Indian J Physiol Pharmacol 1995; 39(3): 271-274

ALLOXAN DIABETES IN SWISS MICE: ACTIVITY OFNA+-K+-ATPASE AND SUCCINIC DEHYDROGENASEG. MISHRA, R. ROUTRAY, S. R. DAS AND H. N. BEHERA*

P.G. Departmerit of Zoology,Berhampur University,Berhampur - 760 007 (Orissa)

( Received on June 27, 1994 )

Abstract: The activities of two enzymes viz: Na+-K+-ATPase and SUCCInICdehydrogenase (SDH) in brain and liver of alloxan diabetic Swiss albino miceare reported. Alloxan diabetes caused significant decrease in the activity ofNa+-K+-ATPase reflecting reduced glucose transport across the cell membrane.On the contrary, the observed enhanced activity of the enzyme SDH isattributed to increased supply of TeA cycle substrates from accelaratedoxidation of fatty acids.

Key words: alloxan diabetesNa+-K+-ATPase

INTRODUCTION

Disorders in glucose homeostasis duringdiabetic syndrome are chiefly due to poortransport of glucose across the cell membrane.Glucose enters the cell by a specific transportsystem that is regulated by the hormone insulin(1). This possibly leads to lower intracellularglucose concentration making the cell deficientof this primary energy currency for furtherdegradation and energy output. As might beexpected, the activity of glycolytic enzymesdecreased (2). Likewise, the enzyme pyruvatedehydrogenase responsible for irreversiblefunneling of glycolytic product i.e. pyruvate intoTCA cycle showed lower activity (3) thus limitingfree entry of substrates into TCA cycle. In viewof the above facts, in the present study theactivity of two enzymes, viz. Na+-K+-ATPaseresponsible for glucose symport and succinicdehydrogenase (SDH) responsible in part forthe mitochondrial oxidation of fuel moleculeswas studied in liver and brain of alloxan diabeticSwiss mice.

METHODS

Swiss albino mice (Mus musculus) body wt.range 15-33 g (approximate age 2-3 months) of

Swiss micesuccinic dehydrogenase

both sexes were procured from a commercialfirm at Calcutta and were maintained at roomtemperature (30 ± 2°C) on a freshly prepareddiet (500 g semolina, 50 g milk powder, 20 yeasttablets, NaCI salt 5 g, boiled to make paste toserve 20 animals) and water was providedad libitum. A minimum acclimation period of 7days was always allowed before the beginningof the experiments.

Alloxan treatment: After laboratoryacclimation, 20 animals were starved for 48hours and divided into control and experimentalgroups (ten animals each). Diabetes was inducedin experimental groups of mice throughintraperitoneal injection of alloxan (Fischameno,Loba Chemie, Wien, Austria) dissolved indistilled water at a dose of 100 mg/kg body wt(4), while the control group received an equalvolume of distilled water. Diabetic state wasmaintained by administration of repeated dosesof alloxan on every alternate day for 7 days.

Blood glucose level: On the 8th day oftreatment, blood drawn from the subclavianvein of mice of both control and experimentalgroups under mild ether anaesthesia andcollected in graduated centrifuge tubescontaining 2 ml of 2% sodium citrate (E Merck)

"Corresponding Author

272 Mishra et al

solution. Glucose contents of the sample wasdetermined colorimetrically (5).

Tissue processing: The brain and the liverwere quickly dissected out in precooledmammalian Ringer (Kreb's Ringer phosphate)and the adherent tissues were cleaned. A partof the liver was used for the estimation ofglycogen content following standard procedures(6, 7). The entire brain and rest of the liverwere blotted off in Whatman filter paper No.1and weighed. A 5% homogenate of each tissue

. was prepared in 0.25M sucrose solution usinga REMI homogenizer (Bombay) at a mediumspeed for 1 min. This homogenate was used forthe assay of Na+-K+-ATPase and succinicdehydrogenase activity as described in ourearlier publication (8). Significance of the resultwas analyzed using Students 't' test (9).

RESULTS

Blood glucose and liver glycogen level:Alloxan treatment for 7 days to mice increasedthe blood glucose content significantly ascompared to the values of controls. On theotherhand, the liver glycogen levels declinedsignificantly following alloxanization (Fig. 1).Such observations confirmed the induction ofalloxan diabetes in mice as reported earlier (4).

Na+-K+-ATPase activity: The enzyme(A'I'Pase) activity in brain, and liver of micedecreased significantly following 7 days ofalloxan treatment (Fig. 2).

SDH activity: Succinic dehydrogenaseactivity in the brain and the liver homogenatesof Swiss mice increased significantly following 7days of alloxan treatment (Fig. 2).

DISCUSSION

Alloxan, a B-cytotoxin, induces a "chemicaldiabetes" (alloxan diabetes) in a wide variety ofanimal species through damage of the insulinsecreting cells (10). Induction of diabetes inthese animals was confirmed by a significantrise in blood-glucose and fall in the liver glycogenlevel (Ll ), as shown in other animal species.The lower activity of the glycolytic enzymes

Indian J Physiol Pharmacal 1995; 39(3)

such as glucokinase, phosphofructokinase,glyceraldehyde-3- phosphate dehydrogenase (2)and of various isoenzymes of LDH (12) within asingle metabolic pathway possibly points todecreased supply of glucose to the cells duringinduced diabetes. The entry of glucose into cellis mediated by specific transport ATPase suchas Na+-K+-ATPase in conjuction with themovement of Na" and K+ ions across the cellmembrane (13) and the activity of this enzymeis largely influenced by the concentration of thehormone insulin in the blood (1). The resultsindicate a decreased activity of this enzyme inthe brain and the liver of alloxan diabetic Swissmice. Such an observation conforms to the earlierreports that the transport of ions and glucoseacross the cell membrane is reduced duringdiabetes (14). Most transport ATPases, so alsothe Na+-K+-ATPaserequire complete integrationof cell membrane for their activity (15). Acertain degree of fluidity seems essential for

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Na'-K'-ATPase and the fluidity of thephospholipids bilayer of the membranes, to alarge extent, is determined by the fatty acids(16). Decreased enzyme activity was reportedwith the decrease in phospholipid molecules(17), It is possible that the reductions in theactivity of this enzyme in tissues of alloxandiabetic Swiss mice is chiefly due to decreasesin both fatty acid and phospholipid levels sincereductions in the contents of these liquidfractions has already been observed inerythrocyte membrane (8) and cardiac tissue(19) during diabetes mellitus. On the other hand,the possibility of nonenzymatic glycosylation ofthe above enzyme, as it occurs in human diabetes(20) leading to its malfunction (21) cannot beignored.

Consequent upon the decreased activity ofglycolytic enzymes, the concentration of the endproduct of glycolysis, i.e. pyruvate entering theoxidative pathway is low as evident by decreased

NaT-KT-ATPase and SDH Activity during Alloxan Diabetes 273

rate of oxidative decarboxylation of pyruvate toAcetyl CoA through a reduction in the activityof pyruvate dehydrogenase complex (3). As suchone might expect a low yield of TCA cycleenzymes and low energy output unless adequateacetyl CoA is derived from oxidation of lipids,Therefore the present study also deals with theactivity of TCA cycle enzyme succinicdehydrogenase (SDH) in tissues of alloxandiabetic Swiss mice. This enzyme directlyinvolved in the aerobic oxidation of food stuffand is probably the best candidate for suchstudies because it is tightly bound to the innermitochondrial membrane and the oxidation ofsuccinate to fumarate in animal tissues is linkedto 02 via cytochrome and cytochrome oxidase(22).

The results indicate that the activity of theenzyme SDH in brain and liver homogenates ofalloxan diabetic Swiss mice increasedsignificantly over the respective control values(Fig. 2) in conformity with the earlier observation(23). Such observations make the availability ofacetyl CoA from non-carbohydrate precursorsmore plausible and free entry of this metaboliteinto TCA cycle in sufficient amounts so as toreflect an increase in the rate of aerobicoxidation. It is known that such a metabolitecan be derived from oxidation of non-carbohydrate precursors such as lipids.Moreover, diabetes is invariably associatedwith increased fatty acid oxidation (18).Therefore, one might suggest that the increasedactivity of SDH in tissues of diabetic Swissmice on the face of decreased rate of glycolysis,is mediated through increased supply ofsubstrates from accelerated oxidation offatty acids, as has also been pointed outearlier (23).

ACKNOWLEDGEMENTS

Thanks are due to the Council of Scientificand Industrial Research (CSI:R) New Delhi forproviding Dr. (Mrs) Gitanjali Mishra withResearch Associateship and to Universityauthorities for making the laboratory facilitiesavailable during the course of this work.

274 Mishra et al Indian J Physiol Pharmacol 1995; 39(3)

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