variables were compared using ANOVA followed by Newman-Keuls test; for all analyses per-formed using Graph Pad Prism 4 (Version. 4) p<0.05 was considered to be statistically sig-nificant.

Results

35th day blood analysis Pups treated with streptozotocin per se and in combination with aspirin for one month and for two months showed significantly raised body weight, fasting blood glucose and insulin resistance levels when compared to the normal control group of pups (p=0.0005, p<0.0001, p<0.0001) respectively. Pups treated with streptozotocin alone and in com-bination with aspirin for one month and two months showed significantly lowered fasting insulin and insulin sensitivity levels when compared to the normal control group of pups (p<0.0001, p<0.0001 respectively). Pups treated with aspirin for one month had significantly raised IGF-1 levels but the two month-treatment group showed significantly lowered IGF-1 levels when compared to normal pups (p<0.0001) (Table 1). 65th day blood analysis Pups treated with streptozotocin per se and in combination with aspirin for one and two months had significantly raised body weight, fasting blood glucose and insulin resistance levels when compared to the normal control group of pups (p=0.0006, p<0.0001, p=0.0030) respectively; those treated with streptozotocin alone and in combination with aspirin for one month and for two months showed significantly lowered fasting insulin and insulin sensitivity levels when compared to the normal control group of pups (p<0.0001, p=0.0068) respectively. Pups treated with aspirin for one month had signif-icantly raised IGF-1 levels, while the two month treatment pups had significantly lo-wered IGF-1 levels when compared to nor-mal pups (p<0.0001) (Table 2).

Table 1. Effects of aspirin on various biochemical parameters at 5th week

Parameter

Normal

Diabetic

Aspirin treatment

P value

One Month

Two Months

Sex (M/F)

4/8

3/5

2/5

4/5

 

Body weight(g)

104.5±14.3

118.7±15.5

138.5±18.6

106.6±16.5

0.0005

Glucose (mg/dL)

77.0±14.1

207.2±14.3

117.1±28.0

112.8±10.5

<0.0001

Insulin (µg/L)

1.8±0.1

1.2±0.1

1.5±0.0

1.6±0.1

<0.0001

IGF-1 (ng/ml)

681.7±40.7

381.0±32.1

884.7±67.9

522.8±87.0

<0.0001

HOMA-IR

8.6±1.2

14.8±1.9

11.2±3.1

10.9±1.1

<0.0001

HOMA-S

0.12±0.02

0.07±0.01

0.10±0.03

0.09±0.01

<0.0001

All variables are expressed as means ± SD. Group differences of continuous variables were compared using ANOVA fol-lowed by Newman Keuls test. For all analyses, a P value < 0.05 was considered to be statistically significant.

Table 2. Effects of aspirin on various biochemical parameters at 10th week

Parameter

Normal

Diabetic

Aspirin treatment

P value

One month

Two months

Sex (M/F)

4/8

3/5

2/5

4/5

 

Body weight (g)

99.5±20.9

130.6±29.9

144.2±19.2

111.1±15.1

0.0006

Glucose (mg/dL)

77.7±7.3

170.4±26.6

127.7±34.1

115.7±23.4

<0.0001

Insulin (µg/L)

1.8±0.1

1.1±0.1

1.5±0.0

1.5±0.1

<0.0001

IGF-1 (ng/ml)

670.5±37.3

393.6±18.8

868.7±65.3

497.2±76.4

<0.0001

HOMA-IR

8.6±0.8

12.0±2.4

12.0±3.4

10.9±1.8

0.0030

HOMA-S

0.12±0.01

0.09±0.02

0.09±0.03

0.09±0.02

0.0068

All variables are expressed as means ± SD. Group differences of continuous variables were compared using ANOVA fol-lowed by Newman Keuls test. For all analyses, a P value < 0.05 was considered to be statistically significant.

 Discussion

Results of our study are consistent with those of the Yaun et al. study that reported salicylates inhibit IKK-B activity and restore insulin sensitivity, both in vitro and in vivo.5 Hundal et al. reported that treatment of nine type 2 diabetic patients for 2 weeks with high dosages of aspirin (7 g/day), resulted in re-duced hepatic glucose production and fasting hyperglycemia and increased insulin sensitiv-ity.12 Micossi et al. reported aspirin stimu-lates insulin and glucagon secretion and in-creases glucose tolerance in normal and di-abetic subjects.13 Seino et al. reported that acetyl salicylic acid (ASA) alleviates glucose intolerance in maturity onset diabetics by a direct enhancement of insulin secretion.14 Our study also indicates that following one month treatment with asprin, blood glucose levels and insulin resistance levels were sig-nificantly reduced and levels of insulin and insulin sensitivity improved significantly.

There is a great deal of evidence that aspi-rin / NSAIDs affect insulin resistance; it has been long known that salicylates have a hy-poglycemic effect and reduce fasting blood glucose in diabetic persons.15-19 High doses of salicylates have been shown to reverse hyperglycemia, hyperinsulinemia, and dysli-pidemia in obese rodents by sensitizing insu-lin signaling.20 In patients with type 2 di-abetes, aspirin treatment has been shown to reduce fasting plasma glucose, total choles-terol, C-reactive protein, triglycerides, and insulin clearance; aspirin reduced hepatic glucose production and improved insulin-stimulated peripheral glucose uptake by 20%.21 The influence of aspirin/NSAID on insulin resistance appears to be independent of COX-2 inhibition, involving instead inhi-bition of nuclear factor-nB and InB and/or activation of peroxisome proliferator-activated receptors.20 An interaction between aspirin and IRS1 in antagonizing effects of tumor necrosis factor- α (TNF- α) has also been reported. TNF- α, a major cause of insulin resistance in obesity and inflammation, has been reported to inhi-bit insulin-induced glucose uptake by target-ing components of the insulin signaling cas-cade, one of which is insulin receptor sub-strate.22-26 IRS1 is the major cytoplasmic sub-strate of the insulin receptor in most insulin sensitive tissues and is necessary for main-tenance of metabolic homeostasis. Aspirin has been shown to inhibit the TNF- α-induced serine phosphorylation of IRS1 through inhibition of multiple serine kinases, including IB kinase.21

Our findings are however consistent with other animal studies, demonstrating low insu-lin sensitivity in mice with liver specific dele-tion of the IGF-1 gene that is reversed by treatment with recombinant human IGF-127,28 IGF-1 has hypoglycemic effects and en-hances insulin sensitivity in both experimen-tal and human subjects, due to its type-1 re-ceptors and/or hybrid insulin / IGF-1 recep-tors.29 High levels of IGF-1 in the 1-month treated aspirin group when compared to the two month treated group, demonstrate that short term therapy seems to be beneficial. A short course of preconditioning of hepatocytes with aspirin is better than long term treatment, in the production of IGF-1. Mechanisms of in-creased levels of IGF-1 in the one month as-pirin treated group however are not very clear .

In conclusion, the present study indicates that aspirin pretreatment seems to protect the pancreas from damage caused by STZ, main-tains glucose levels in diabetic rats, increases insulin sensitivity and reduces insulin resis-tance. The insulin like pathway, particularly IGF-1, may be involved in the protection provided by aspirin treatment in type-2 di-abetes. The mechanism of increased levels of IGF-1 in the one-month treated group when compared to the two-month treated group is not clear and further studies are required to prove this hypothesis.

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