Changes in metabolism of rat kidney and liver caused by experimental diabetes and by dietary sucrose

Diabetologia, Apr 1982

S. S. Kang, R. Fears, S. Noirot, J. N. Mbanya, J. Yudkin

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Changes in metabolism of rat kidney and liver caused by experimental diabetes and by dietary sucrose

Diabetologia Changes in Metabolism of Rat Kidney and Liver Caused by Experimental Diabetes and by Dietary Sucrose S. S. K a n g I 0 R. Fears 0 S. N o i r o t ~ 0 J. N. M b a n y a 0 a n d J. Y u d k i n I 0 0 Department of Nutrition, Queen Elizabeth College, London and 2Beecham Pharmaceuticals Research Division , Epsom, Surrey , UK 0 0 1 2 - 1 8 6 X / 8 2 / 0 0 2 2 / 0 2 8 5 / $ 0 1 . 0 0 Streptozotocin-diabetes; sucrose-diet; alloxan-diabetes; lipogenesis; lipogenic enzymes; g l u c o n e o g e n i c enzymes; kidney; liver; rat; k i d n e y lipids - Summary. T w o g r o u p s o f rats were fed diets in which the c a r b o h y d r a t e c o m p o n e n t was either starch or sucrose. A third g r o u p was fed on a stock diet. H a l f o f the animals in each g r o u p were m a d e diabetic b y injection o f either streptozotocin, in two o f the groups, or alloxan, in the third group. Both di~abetes a n d sucrose-feeding increased renal gluconeogenesis as indicated b y increased activities o f f r u c t o s e - l , 6 - d i p h o s p h a t a s e a n d glucose-6p h o s p h a t a s e . Sucrose-feeding increased fatty acid synthesis b o t h in the liver a n d kidney. H o w e v e r , the effect o f diabetes on fatty acid synthesis was different at the two tissue sites. Diabetes, w h e t h e r i n d u c e d b y streptozotocin or alloxan, d e c r e a s e d fatty acid synthesis in the liver but increased the rate in the kidney. T h e latter r e s p o n s e was o b t a i n e d for e a c h diet but was additive with the effect o f sucrose. We c o n c l u d e that the effect o f diabetes on renal lipid m e t a b o l i s m m a y reflect, in part, the accelerated glucose flux. The r e s p o n s e to b o t h diabetes a n d sucrose-feeding is also possibly associated with the increased lipid required for the m e m b r a n e synthesis rep o r t e d previously. We h a v e s h o w n previously that the c o n s u m p t i o n o f sucrose-rich diets b y rats results in k i d n e y g l o m e r u l a r b a s e m e n t m e m b r a n e thickening that resembles the effect o f diabetes [ 1 ]. C o m p a r a t i v e l y little is k n o w n concerning the relative effects o f sucrose-feeding a n d diabetes on i n t e r m e d i a r y m e t a b o l i s m in the kidney, whereas it is established that dietary sucrose a n d diabetes h a v e converse effects on hepatic lipogenesis [ 2-5 ] but qualitatively similar effects on gluconeogenesis [ 6-8 ]. It was the p u r p o s e o f the present experim e n t s to e x a m i n e the effects o f diabetes a n d sucrosefeeding o n renal c a r b o h y d r a t e a n d lipid m e t a b o l i s m , c o m p a r i n g the r e s p o n s e with changes in the liver w h e r e a p p r o p r i a t e , in an a t t e m p t to obtain further inf o r m a t i o n concerning the deterioration in kidney function o b s e r v e d in these m e t a b o l i c states. Materials and Methods Animals Three experiments were performed. In the first and second experiment, male Wistar rats weighingbetween 170and 200g were fed on a reconstituted diet containing corn starch (68%)or sucrose (68%) [ 2 ] for a period of 25-30 days. In the third experiment, male Sprague-Dawley rats (180-200 g) were fed on a stock pelleted diet (Oxoid Breeding diet, Betchworth, Surrey). Half the rats in each dietary group were made diabetic. In experiments 1and 2, this was done 2 days after the beginning of the experiment by injection of streptozotocin into the tail vein (65 mg/kg body weight); these rats were thus diabetic for 23-28 days before the experiments were ended. In experiment 3, diabetes was induced by the injection of alloxan (45 mg/kg body weight) 2 days before the experiment was ended. The conditions for the third experiment were chosen to be different from the first two experiments to determine the consistency of the effect of diabetes on lipid synthesis.Food and water were administered ad libitum and rats were killed between 10.00 and 12.00h. Analytical Methods Serum glucose was analysed by a glucose oxidase method (GODPAP method, Boehringer Mannheim, FRG), insulin by the doubleantibody method of Hales and Randle [ 9 ] using a human insulin standard. Total lipids were estimated in the kidney homogenate by the sulfovanillin reaction (Total lipids, Boehringer Mannheim, FRO). Values are expressed as mean _+ SEM, a Values for sucrose-fedrats significantlydifferent from starch-fed rats (p < 0.005), b(p < 0.01) Enzyme activities:Theleft kidney was homogenisedin five volumes of ice-cold 0.15 mol/l KC1 and the homogenates were centrifuged at 15,000 g for 30 min at 4 ~C. The supernatant was used to measure the activities of glucose-6-P dehydrogenase (G-6-PDH) [ 10 ], pyruvate kinase (PK) [ 11 ], fructose-l-6-diphosphatase (FDPase) [12] and glucose-6-phosphatase(G-6-Pase) [ 13 ]. Lipogenesisin vivo:Synthesis of fatty acids were measured 1 h after an IP injection of 3H20 (1.5 mCi/100 g body weight in 0.9% NaC1, 0.1 ml/100 g body weight) [ 14 ]. Statistical analysis:The data were subjected to a two-way analysis of variance (ANOVA) to assess the effect of diet and of diabetes, and the interaction between the two. Due to non-homogeneity of variance, some data were transformed into common logarithms prior to analysis. The significance of differences between individual means was tested by Student's t-test. Results In agreement with results presented previously [ 3 ] in two separate experiments, the feeding of sucrose did not affect gain in body weight by the rats or their apparent well-being compared with rats fed starch. However, sucrose produced heavier kidneys than did starch (Table 1). Streptozotocin-induced diabetes led to impaired growth and kidney weights relative to body weight were increased. Analysis of variance disclosed a significant interaction (p < 0.05) between the effects of sucrose and diabetes on kidney weights. The streptozotocin-treated rats remained diabetic as judged by the presence of hyperphagia, polydypsia, polyuria and glucosuria, and were markedly hyperglycaemic at the end of the experiment (Table 1), blood glucose concentrations being negatively correlated with insulin concentrations (r = -0.83, p < 0.001). EnzymeActivities In the first experiment (Table 1), we compared the influence of diet and diabetes on individual enzymes in the kidney whose activities provide either an index of the potential rate of provision of substrate (pyruvate kinase) or reducing equivalents (glucose-6-P dehydrogenase) for lipogenesis, or an index of the potential rate of gluconeogenesis (fructose-1,6-diphosphatase, glucose-6-phosphatase). Our results are expressed as enzyme units per 100 g body weight, a measure that relates biochemical activity to the functional requirement of the whole animal [ 15 ]. It is particularly necessary to consider the results in this manner when comparing diabetic rats whose body weight gain is impaired and where liver weight shows a moderate decrease but kidney weight an increase. Diabetes significantly increased the activities of FDPase (p < 0.001) and G-6-Pase (p < 0.001); G-6P D H and PK activities were also enhanced (p < 0.001; p < 0.001). The response was qualitatively similar when enzyme activities were expressed per unit weight of protein but the physiological effect of diabetes was then underestimated because the use of unit weights cannot take into account the large increment in kidney size. The effect of sucrose feeding was to increase all enzyme activities although the enhancement of G-6-PDH and G-6-Pase did not attain statistical significance in normal animals and the change in G-6-PDH and FDPase activities was not significant in diabetic rats. The enhancement of PK activity by sucrose was greater in the diabetic rats as reflected by the significant interaction between sucrose and diabetes (p < 0.01). Fatty Acid Synthesis In the second long-term experiment, we attempted to confirm the changes observed for the key enzymes potentially indicative of the rate of lipogenesis (G-6PDH, PK) by measuring the total mass of fatty acid synthesised in vivo, 1 h after the administration of 3 H 2 0 . Sucrose-feeding enhanced hepatic fatty acid synthesis in both normal (p < 0.05) and diabetic rats (p < 0.01), the rate was decreased by diabetes using either diet. The response measured in the kidney was in agreement with the results of the enzyme activities; fatty acid synthesis was increased by diabetes, although the change reached statistical significance only in the sucrose-fed rats. Fatty acid synthesis was also increased by sucrose-feeding although the change reached statistical significance only in the diabetic rats. The interaction between sucrose and diabetes was significant in liver (p < 0.01) and kidney (p < 0.05). Thus the combination of the two factors appeared synergistic and was associated with a large increase in the accumulation of total lipid in the kidney (Table 2). Acute Effects To determine whether the effect of the diabetic state on the rate of fatty acid synthesis in the kidney relative to the liver is maintained under different conditions, we also examined, in experiment 3, the acute response to alloxan in stock-fed rats of a different strain. Diabetes was characterised by hyperglycaemia (Table 3) and by polyuria, glycosuria and by an increased intake of water. In this acute experiment, diabetes had little influence on liver and kidney weight relative to total body weight, so that lipid synthesis has been expressed per unit wet weight of tissue. Similar changes were noted when tissue activities were expressed per 100 g body weight. In agreement with the longer-term experiments, diabetes significantlyinhibited fatty acid synthesis in the liver and increased the rate in the kidneys. Discussion The results for enzyme activity and rate of lipogenesis for the long-term experiments are expressed per 100 g body weight, a procedure which has been considered to provide the best means of comparing diabetic animals with their age-matched controls [ 15 ]. If the results are expressed, for example, per unit wet weight then there is no effect of diabetes on either the potential or actual rate of renal lipogenesis but we feel that Rats Starch-fed ( n = 8 ) Sucrose-fed ( n = 8 ) Starch-fed diabetic (n = 8) Sucrose-fed diabetic ( n = 8 ) Diet this method of calculation underestimates the response and leads to a conclusion at variance with the effect of acute diabetes (Table 3). As the physiological use of circulating substrates by the whole kidney depends on the tissue weight with respect to body weight, we agree with Sochor and co-workers [ 15 ]that it is correct to take account of the changes both in tissue weight and body weight. Similarly, the increase in gluconeogenesis is only of physiological significance in terms of the contribution made by the kidney to metabolism overall. In the kidney, the increased potential for gluconeogenesis after streptozotocin-induced diabetes is in agreement with other findings [ 4 ] and suggests a role for the kidney in the overproduction of glucose. A similar effect on renal gluconeogenesis was p r o d u c e d by sucrose-feeding, resulting from the ability o f the kidney to convert fructose to glucose [ 16 ]. Whereas sucrose-feeding also increased renal lipogenesis in parallel with the changes in the liver, we f o u n d that the effect of diabetes on fatty acid synthesis was dissimilar at the two sites. This novel finding applied both to acute and, to a smaller extent, chronic diabetes and the qualitative response was independent of diet. The increase in fatty acid synthesis, measured by 3H incorporation, tended to be smaller than the changes observed in individual enzyme activities. This may be related to a simultaneous increase in fatty acid oxidation [ 17 ] resulting in a smaller net accumulation of radiolabelled fatty acid. Whilst the effect of diabetes on the liver can be attributed to the insulin sensitivity of that organ and the effect of sucrose-feeding attributed to substrate flux, the control of events in the kidney is less clearcut. Hormonal control is probably of greater importance in hepatic gluconeogenesis [ 18 ] than in the kidney, where the availability of specific substrate determines the rate of gluconeogenesis [ 19 ]. Moreover, insulin is not required for renal phosphorylation of glucose by hoxokinase [ 20 ] so that the rates ofglycolysis and lipogenesis may reflect the high blood glucose concentration. Furthermore, the diabetic kidney has a greater requirement for fatty acid to synthesise phospholipid for new membrane during hypertrophy and, perhaps, also for the thickening basement membrane. The additive, possibly synergistic, effects of dietary sucrose and diabetes on fatty acid synthesis and lipid accumulation are of particular interest and may be explained, in part, by the hyperphagia found in diabetes with the consequent increased requirement for disposal of fructose. We conclude from the present results that sucrosefeeding and diabetes have qualitatively similar effects on renal lipogenesis in support of previous findings [ 1 ] for similarity as regards the modification of glomerular basement membrane. The potency of the interaction between sucrose and diabetes underlines the usefulness of this carbohydrate in models for the study of biochemical mechanisms in diabetes. 1. 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B0cher T , Pfleiderer G ( 1955 ) Pyruvate kinase from muscle . Methods Enzymol 1 : 435 - 440 12. Latzko E , Gibbs H ( 1974 ) Alkaline Cl-fructose-l,6-diphosphatase . In: Bergmeyer HU (ed) Methods of enzymatic analysis . Academic Press, New York, pp 881 - 884 13. Baginski ES , Foil PP , Zak B ( 1974 ) Glucose-6-phosphatase . In: Bergmeyer HU (ed) Methods of enzymatic analysis . Academic Press, New York, pp 876 - 880 14. Fears R , Umpleby AM ( 1979 ) Evidence for the operation of the extrahepatic lipoprotein receptor system in vivo in rats . Biochem J 182 : 803 - 809 15. Sochor M , Baquer NZ , McLean P ( 1979 ) Glucose overutilization in diabetes: evidence from studies on the changes in hexokinase, the pentose-phosphate pathway and glucuronate-xylulose pathway in rat kidney cortex in diabetes . Biochem Biophys Res Commun 86 : 32 - 39 16. Flinn RB , Leboeuf B , Cahill GF ( 1961 ) Metabolism of 14C-labelled substrates in kidney cortical slices from normal and alloxan-diabeticrats . 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S. S. Kang, R. Fears, S. Noirot, J. N. Mbanya, J. Yudkin. Changes in metabolism of rat kidney and liver caused by experimental diabetes and by dietary sucrose, Diabetologia, 1982, 285-288, DOI: 10.1007/BF00281307