a-lipoic acid ameliorates altered colonic contractility and...
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Indian Journal of Experimental Biology Vol. 42, March 2004, pp. 279-282
a-Lipoic acid ameliorates altered colonic contractility and intestinal transit in STZ-diabetic rats
M Satish Kurnara & K V Harish Prashanthb
Department of Studies in Biochemistry, University of My sore, Manasagangotri, Mysore 570 006, India
Received 17 June 2003; revised 31 December 2003
a-Lipoic acid treatment (100 mg/kg/day for 2 weeks after 6 weeks of untreated diabetes) of streptozotocin diabetic rats partially but significantly reversed both reduced contractile response of distal colon to acetylcholine and delayed transit of charcoal meal in small intestine compared to diabetic control. These effects of a-Lipoic acid were associated with complete reversal of diabetes induced increased plasma lipid peroxidation level. a-Lipoic acid had no effect on any of the parameters measured in non-diabetic rats. These findings demonstrate contribution of oxidative stress in the development of physiological changes of gut in diabetes.
Keywords: Lipoic acid (a), Colonic contractility, Intestinal transit, STZ-diatetes, Rat
The gastrointestinal tract is frequently affected in diabetes mellitus. The changes include reduced peristalsis and dilation of esophagus, delayed gastric emptying, disordered small intestinal movement, and colonic atony or mega colon and these may be manifested as constipation, diarrhoea or fecal incontinence l
. Reduced contractile response of colon to acetylcholine2
, delayed small intestinal transie together with a deficit of cholinergic innervation of the colon4
and small intestine5 have been demonstrated in experimental diabetes.
The diabetic state, in both human and experimental animals, is associated with oxidative stress6 and oxidative damage has been suggested to be contributory factor in the development and complications of diabetes? General increase in the antioxidant state achieved by dietary supplementation can help to diminish oxidative stress associated with diabetes mellitus8
• a-Lipoic acid (ALA) is a powerful lipophilic free radical scavenger in vitro and in vivo9
. ALA has potential preventive or ameliorative effect in both type 1 and type 2 diabetic complications lO
. ALA supplementation has shown to prevent cataract formation II, reduce the symptoms of diabetic peripheral neuropathyl2 and improve cardiac dysfunction 13.
Further, ALA administration prevented rat intestinal
bpresent address: Department of Biochemistry and Nutrition, Central Food
Technological Research Institute, Mysore 570 013, India. ·Phone: 91-821-2515525; Fax: 91-821-2421263 E-mail: mskgowda@yahoo.com
short term ischemia-reperfusion 14. However, the involvement of oxidative stress in the development of functional changes in gastrointestinal tract anj effect of ALA on such changes are less documented.
The aim of the present study is to examine the effects of ALA treatment on altered response of distal colon to exogenous acetylcholine and small intestinal transit of charcoal meal in streptozotocin induced diabetic rats.
Materials and Methods Animals and in vivo treatment-Animal ex
periments were approved by the Institutional Animal Ethical Committee, Mysore, India. Wistar rats (150-180 g) of either sex were fasted overnight and then injected, ip, with 45 mg/kg streptozotocin (Himedia Ltd., Mumbai, India) in 0:5 ml of 0.05 M citrate buffer (PH 4.5). Diabetes was defined as a non fasting plasma glucose level more than 200 mg/dl in tail vein blood (Pulsatum Health Care Pvt. Ltd., Calibration Code 1000, Bangalore, India) 48 hr after streptozotocin injection and again on the day before death. Uninjected non-diabetic (ND) and streptozotocin injected diabetic (STZ-D) rats were maintained on commercial pelle ted food and water ad libitum till the end of the experiment. ND rats were randomly assigned to receive no treatment or after 6 weeks to begin receiving, ip,lOO mg kg" I day"l. ALA (NDIL) (Himedia Ltd., Mumbai, India) for 2 weeks. Similarly, STZ-D rats were randomly assigned after 6 weeks to receive no treatment or to receive, ip,lOO mg kg-I day" 1 ALA (STZ-DIL) or 4 IU of NPH insulin subcutaneous
280 INDIAN J EXP BIOL, MARCH 2004
. (STZ-DII) (Eli Lilly and Ranbaxy, New Delhi, India) for 2 weeks.
Charcoal meal transit in small intestine-At the end of the treatment period, overnight fasted animals of different groups were administered, po, 2ml/rat with charcoal meal (10% charcoal in S% gum acacia) and 20 min later the rats were killed by cervical dislocation. The abdomen was opened and the intestine was removed from pyloric junction to caecal end. Then colon was separated and kept in continuously aerated Tyrode's solution. The farthest distance travelled by the charcoal meal through the small intestine and total length of the intestine were measured. Gastrointestinal transit was expressed as the percentage of the distance travelled by the charcoal meal relative to the total length of small intestine l5.
Contractile response of colonic smooth muscle---Immediately after cleansing the colon, 1 cm of distal colon was mounted under a resting tension of O.S g in an organ bath (40 ml) containing continuously aerated Tyrode's solution with the composition (mmol/l) NaCl :136.9, KCl : 2.7, CaCh : 1.8; MgCh: 10, NaHP04 :0.4, NaHC03 : 11.9, and dextrose: S.6 (PH 7.4S). The temperature was maintained at 37°±loC throughout the experiment and the tissue was allowed to equilibrate for 30 min before exposing to acetyl choline (Hi Media Ltd., Mumbai, India). A primary dose 100 ng of acetylcholine was tested before starting the actual concentration response curve. The contractile responses were recorded isotonically on a Smoked Kymograph drum. At the end of the initial equilibration period dose response curves were obtained for ascending dose of acetylcholine. ED 50 values of acetylcholine were calculated from the graph plotted using percent response against log dose.
Lipid peroxidation in plasma-Lipid peroxidation in plasma was estimated by measuring malondialdehyde (MDA) level in plasma. Amount of malondialdehyde formed was quantitated by reaction with thiobarbituric acid as reported previously'6.
Statistical analysis-Data are presented as the mean±SE from 7 rats per group. Comparison of mean values among the various groups was performed by one way ANOY A. For the single comparison between the groups unpaired Student's t- test was used. P values less than O.OS were considered significant.
Results and Discussion Baseline body weights were similar in all groups.
Table I shows the mean weights for the animals and
the mean blood glucose concentration at death. Eight weeks after injection of streptozotocin, diabetic rats had significantly lower body weights and increased plasma glucose levels when compared with their age matched non-diabetic controls. Treatment of streptozotocin diabetic rats with ALA or insulin had no significant effect on body weights and blood glucose level when compared with diabetic controls. Similarly, treatment of non-diabetic rats with ALA had no significant effect on body weight and blood glucose level compared to non-diabetic controls.
The ED50 of acetylcholine, percent transit of charcoal meal in small intestine and plasma MDA levels all shown significant difference among groups. [F(4,30) = P<O.OI, ANOYA; Table 2]. Streptozotocin induced diabetic rats resulted in significant increase in ED50 of acetylcholine (P<O.OOI), plasma MDA level (P<O.OOl) and significant reduction of transit of charcoal meal (P<O.OOI) compared to normal controls.
ALA treatment of diabetic rats significantly reduced EDso of acetylcholine (P<O.Ol) and increased the percent distance travelled by charcoal meal (P<O.OOl) compared to streptozotocin diabetic controls. But these parameters were still higher than those of non diabetic control rats. Effect of ALA in diabetic rats were associated with reduction in plasma lipid peroxidation level which was significantly less (P<O.OOl) compared to diabetic controls but was not significantly different compared to non~diabetic control rats.
Insulin treatment of diabetic rats significantly reduced ED50 of acetylcholine (P<O.OS), increased percent distance travelled by charcoal meal (P<O.OOI) and reduced the plasma MDA level (P<O.OS) compared to diabetic control. But these parameters were still higher compared to non-diabetic control rats. No
Table l-Characteristics of experimental groups
[Values are mean ± SE]
Groups End weight End blood glucose (g) (mg/dl)
ND 255±6 96± 3 NDIL 260±5 90±5 STZ-D 157± 7 * 297 ± 16 * STZ-DIL 159± 5 286±24 STZ-DIl 165±7 272 ±20
*P<O.OOI when compared with ND. Baseline body weight is 231± 12g. ND= uninjected non-diabetic, STZ-D= streptozotocin injected diabetic, L= a-Lipoic acid treated, and 1= Insulin treated
KUMAR & PRASHANTH: ALA EFFECf ON DIABETIC INTESTINAL TRANSIT 281
Table 2-Effect of ALA or Insulin on lipid peroxidation, contractile response of distal colon to exogenous acetylcholine and small intestinal transit of charcoal meal in non-diabetic and STZ-diabetic rats
[Values are mean±SE]
Groups
NO NOlL STZ-O STZ-OIL STZ-Ofl
MOA (n moUlOO ml of plasma)
409.75± 12.98 417.81± 31.96 801 .24±31.44a 499.74±45.09b
574.04±17.36b
Contractile resposne of distal colon to exogenous acetylcholine
EOso (J,lg) 1.21± 0.02 0.63±O.23
13.61±2.91a 3.35±O.42 c 5.75±O.63 d
Charcoal meal transit in small intestine (%)
84.12 ± 1.76 85.21±0.41 62.58±1.l8a
71.86±1.03b
68.48± 1.98b
P values: ap<O.OOI when compared to NO rats, bp<O.OOl, cp<O.OI, dp<0.05 when compared to STZ-O rats. NO= uninjected non-diabetic, STZ-O= streptozotocin injected diabetic, L= a-Lipoic acid treated, and 1= Insulin treated
significant change in any of the parameters measured was observed after treating non diabetic rats with ALA compared with non-diabetic controls.
Distal colon from untreated diabetic rats were found to be less sensitive to acetylcholine, further, delay in transit of intestinal content was also observed. These observations are in agreement with pre-
vious reports2•3
• There was parallel increase in lipid peroxidation level in diabetic rats. This observation confirms the fact that hyperglycemia per se results in lipid peroxidation6
• Treatment of diabetic rats with ALA significantly reversed all the parameters measured, suggesting oxidative stress may be contributory factor causing changes in these parameters in diabetic rats. This theory is supported by the present observations in non-diabetic rats treated with ALA and diabetic rats treated with insulin. ALA treatment of nondiabetic rats had no significant effect on plasma lipid peroxidation level, contractile response of colon to acetylcholine and transist of charcoal meal in intestine compared to normal controls, which suggest that DLa-lipoic acid effects are diabetes dependent. Further, improvement of contractile response of distal colon to acetylcholine and propulsive movement of small intestine along with reduction of lipid peroxidation after treatment of diabetic rats with insulin indicate that hyperglycemia stimulated oxidative stress may contribute physiological changes in colon and small intestine.
The reduced contractile response of colonic smooth muscle to exogenous acetylcholine may be the result of excessive degradation of acetylcholine by tissue acetylcholine esterase, diminished muscaranic receptor sensitivity or density or defective interaction between muscaranic receptor and intracellular contrac-
tile process. The myogenic-phenomenon in distal small intestine of diabetic rats is not affected5
• Studies of the responsiveness of diabetes colonic smooth muscle to acetylcholine are limited, whereas vascular17
, 18 and cardiac muscle19 in experimental diabetes does show altered sensitivity to acetylcholine. Decrease in muscarinic receptor density in atrial muscle has been suggested for reduced cardiac sensitivity to cholinergic agents20
• Therefore, it appears that oxidative stress may induce changes in muscaranic receptor density and binding affinity leading to reduced cholinergic response and ALA treatment may abolishes such changes.
Impaired cholinergic response of distal small intes
tinal smooth muscle has been reported5• Further, in
crease in noradrenaline level and in its turnover have also been observed in ilea form 6-week streptozotocin diabetic rats21
• ALA therapy improved the cholinergic response of colonic smooth muscle in diabetes rats in the present study. If this would also happen in small intestinal smooth muscle, it may provide one mechanism to explain enhanced intestinal transist of charcoal meal after ALA therapy. Other possible mechanisms include decrease in noradrenaline concentration and its turnover.
In conclusion, this study suggests that the role of oxidative stress in the development of gastrointestinal functional changes associated with experimental diabetes. Beneficial effects of ALA on gastrointestinal functional changes in diabetes of long duration needs to be investigated.
Acknowledgement The authors are grateful to the Chairman, Depart
ment of Studies in Biochemistry, University of Mysore, Mysore for facility.
282 INDIAN J EXP BIOL, MARCH 2004
References 1 Clarke B F, Ewing D J & Campbell I W, Diabetic autonomic
neuropathy, Diabetologia. 17(1979) 195. 2 Jaiprakash R, Nagarani M A & Venkataraman B V, Effect of
felodipine on cholinergic responses of the colonic smooth muscle of streptozotocin induced diabetic rats, Indian J. Exp. Bioi, 33 (1995) 297.
3 Patil B M & Thomas K M. Inhibition of nitric oxide synthase by N-nitro-L-arginine overcomes delayed intestinal transit in streptozotocin induced diabetic mice, Illdian J. Pharmacol, 31, (1999) 146.
4 Schmidt R E, Nelson J S & Johnson E M, Experimental diabetic autonomic neuropathy, Am J Pathol. 103 (1981) 210.
5 Nowak T V, Harrington B, Kalbfleisch J H & Amatruda J M, Evidence for abnormal cholinergic neuromuscular transmission in diabetic rat small intestine, Gastroenterology, 91(1986) 124.
6 Androne L, Gavan N A, Veresiu I A & Ovasan R, III vivo effect of lipoic acid on lipid peroxidation in patients with diabetic neuropathy, Diabetes Nutr Metab. 13 (2000) 308.
7 Dincer Y, TeIci A, Kayali R, Yilmaz I A, Cakatay U & Akeay, T, Effect Of alpha-lipoic acid on lipid peroxidation and anti-oxidant enzyme activities in diabetic rats, Clill Exp Phannacol Physiol, 29 (2002) 281.
8 Ruhe R C & McDonald R B , Use of antioxidant nutrients in the prevention and treatment of type 2 diabetes, J Am Call Nutr, 20 (2001) 365S .
9 Phillip A, The roles of oxidative stress and antioxidant treatment in experimental diabetic neuropathy, Diabetes, 46 (Suppl. 2) (1997) S38.
10 Packer L & Tritsciler H, Alpha lipoic as a biological antioxidant, Free Rad Bioi Med. 19 (1995)227.
11 Maitra I, SerbinovaE, Tritschler H & Packer L, Alpha-lipoic acid prevents buthionine sulfoximine-induced cataract formation in newborn rats, Free Rad Bioi Med, 18(1995) 823.
12 Stevens M J, Obrosova I, Cao X, Huysen C V & Greene D A, Effects of DL-alpha-Iipoic acid on peripheral nerve conduction, blood flow, energy metabolism, and oxidative stress in experimental diabetic neuropathy, Diabetes, 49 (2000) 1006.
13 Ziegler & Gries F A, Alpha-lipoic acid in the treatment of diabetic peripheral and cardiac autonomic neuropathy, Diabetes. 46 (Supp. 2) (1997) S62.
14 Boveris A, Roldan E J A & Alvarez S, Effect of lipoic acid on short-term ischemia-reperfusion in rat intestine, in Biological oxidants and antioxidants: New developments ill research alld health effects, Vol. 10, edited by L Packer and E Cadenas (Styttgart Hippokrates Verlag) 1994,77.
15 Janseen PAS & Jagenerous A H, New series of potent analysis, J Pharma Pharmacal, 6 (1957) 38.
16 Dillard C J, Kunnert K J & Tappel A L, Effects of viitamin E, ascorbic acid and mannitol on alloxan induced lipid peroxidation in rats, Arch Biochem Biophysics. 216 (1982) 204.
17 Brody M J & Dixon RL, Vascular reactivity in experimental diabetes mellitus, Circ Res, 14 (1964) 694.
18 Head R J, Longhurst P A, Panek, R L & Stitzel R E, A contrasting effect of the diabetic state upon the contractile responses of aortic prepara- tions from the rat and rabbit, Br J Pharmacol, 91 (1987) 275.
19 Carrier G 0 & Aronstam R S, Altered muscarinic receptor properties and funciton in the heart in diabetes, J Pharmacal Exp Ther, 242 (1987) 531.
20 Abayomi K A & Lucas PD, Muscarinic receptor density is reduced in diabetic rat atria, an effect prevented by the aldose reductase inhibitor, Statil, J Pharm Pharmacal, 39 (1987) 1019.
21 Lucas P D & Qirbi A, Tissue noradrenaline and the polyol pathway in experimentally diabetic rats, Br J Pharmacal, 97 (1989) 347.
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