The Activity of the Pyruvate Dehydrogenase. Complex in Heart Muscle in the Previously. Obese Mouse Model. Kate Steinbeck, Ian D. Caterson x and John R.
Bioscience Reports, Vol. 6, No. 12, 1986
The Activity of the Pyruvate Dehydrogenase Complex in Heart Muscle in the Previously Obese Mouse Model Kate Steinbeck, Ian D. Caterson x and John R. Turtle Received January 26, 1987 KEY WORDS: pyruvate dehydrogenase; obese mice.
Obese gold thioglucose injected mice were reduced to lean control weight by food restriction. When pair fed with lean controls these animals then gained weight (were metabolically more efficient). Serum glucose was also elevated in this group (14.5 _ 0.4 (14) v s 12.1_ 0.3 mmol/L, p 30% lean control weight. At 42 days post injection GTG animals kept in individual cages were meal fed 40 % control intake of standard laboratory chow daily until control weight was reached (16 days). Controls maintained weight on 5.7 g chow per day (Group I). The previously obese GTG animals were either pair fed with the controls (Group II) or maintained at lean control weight by being fed 4.5 g chow per day (Group III). This feeding was continued for 30 days when all animals were sacrificed after an overnight fast. GTG animals at the end of the weight reduction period (Group IV), control animals fed 80 % of normal control intake for 10 days (Group V), and GTG animals 12 weeks post injection fed ad lib. (Group VI) were also sacrificed. Animals were anaesthetised with sodium pentobarbitone (60 mg/kg body weight IP) and the heart removed and immediately frozen with a tissue clamp precooled in liquid nitrogen. A sample of blood was withdrawn from the chest cavity and centrifuged (10,000g, 15 min, 4~ Extraction of frozen tissue for measurement of PDHa and citrate synthase activity (CSa) was performed as described previously (1).
Assays PDHa and CSa in heart muscle were assayed spectrophotometrically as described by Kerbey et al. (1). One Unit of enzyme activity converts 1 #mole of substrate into product/minute at 30~ Serum glucose was determined using a glucose oxidase method in an YSI 23AM analyser. Serum immunoreactive insulin was measured by double-antibody radioimmunoassay. Source of animals, chow and laboratory reagents was as described in Kerbey et al. (10).
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Statistics
Differences were analysed using Student's t test. Results are given as mean __+SEM with the number of observations in parentheses.
RESULTS AND DISCUSSION
Animal weights and biochemical values are shown in Table 1. The previously obese GTG animals were metabolically more efficient, requiring only 80 % of control food intake to maintain weight. Pair feeding produced a 6 ~o weight gain (1.7 g) over lean control weight in 30 days (p < 0.02). The results were similar to those reported in rats with VMH lesions (11,12), and confirmed weight gain in the absence of hyperphagia. The previous period of obesity may have made the animal metabolically more efficient through inhibition of activity of brown adipose tissue (13). PDHa was decreased in the previously obese animals pair fed with lean controls (Group II) to a level similar to that of obese animals feeding ad lib. (Group VI), but body weights were different. The animals previously obese but weight maintained with lean controls (Group III) had PDHa values similar to lean controls of identical age (Group I). In food restricted lean controls (Group V) there was a marked increase in PDHa. In dieted obese animals (Group IV), PDHa was higher than in the obese
Table 1. Pyruvate dehydrogenase complex activity in heart muscle, body weight and serum insulin and glucose levels in control and obese CBA/T6 mice Group
I I1 Ili IV V VI
Lean controls n=9 Previously obese Pair fed with controls n=14 Previously obese Weight maintained n=13 Obese animals Acutely dieted n=5 Lean animals Restricted intake n=5 Obese animals n=15
Body weight (g)
S. Glucose (ram/L)
S. Insulin (~Units/ml)
27.1 ___0.5
12,1 +0.3
28.8 _+0.4
14.5 + 0.4*
183 -+ 26*
1.57 -+ 0.23****
26.9 -+ 0.5
11.8 _+0.3
171 + 24**
3.71 _+ 0.45
27.0 _+ 1.5
11.2 _+0.4
209 _+ 61"
4.90 _+0.42****
23.4 + 0.6
12.0-+ 0.7
41.4 _+0.4*
23.7 + 0.8*
7 4 + 10
Pyruvate dehydrogenase complex Active from (PDMa) (U/g dry wt) 3.34_+0.62
42 -+ 2 * * * 9 1 4 9i 1.53 -+ 1.80* * 9 1 4 9 262 _+ 30"+
1.55 -+ 0.35****
For details of induction of obesity, dietary intake of animals, tissue extraction and enzyme assay see Materials and Methods section. Statistical significance compared to lean controls. *p < 0.001, **p < 0.005, ***P < 0.01, ****p < 0.05. Compared to dieted animals 9 p < 0.05, 9 1 4p9< 0.005. Compared to food restricted animals t p < 0.001.
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animal and lean controls. These results indicate that the activity of the PDH complex can vary with body weight, food intake and the presence or absence of a VMH lesion. Serum insulin was elevated in all animals with a VMH lesion irrespective of body weight or food intake. In these animals therefore a state of insulin resistance exists. With food restriction in control animals serum insulin fell (p < 0.01) yet PDHa increased (p< 0.005). This indicates that factors other than insulin alone are controlling PDH complex activity in this situation. Serum glucose levels responded to amount of food intake, being highest in those animals with greatest intake (Groups II, III and IV). The activity of the PDH complex is the major determinant of glucose disposal by oxidation in animal cells and it is reduced in diabetes, starvation and obesity (1, 3, 10). In these situations the reduction is thought to be due to increased oxidation of fatty acids in cells (2, 14). Fatty acid oxidation is increased due to insulin deficiency (diabetes and starvation) or increased availability (insulin resistance). In this study it was expected that food restricted animals would have a decrease in PDHa due to increased serum NEFA; paradoxically there was an increase in PDHa in both VMH lesion animals and in lean controls. It is probable that in these animals true starvation has not occurred and that insulin levels are adequate to inhibit lipolysis. There is an increase in the sensivity of PDH to insulin in both obese and control animals upon food restriction and yet the animals do not become hypoglycaemic. It is therefore possible that alternate routes of glucose disposal (glycogen synthesis and lipogenesis) may be reduced or inhibited in this situation. In previously obese animals pair fed with lean controls there is a small weight increase (6 7o) and a 53 % reduction in PDHa. If this change is due to increased nonesterified fatty acid (NEFA) availability and oxidation then this must be due to the change in body weight and/or composition and increased insulin resistance. The changes in both PDHa, insulin and glucose described in this study would argue for a persistent alteration in the transmission of the insulin signal (post-binding) in the control of the activity of PDH to which is added alteration in body weight and/or composition as a control factor. These latter factors may act through alteration in circulating or tissue NEFA or triglyceride levels. The weight maintained previously obese GTG mouse is a model for long term insulin resistance in the absence of obesity and hyperglycaemia and in the presence of metabolic efficiency. As such it may have implications for weight maintenance after weight loss in both human and experimental obesity. This model needs further definition with tissue glucose uptake and insulin receptor studies together with measurement of serum and tissue NEFA. ACKNOWLEDGEMENTS
This study was supported by the N.H. and M.R.C. of Australia. REFERENCES 1. Kerbey, A. L., Randle, P. J., Cooper, R. H., Whitehouse, S., Pask, H. T. and Denton, R. M. (1976). Regulation of pyruvate dehydrogenase in rat heart. Biochem. J. 154:327 348.
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2. Kerbey, A. L., Radcliffe, P. M. and Randle, P. J. (1977). Diabetes and the control of pyruvate dehydrogenase in rat heart mitochondria by concentration ratios of adenosine triphosphate/adenosine diphosphate, of reduced/oxidised nicotinamide-adenine dinucleotide and of acetylcoenzyme A/coenzyme A. Biochem. J. 164:509-519. 3. Kerbey, A. L. and Randle, P. J. (1981). Thermolabile factor accelerates pyruvate dehydrogenase kinase reaction in heart mitochondria of starved and alloxan-diabetic rats. FEBS Lett. 127:188-192. 4. Van Putten, L. M., van Bekkum, D. W. and Querido, A. (1955). Influence of hypothalamic lesions producing hyperphagia and of feeding regimes on carcass composition in the rat. Metab. Clin. Exp. 4:68-74. 5. Bray, G. A. and York, D. A. (1979). Hypothalamic and genetic obesity in experimental animals: an autonomic and endocrine hypothesis. Physiological Rev. 59:719-809. 6. Goldman, J. K., Bernardis, L. L. and Frohman, L. A. (1974). Food intake in hypothalamic obesity. Am. J. Physiol. 227:88-91. 7. Cox, J. E. and Powley, T. L. (1981). Intragastric pair feeding fails to prevent VMH obesity or hyperinsulinaemia. Am. J. Physiol. 240:E566-E572. 8. Katsuki, S., Flirata, Y., Horino, M., Ito, M., Ishimoto, M., Makino, N. and Hososako, A. (1962). Obesity and hyperglycaemia induced in mice by gold thioglucose. Diabetes 11:209-215. 9. Debons, A. F., Krimsky, I., Maayan, M. L., Fani, K. and Jimenez, F. A. (1977). Gold thioglucose obesity syndrome. Fed. Proc. 36:143-147. 10. Kerbey, A. L , Caterson, I. D., Williams, P. F. and Turtle, J. R. (1984). Proportion of active dephosphorylated pyruvate dehydrogenase complex in heart and isolated heart mitochondria is decreased in obese hyperinsulinaemic mice. Biochem. J. 217:117-121. 11. Walks, D., Lavau, M., Presta, E., Yang, M-U. and Bjontorp, P. (1983). Refeeding after fasting in the rat: Effects of dietary induced-obesity on energy balance regulation. Am. J. Clin. Nutr. 37:387-395. 12. Walgren, M. C. and Powley, T. L (1985). Effects ofintragastric hyperalimentation on pair-fed rats with ventromedial hypothalamic lesions. Am. J. Physiol. 248:R172-R180. 13. Himms-Hagen, J. (1984). Thermogenesis in brown adipose tissue as an energy buffer. N. Engl. J. Med. 311 : 1549-1558. 14. Caterson, I. D., Williams, P. F., Kerbey, A. L., Astbury, L. D., Plehwe, W. E. and Turtle, J. R. (1984). The effect of body weight and the fatty acid oxidation inhibitor 2-tetradecylglycidic acid on pyruvate dehydrogenase activity in mouse heart. Biochem. J. 224:787-791.
