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

ALLOXAN DIABETES IN SWISS MICE: ACTIVITY OFNA+-K+-ATPASE AND SUCCINIC DEHYDROGENASE

G. lVIISHRA, R. ROUTRAY, S. R. DAS AJ.'JD H. N. BEHERA'"

P.G. Department 0/ Zoology,Berlwmpur Uniuersity,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 rel1ecting reduced glucose transport across the cell membrane.On the contrary, the observed enhanced activity of the enzyme SDH isattributed to increased supply of TC \ cycle substrates from accelaratedoxidation of fatty Gcids.

Key words: alloxan diabetes

Na'-K'-ATPase

INTRODUCTION

Disorder 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 deficient)f this primary energy currency for further

degradation 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 int9 TCA cycle. In viewf the above facts, in the present study the

activity of t\VO enzymes, viz. Nat-K'-ATPaseresponsible for glucose symport and succinicdehydrogenase (SDH) responsible in part forthe mitochondrial oxidation of fuel moleculeswas studi d in liver and brain of aUoxan diabeticS\oviss mice.

METHODS

Swiss albino mice (Mus musculus) body wt.range 1:5-33 g (approximate age 2-3 months) of

~Corr spuoding Author

Swiss mice

succinic dehydrogenase

both sexes were procured from a commercialfirm at Calcutta and were maintained at roomtemperature (30 ± 2CC) 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 48hClUrs 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 ater at a dose of 100 mglkg body wt(4), while the control group received an qualvolume of distilled water. Diabetic state wasmaintained by administration of repeated closeof 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)

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olurion. Glucose contents of the sample wasdetermined c;olorimetrically (5).

Tissue processing: The brain and the liverW('!" , quickly dissected out in precooledmaml1lalian 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 bloLLcd off in Whatman filter paper No.1and weighed. A 5% homogenate of each tissue\ a· pre! ared in 0.2;jM sucrose solution usinga RE iII homogenizer (Bombay) at a mediumspeed for 1 min. This homogenate was used forthe assay of Na'-K'-ATPase and succinicdehyd rogenase activity as described in ourearl ier publication (8). Significance of the resultwa 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 theothcrhand, the liver glycogen levels declinedsi!!l1ificantly following al1oxanization (Fig. 1).

Such observations confirmed the induction ofal10xan diabetes in mice as reported earlier (4).

Na '-K'-A TPase activity: The enzyme(ATPase) activity in brain, and liver of micedecreased significan Hy following 7 days ofal oxan 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 "chemicaldiabete " (alloxan diabetes) in a wide variety ofanimal species through damage of the insulinsecreting cells nO). Induction of diabetes inthese animals was confirmed by a significantrise in blood-glucose and fall in the liver glycogenlev 1 (11), as shown in other animal species.Th(~ lower actIvity of the glycolytic enzymes

Indian J Physial Pharmacal 1995; 39(3)

such as glucokinase, phosphofructokinase,glyceraldehyde-3- phosphate dehydrogenase (2)and of various isocnzymes of LDH (12) within asingle metabolic pathway possibly points todecreased supply of glucose to the ceBs 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 III

the brain and the 1iver 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'-ATPase require complete integrationof cell membrane for their activity (15). Acertain degree of fluidity seems essential for

Indian J Physiol Pharmacal 1 95: 39(3) Na+·K+·ATPa e and SDH Activity dunng Alloxan Diabetes 273

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rate of oxidative decal'box lation of pyruvate toAcetyl CoA through a reduction in the activityof pyruvate dehydrogenase complex (3). As uelon might expect a low ield of TCA cycleenzymes and low energy output unless adequat

cetyl CoA is derived from oxidation of lipids,Therefore the present study also deals with theactivity of TCA cycle enzyme succinicdehydrogenase (SDH) in tissue: of alloxandi:1betic Swiss mice. Thi enzyme directl'involved in the aerobic oxidation of food stuf'and is probably the best candidate fat suchstudie3 because it is tightly bound to the innermitochondrial membran and th oxidation ofuccinat' to fumarate in ani nal ti ue is linked

to 0" via cytochrome anc cytochr me oxida ~

(22). -

a'·K· TPase and the fluidity of thephospholi pids bilayer of the membranes, to alarg- extent, is determined by the fatty acids(16). Decreased enzyme activity was reportedwith th decrease in phospholipid molecules(171. It is possible that the reductions in theactivity of this enzyme in tis 'ues of alloxandiabetic Swiss mice is chiefly du to decreasesin both fatty acid and phospholipid levels sincereductions in the contents of these liquidfractions has already been observed incryLhroc t membrane (18) and cardiac tissue(19) during diabetes mellitus. On the other hand,the po 'sibility of nonenzymatic glycosylation oftIl above enzyme, as it occurs in human d' abetes(20) leading to its malfunrtion (21) cannot beignored.

Consequent upon the decreased activity ofglycolytic enzymes, th concentration of the endproduct of glycolysi , i.~. pyruv te ntering theo. idative pathway is low' s evident by decreased

The results indicate that he activity of th _enzyme SDH in brain and liver homogenate ofalloxan diabetic Sis mic' mcreasedsignificantly over the l' p dive ontrol lue( •ig. 2) in conformity with the earlier observation(23), Such observations make the a ailabilityoface I CoA from non-carbohydrate precursormol' plausible and free en ry of this metaboliteinto TCA cycle in sufficient amounts so a toreflect an increase in the rate of aerobioxidation. It is known that s ch a metabolitecan be derived from oxidation of non·arbohydrate precursor II h as lipid,

Moreover, diabetes is in ri bly a~sociated

with increased fatty acid oxidation ( 8J.Therefore, one might suggest that the increasedactivity of SDH in tissue of diabetic S i" smice on the face of decreased rate of gl colysis,is mediated through increased supply ofsubstrates from accelerated oxidaion offatty acids, as has also been point d outearlier (23).

ACKNOWLEDGEMENTS

Thanks are due to the Council 0 Scientifiand Industrial Research (CSIR) ew Delhi orproviding Dr. (Mrs) Gitanjali Mishra vithResearch Associateship and to Universitauthorities for makina' th I boratory facilitiesavailable during the co rs of this work.

274 Mishra et al

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