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

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