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Adaptation to underfeeding in young growing rats
NUTRITION RESEARCH, Vol. 5, pp. 1409-1418, 1985 0271-5317/85 $3.00 + .00 Printed in the USA. Copyright (c) 1986 Pergamon Press Ltd. All rights reserved.
ADAPTATION TO UNDERFEEDING IN YOUNGGROWINGRATSI P.F. Mohan, M.Sc. and B.S. Narasinga Rao, B.Sc. (Hon), Ph.D. National Institute of Nutrition Indian Council of Medical Research Jamai Osmania PO, Hyderabad 500007, India
ABSTRACT The involvement of physiological and biochemical processes in adaptation to 50% energy restriction in young and growing rats was studied. The physiological processes like basal 02 consumption, C02 production, respiratory quotient, norepinephrine stimulated 02 consumption, brown adipose tissue mass was unaltered whereas spontaneous physical activity had increased by 54% in diet restricted rats. As compared to Bd l i b fed controls ATP, ADP, AMP and energy change in muscle w e r e ~ a ~ r e d in energy restricted rats. In liver, the levels of ATP decreased by 23% but not of ADP and AMP. Ouabain sensitive ATPase increased in liver by 26% but not in muscle and kidney. Ouabain insensitive AtPase decreased by 21% in kidney but not in muscle or liver. Mitochondrial state 3 oxidation rate was decreased with both glutamate-malate and succinate as substrates by 32 and 23% respectively in liver but not in kidney. Most of the observed changes in the above biochemical parameters do not seem to be involved in the adaptation to energy deficiency in young rats. KEY WORDS: Adaptation, Underfeeding, Oxygen consumption, Brown adipose tissue, Norepinephrine, Physical activity, ATPase, Mitochondria, Energy charge INTRODUCTION I t is well recognised that man and animals can adapt to energy deficiency (1). Several workers have provided evidence for energy conservation through a reduction in basal metabolism, specific dynamic action, energy cost of activities (2) etc. Other energy utilizing processes in the body may also be involved in energy conservation. We have earlier reported (3) evidence for conservation in the energy utilized for maintenance in growing young rats subjected to chronic food restriction (50% of ad l i b intake). The biochemical processes underlying this phenomenon are however, not well understood. I t is also possible that the mechanism underlying the efficient utilisation of energy which, an animal develops certain types of obesity may also operate in an energy deficient animal (3,4). Amongthe processes known to be involved in the conservation of energy in obese rats are, a decrease in the me~Drane bound enzyme, Na+, K+-ATPase (4,5) which is considered to utilize 20-60% of cellular energy (6), and a reduction in the thermogenic brown adipose tissue mass which i Address for reprints:
Mr. P.F. Mohan, National Institute of Nutrition, Hyderabad 500007, India. 1409
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P.J. MOHANand B.S. NARASINGARAO
is known to dissipate energy as heat (7,8) are considered important. Thusan attempt has been made to understand the physiological and biochemical mechanisms of adaptation to underfeeding in rats. In this paper results of a study on the effect of energy restriction on physical a c t i v i t y , basal oxygen consumption, ~carbohdiox~deproduction, norepinephrine (NE) stimulated oxygen consumptilon,$nterscapularbrown adipose tissue (IBAT) mass, adenine nucleotides, ATPase and mitochondrial respiration are presented. MATERIALSAND METHODS The following enzymes and substrates were obtained from Sigma Chemical Company, St. Louis, Missouri, USA: ATP, ADP, NADP, NADH, phosphoenol pyruvate, glutamate, malate, succinate, ouabain, norepinephrine bitartrate, sucrose, hexokinase, glucose,6-phosphate dehydrogenase, myokinase, pyruvate kinase, lactate dehyd?ogenaseand albumin. The rest of the chemicalswere purchased locally. Experiment - I: Male weanling rats (inbred wistar strain of the National Institute of Nutrition, Hyderabad, India) were used in the present study. Twenty rats were equally divided into two groups and were fed 20% protein diet (3). Group I rats were fed ad l i b (control) and group I I rats were fed 50% of the dliet consumedby group-rra-~s (restricted). Feeding was continued for 8 weeks and' at the end of which the following parameters were determined. a) Physical a c t i v i t y : Spontaneousphysical a c t i v i t y of rats was determined for 4 hrs/rat in optovarimex a c t i v i t y meter (Colombus Instruments, Ohio, USA) between 8:00-12:00 hrs. b) Basal and NE stimulated respiration: Basal 02 consumption and' CO2 production of rats were measured with Taylor Servomex Oxygen analyser, (Type OA 272, Sussex, England) and Beckman medical gas analyzer (LB-2, Beckman Instruments, I l l i n o i s , USA) respectively. Readings were noted when the rat was at rest. To the same rat later, norepinephrine (250/~g/kg body weight) as bitartrate was injected intraperitoneally and readings were taken when the rat was at rest again to measure to NE stimulated 02 consumption. Values of O~.and C02 were corrected for standard temperature and pressure (STP) and expressed as ml/min kg 0.75 metabolic body size ~ ~o.7~). c) Adenine nucleotides: Rats were anaesthetised with ether, l i v e r musclewere frozen in situ with a pair of tongs precooled in liquid nitrogen the frozen portions were c u t o u t and were finely ground in a mortar under l i q uid nitrogen. The concentrations of ATP, ADP and AMP were determined by the coupled enzyme assay (9). From these values, the energy charge was computed. TABLE I Composition of 20% Protein Diet Ingredient
g/lO0 g diet
Casein
24 (20)
Starch
65
Groundnut Oil
5 (5)
Salt mixture
4
Vitamin mixture
2
Values in parenthesis indicate the actual protein and fat content of diet determined by Macrokjeldahl method and ether extraction in soxlet apparatus respectively. The composition of salt and vitamin mixtures are according to (10).
ADAPTATION TO ENERGYDEFICIENCY
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Experimen.t-2: Twogroups of inbred wistar/NIN strain rats were fed for 8 weeks one 20% protein diet ad l i b and the other 50% of the diet consumed by by the ad l i b fed groups rats-T.-~r~e rats were killed by cervical dislocation and the i~61ol-6wing parameters were determined, a) Brown adipose tissue: BAT from interscapular region was removed, cleaned free of White adipose tissue and weighed. The values were expressed as mg/IO0 gram body weight, b) ATPase: Liver, kidney and thigh muscles were removed and ATPases were assayed in the microsomal pellet, which was prepared as follows: The tissues were minced and homogenised in 8 volumes of a medium containing 0.25 M sucrose, 30 mMHistidine pH 7.4 and 2 mMEDTA with polytron [Brinkmann Instruments, New York, USA) for 60 seconds discontinuously at 4000 RPM for l i v e r and kidney and for 90 seconds at 5000 RPM for the muscles. The homogenates were centrifuged at 10,000 g for 10 minutes (Sorvall RC 5B Superspeed cold centrifuge, 5534 rotor, DulPont Instruments, New Town, USA). The supernantant was collected and the sediment was resuspended'in the homogenising medium, rehomogenisedfor 15 seconds for l i v e r and kidney and 30 seconds for muscle, and centrifuged at 100,000 g for 10 minutes. The pooled homogenate was centrifuged at 100,000 g for 1 hour (in Sorvall OTD 65 ultra centrifuge, 50 Ti rotor, Du Pont Instruments, New Town, CT, USA). The pellet rich in plasma membrane fragments and microsomes was collected, suspended i n t h e homogenising medium and stored frozen until assayed' for ouabain sensitive and insensitive ATPase by coupled enzyme assay according to the method of Scharschmidt et al (11). All the above steps were carried-out at 0-4~ The protein content of the enzyme extract was determined by the biuret method (12). Experiment-3: Two groups of wistar strain rats were fed in a similar way for ,8 weeks as mentioned in the above two earlier experiments. They were killed by cervical dislocation and mitochondrial respiration determined as follows: Liver and kidneys were removed, minced, homogenised in a mediumcontaining 0.25 M sucrose and 0.2 mMEDTA, in a loose f i t t i n g all glass Potter Elvehjem homogeniser. The homogenate was centrifuged at 600 g for 10 min in cold centrifuge (remi K24 swing out rotor) Remi Udyog, Bombay, India), the supernatant was again centrifuged at IO,O00 xg for 10 min (Sorvall RC 5B superspeed cold centrCfuge, SS 34 rotor, Du Pont Instruments, New Tower, CT, USA). The pellet rich in mitochondria was washed twice resuspending i t in the homogenising medium and centrifuged. The mitochondrial respiration was measured according to the method of Esterbrook (13). The mitochondrial protein content was determined by the biuret method (12). RESULTS Data gilven in Table 2 indicate that basal 02 consumption, C02 production and respilratory quots were not different between the diet restricted group and control groups. In response to NE injection, 02 consumption increased over basal value by 60-70% and the magnitude of this increase was the same in both groups. The spontaneous physical a c t i v i t y of the rats in the underfed group was higher by 54 per cent. Interscapular brown adipose tissue noss expressed as mg/IO0 body weight was also not significantly different between the two groups. Results given in Table 3 show that the levels of adenine nucleotides, ATP, ADP and AMP, expressed as ~moles/gram wet weight of tissue and the energy charge in muscle were unaltered in the energy -restricted group. In the l i v e r , ATP levels decreased by 23% and the energy charge decreased from 0.81 in control to 0.77 in diet restricted group.
1412
P.J. MOHANand B.S. NARASINGA RAO
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TABLE 3 Adenine Nucleotidesand Energy charge ATP
ADP
LMP
Energy charge
Liver Group 1 ad l i b
2.6•
1.00•
0.24•
0.81•
Group 11 Restricted
2.0•
1.10•
0.23•
0.77•
ad l i b 4.5•
0.70•
0.19•
0.90•
0.75•
0.21•
0.90•
**
Muscle Group 1
Group 11 Restricted
5.2•
1.
Values are given as Mean • S.E.M.
n = 10
2.
Values expressed as umoles/gram tissue
3.
Energy charge = ATP +~ADP
4.
**The liver ATP values of group II were compared with group I and was significantly different by paired ' t ' test P< 0.02
The specific ~ctivity~of ouabain sensitive ATPase expressed as umoles ADP formed mg protein- . hour- i (Table 4) increased in liver by 26% but was unaffected in muscle and kidney. The ouabain insensitive ATPase specific in kidney decreased by 21% but not in liver and muscle. The rate of state 3 respiration in liver mitochondria expressed as ug atoms oxygen, mg protein-', hour-i with both substrates, (glutamate-malate and succinate) (Table 5) decreased by 32 and 23 percent respectively. Kidney mitochondria did not show any of these changes. Tha ADP/O ratio was also unaffected. The protein content per gram of tissue was same in control and restricted group. DISCUSSION That basal 02 consumption was not affected and its implications have been reported earlier which indicates the major type of food material did not provide any further information either, between the control and diet restricted groups.
by moderate energy restriction (3). The respiration quotient being oxidased (14) in the body since i t was not different
Nerepinephrine stimulated oxygen consumption is a measure of heat production mostly from BAT (15). The role of BAT has been studied mostly in conditions of cold stress (15) and diet induced thermogenesis (7). A decreased response to norepinephrine injection and reduction in BAT mass have been shown
1414
P.J. MOHANand B.S. NARASINGARAO
TABLE 4 Ouabain sensitive and insensitive ATPase SPECIFIC ACTIVITY IN RAT LIVER, KIDNEY AND MUSCLE Group 1 Ad l i b
Group 11 restricted
Percent charge
Kidney Ouabain sensitive
29.6•
29.6•
Ouabain insensitive
20.0•
15.9t1.33
-21
Liver Ouabain sensitive
3.1•
3.9•
Ouabain insensitive
2.7•
3.0•
5.5•
4.8•
17.0•
16.3•
+26
Muscle Ouabain sensitive Ouabain insensitive 1.
Values given as Mean • S.E.M.
n = 10
2.
Values expressed as specific a c t i v i t y (~moles ADP formed, mg protein -1. hr-1).
3.
*** The kidney ouabain insensitive values of group I I were compared with group 1 and was significantly different by paired 't'test P < 0.001.
4.
*** The l i v e r ouabain sensitive values of group 11 were compared with group 1 and was significantly different by paired ' t ' test P < 0.001.
in obese rat (7), and therefore, this factor has been implicated in the etiology of obesity (8). However, the role of BAT in dissipating energy as heat under normal conditions or under energy deficiency has not been studied so far. In the present study the response to norepinephrine and brown adipose tissue mass were unaltered in energy restricted rats, i t would appear that in growing young rats this parameter may not be an important process involved in energy conservation. Though alteration in physical a c t i v i t y may not constitute a physiological form of adaptation, data on physical a c t i v i t y suggests that the energy restricted rats infact do not save energy by reducing their physical movements either. I f any they appear to be more active than ad l i b fed rats in short term measurements. A long period of 24 hours a c t i v i t y measurement would have been a better indicator of the physical a c t i v i t y of the rats, but because of technical reasons, the measurement was res~cricted to only 4 hours. However, reports in l i t e r a t u r e show that moderate energy restricted rats, do have a higher (16,17) or a similar (18) physical a c t i v i t y as the ad l i b control group.
ADAPTATION TO ENERGY DEFICIENCY
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The energy charge which is given by the equation ATP + 89 is A~AMP an indicator of the thermodynamic state of the tissue cells and is known to vary over a range of 0.7 to 0.9 in biological systems (19). Since there is no significant change of these value in l i v e r and muscle, energy restricted rats, appear to be energetically as normal as control rats. There appears to be a close relationship between ATP levels, ATPase and mitochondrial respiration. This is very c l e a r l y seen in l i v e r where a decrease in ATP concentration is associated with an increase in ATP u t i l i s i n g enzyme and a decrease in the rate of ATP production as indicated by state 3 rates of l i v e r mitochondrial respiration. The reason for these biochemical changes occurring s p e c i f i c a l l y in l i v e r is not clear. I t was reported e a r l i e r that ATP levels did not change in muscle during underfeeding (20), Nat , K§ which may be expected to decrease as an adaptive mechanism to conserve energy as in obese r a t s , actually increased, in l i v e r of under fed rats. This increase in the enzyme a c t i v i t y can not be attributed to any one specific cause since t h i s is one enzyme which is influenced by a number of factors l i k e hormones (21, 22), changes in sodium level (23), potassium levels (24) changes in membrane compos i t i o n such as loss of l i p i d (25, 26) etc. The increase may probably be a non energy conserving adaptive mechanism of the cell to maintain the intra c e l l u l a r Na§ and K+ levels. The a c t i v i t y of this enzyme has been shown to increase considerably in conditions of protein and calorie malnutrition (27). Although i t is not our interest to look into the mechanism of increased energy u t i l i z a tion as a consequence of increased a c t i v i t y Na+, K+-ATPase in l i v e r may be i n s i g n i f i c a n t because i t forms a small component of total ATPases, compared to kidney and may not be involved in the energy conserving mechanisms of adaptation to energy deficiency. On the other hand, kidney which has high a c t i v i t y of ouabain insensitive component, is decreased in restricted rats, indicating a significant reduction in energy expenditure. The physiological significance of the decrease in the rate of state 3 respiration, an indicator of ATP production in l i v e r mitochondria, is d i f f i c u l t to assess. A decrease of state 3 and 4 respiration rates in rat l i v e r mitochondria, in protein deficiency has been reported (28). Ramanadhamet al (29) reported a decrease in state 3 respiration rate at 3rd week and increase at 7th week in experimental protein and calorie malnourished rats. Thus, an underfed young growing rat does not seem to conserve energy by any one of the physiological or biochemical mechanisms studied, except for small adaptive changes observed in kidney ATPases. However, other processes of wasteful energy expenditure like f u t i l e cycles of substrate cycles (30) needs to be looked into. I t may be that as reported in our earlier study (3) besides growth retardation which is important and a major response to food restriction, decreased fat content of the body and a reduction in maintenance energy expenditute may constitute the principle mechanisms by which young growing rats conserve energy when subjected to semistarvation. A decreased protein turn over may be one of the mechanisms by which energy expended for maintenance is decreased as has been observed in fasted rats (31}. This aspect is also being explored further. REFERENCES I.
Francisco Grande. Many under caloric deficiency IN: Adaptation to the environment, Hand book of physiology. D i l l DB, Adolph EF, and Fole CG., Waverley Press, Inc., BaltCmore, Maryland, 1964; pp 911-938.
2.
Apfelbaum M. Adaptation to changes in caloric intake. Sci. 1978; 2:543-559.
Prog. Food Nutr.
ADAPTATION TO ENERGYDEFICIENCY
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3.
MahonPF, Narasinga Rao BS. Adaptation to underfeeding in growing rats. Effect of energy r e s t r i c t i o n at two dietary protein levels on growth, feed efficiency, basal metabolism and body composition. J. Nutr. 1983; 113:79-85.
4.
Lin MH, Romsos DR, Akera T, Leveille GA. Na+, K+-ATPase enzyme units in skeletal muscle from lean and obese mice. Biochem. Biophy. Res. Commun. 1978; 80:398-404.
5.
York DA, Bray GA, Yuikimura Y. An enzymatic defect in the obese [ob/ob) mouse: loss of thyroid-induced sodium-potassium dependent adenosinetriphosphatase. Proc. Natl. Acad. Sci., USA 1978; 75:477-481.
6.
Deluise M, Blackburn GL, F lier JS. Reduceda c t i v i t y of the red cell sodium-potassium pump in human obesity. N. Engl. J. Med. 1980; 303: 1017.
7.
CawthorneMA. Metabolic aspects of obesity. Molecular aspects of medicine. BaumH, Gergely, J, Franburg BL. (eds) 1982: 352-400.
8.
Ashwell M. Brown adipose tissue-relevant to obesity. Applied Nutrition. 1983; 374:232-244.
9.
Bergmayer HU (ed) Methodsof enzymatic analysis vol 4, pp 2101-2127, Academic Press, Inc. New York, 1974.
10.
Horowitz W (ed) Vitamins and other nutrients. In: Official methods of analysis of the Association of o f f i c i a l analytical chemists. AOAC Publications 1975; pp 816-851.
11.
Scarchmidt BF, Keefe EB, Blankenship NM, Ockner RK. Validation of a recording spectrophotometric method for measurement of membrane associatedMg and Na, K-ATPase a c t i v i t y . J. Lab. Clin. Med. 1979; 93: 790-799.
12.
Gornall AG, Bardawill CJ, David MM. Determination of serum proteins by means of the Biuret reaction. J. Biol. Chem. 1947;177:751-766.
13.
Estabrook RW. Mitochondrial respiratory control and the polarographic measuremement of ADP: 0 Ratio, Methods in Enzymology (ed) Estabrook RW and Pullman HH. Vol X, Academic Press, New York 1961; 41-47.
14.
Kleiber M. The respiratory quotient. In: The f i r e of l i f e - An i n t r o duction to animal energetics, John Wiley & Sons, Inc., New York, 1961 82-93.
15.
CannonB, Johannson BW. Non shivering thermogenesis in newborn. In: Molecular aspects of Medicine, Baum H, Gergely J (eds) 1980: 119-223.
16.
Ahrens RA, Wilson JE Jr. Carbohydrate metabolism and physical a c t i v i t y in rats fed~ diets containing purified casein versus a mixture of amino acids stimulating casein. J. Nutr. 1966; 90: 63-70.
17.
Miller DS, Payne PR. Weight maintenance and food intake. 78:255-262.
18.
Frosum E, Hillman PE, Nesheim MC. Effect of energy r e s t r i c t i o n on total heat production basal metabolic rate and specific dynamic action of foods in rats. J. Nutr. 1981; 111:1691-1697.
HumanNutrition:
J. Nutr. 1962
1418
P.J. MOHANand B.S. NARASINGARAO
19.
Atkinston DE. Adenylate control and the adenylate energy charge In: Cellular energy metabolism and its regulation. Academic Press 1977; pp 85-107.
20.
Howarth RW, Baldwin RL. Concentration of selected enzymes and metabolites in rat skeletal muscle. Effects of food restriction. J. Nutr. 1971; 101:485-494.
21.
Lin MH, Akera T. Increased (Na+, K+) ATPase concentrations in various tissues of rats caused by thyroid hormones treatment. J. Biol. Chem. 1978; 253:723-726.
22.
Schmidt U, Schmid J, Schmid H, Dubach UC. Sodiumand potassium Activated ATPase. A possible target of aldosterone. J. Clin. Invest. 1975; 55:655-660.
23.
Katz AT, Epestein FH. The role of sodium potassium activated adinosine Triphosphatase in the reabsorption of sodium by the kidney. J. Clin. Invest. 1967; 46:199-2011.
24.
Silva P, Hayslett JP, Epstein H. The role of Na-K Activated Adenosine Triphosphatase in potassium adaptation. J. Clin. Invest. 1973; 52: 2665-2671.
25.
Farais RN, Bloj B, Morero RD, Sineriz F, Trucco RE. Regulation of allosteric membrane bound enzymes through changes in membrane l i p i d composition Biochem. Biophys. Acta. 1975; 414:235.
26.
Lin MH, Romsos DR, Akera T, Leveille GA. Increase in Na, K-ATPase enzyme units in l i v e r and kidney from essential fatty acid deficient rats. Experientia. 1979; 35:735-736.
27.
Pimplikar SW, Kaplay SS. Kidney, l i v e r and erythrocyte membrane Na, KAdenosine Triphosphatase in protein energy malnourished rats. Biochem. Med. 1981; 26:12-19.
28.
Tyebir RS, Kunina S, Sims NM, Dandorth EJR. Influence of diet composition on serum Tri-iodothyronine (T3) concentration, hepatic mitochondrial metabolism and shuttle system a c t i v i t y in rats. J. Nutr. 1981;111:252-259.
29.
Ramanadham M, Kaplay SS. Responseof oxidative phosphorylation in l i v e r of protein-energy malnourished rats. Nutr. Metab. 1979; 23:235-240.
30.
Newsholme EA, Crabtree B. Substrate cycles in metabolic regulation and in heat generation. In: Biochemical Adaptation to environmental charge; Smellie, RMS and Pennock JF (eds) The Biochemical Society, London, 1976 pp 61-109.
31.
McNurlan MA, Tomkins AM, Garlick PJ. The efect of starvation on the rate of protein synthesis in rat l i v e r and small intestine. Biochem. J. 1979; 178:373-379. Accepted for publication October 30, 1985.
ADAPTATION TO UNDERFEEDING IN YOUNGGROWINGRATSI P.F. Mohan, M.Sc. and B.S. Narasinga Rao, B.Sc. (Hon), Ph.D. National Institute of Nutrition Indian Council of Medical Research Jamai Osmania PO, Hyderabad 500007, India
ABSTRACT The involvement of physiological and biochemical processes in adaptation to 50% energy restriction in young and growing rats was studied. The physiological processes like basal 02 consumption, C02 production, respiratory quotient, norepinephrine stimulated 02 consumption, brown adipose tissue mass was unaltered whereas spontaneous physical activity had increased by 54% in diet restricted rats. As compared to Bd l i b fed controls ATP, ADP, AMP and energy change in muscle w e r e ~ a ~ r e d in energy restricted rats. In liver, the levels of ATP decreased by 23% but not of ADP and AMP. Ouabain sensitive ATPase increased in liver by 26% but not in muscle and kidney. Ouabain insensitive AtPase decreased by 21% in kidney but not in muscle or liver. Mitochondrial state 3 oxidation rate was decreased with both glutamate-malate and succinate as substrates by 32 and 23% respectively in liver but not in kidney. Most of the observed changes in the above biochemical parameters do not seem to be involved in the adaptation to energy deficiency in young rats. KEY WORDS: Adaptation, Underfeeding, Oxygen consumption, Brown adipose tissue, Norepinephrine, Physical activity, ATPase, Mitochondria, Energy charge INTRODUCTION I t is well recognised that man and animals can adapt to energy deficiency (1). Several workers have provided evidence for energy conservation through a reduction in basal metabolism, specific dynamic action, energy cost of activities (2) etc. Other energy utilizing processes in the body may also be involved in energy conservation. We have earlier reported (3) evidence for conservation in the energy utilized for maintenance in growing young rats subjected to chronic food restriction (50% of ad l i b intake). The biochemical processes underlying this phenomenon are however, not well understood. I t is also possible that the mechanism underlying the efficient utilisation of energy which, an animal develops certain types of obesity may also operate in an energy deficient animal (3,4). Amongthe processes known to be involved in the conservation of energy in obese rats are, a decrease in the me~Drane bound enzyme, Na+, K+-ATPase (4,5) which is considered to utilize 20-60% of cellular energy (6), and a reduction in the thermogenic brown adipose tissue mass which i Address for reprints:
Mr. P.F. Mohan, National Institute of Nutrition, Hyderabad 500007, India. 1409
1410
P.J. MOHANand B.S. NARASINGARAO
is known to dissipate energy as heat (7,8) are considered important. Thusan attempt has been made to understand the physiological and biochemical mechanisms of adaptation to underfeeding in rats. In this paper results of a study on the effect of energy restriction on physical a c t i v i t y , basal oxygen consumption, ~carbohdiox~deproduction, norepinephrine (NE) stimulated oxygen consumptilon,$nterscapularbrown adipose tissue (IBAT) mass, adenine nucleotides, ATPase and mitochondrial respiration are presented. MATERIALSAND METHODS The following enzymes and substrates were obtained from Sigma Chemical Company, St. Louis, Missouri, USA: ATP, ADP, NADP, NADH, phosphoenol pyruvate, glutamate, malate, succinate, ouabain, norepinephrine bitartrate, sucrose, hexokinase, glucose,6-phosphate dehydrogenase, myokinase, pyruvate kinase, lactate dehyd?ogenaseand albumin. The rest of the chemicalswere purchased locally. Experiment - I: Male weanling rats (inbred wistar strain of the National Institute of Nutrition, Hyderabad, India) were used in the present study. Twenty rats were equally divided into two groups and were fed 20% protein diet (3). Group I rats were fed ad l i b (control) and group I I rats were fed 50% of the dliet consumedby group-rra-~s (restricted). Feeding was continued for 8 weeks and' at the end of which the following parameters were determined. a) Physical a c t i v i t y : Spontaneousphysical a c t i v i t y of rats was determined for 4 hrs/rat in optovarimex a c t i v i t y meter (Colombus Instruments, Ohio, USA) between 8:00-12:00 hrs. b) Basal and NE stimulated respiration: Basal 02 consumption and' CO2 production of rats were measured with Taylor Servomex Oxygen analyser, (Type OA 272, Sussex, England) and Beckman medical gas analyzer (LB-2, Beckman Instruments, I l l i n o i s , USA) respectively. Readings were noted when the rat was at rest. To the same rat later, norepinephrine (250/~g/kg body weight) as bitartrate was injected intraperitoneally and readings were taken when the rat was at rest again to measure to NE stimulated 02 consumption. Values of O~.and C02 were corrected for standard temperature and pressure (STP) and expressed as ml/min kg 0.75 metabolic body size ~ ~o.7~). c) Adenine nucleotides: Rats were anaesthetised with ether, l i v e r musclewere frozen in situ with a pair of tongs precooled in liquid nitrogen the frozen portions were c u t o u t and were finely ground in a mortar under l i q uid nitrogen. The concentrations of ATP, ADP and AMP were determined by the coupled enzyme assay (9). From these values, the energy charge was computed. TABLE I Composition of 20% Protein Diet Ingredient
g/lO0 g diet
Casein
24 (20)
Starch
65
Groundnut Oil
5 (5)
Salt mixture
4
Vitamin mixture
2
Values in parenthesis indicate the actual protein and fat content of diet determined by Macrokjeldahl method and ether extraction in soxlet apparatus respectively. The composition of salt and vitamin mixtures are according to (10).
ADAPTATION TO ENERGYDEFICIENCY
1411
Experimen.t-2: Twogroups of inbred wistar/NIN strain rats were fed for 8 weeks one 20% protein diet ad l i b and the other 50% of the diet consumed by by the ad l i b fed groups rats-T.-~r~e rats were killed by cervical dislocation and the i~61ol-6wing parameters were determined, a) Brown adipose tissue: BAT from interscapular region was removed, cleaned free of White adipose tissue and weighed. The values were expressed as mg/IO0 gram body weight, b) ATPase: Liver, kidney and thigh muscles were removed and ATPases were assayed in the microsomal pellet, which was prepared as follows: The tissues were minced and homogenised in 8 volumes of a medium containing 0.25 M sucrose, 30 mMHistidine pH 7.4 and 2 mMEDTA with polytron [Brinkmann Instruments, New York, USA) for 60 seconds discontinuously at 4000 RPM for l i v e r and kidney and for 90 seconds at 5000 RPM for the muscles. The homogenates were centrifuged at 10,000 g for 10 minutes (Sorvall RC 5B Superspeed cold centrifuge, 5534 rotor, DulPont Instruments, New Town, USA). The supernantant was collected and the sediment was resuspended'in the homogenising medium, rehomogenisedfor 15 seconds for l i v e r and kidney and 30 seconds for muscle, and centrifuged at 100,000 g for 10 minutes. The pooled homogenate was centrifuged at 100,000 g for 1 hour (in Sorvall OTD 65 ultra centrifuge, 50 Ti rotor, Du Pont Instruments, New Town, CT, USA). The pellet rich in plasma membrane fragments and microsomes was collected, suspended i n t h e homogenising medium and stored frozen until assayed' for ouabain sensitive and insensitive ATPase by coupled enzyme assay according to the method of Scharschmidt et al (11). All the above steps were carried-out at 0-4~ The protein content of the enzyme extract was determined by the biuret method (12). Experiment-3: Two groups of wistar strain rats were fed in a similar way for ,8 weeks as mentioned in the above two earlier experiments. They were killed by cervical dislocation and mitochondrial respiration determined as follows: Liver and kidneys were removed, minced, homogenised in a mediumcontaining 0.25 M sucrose and 0.2 mMEDTA, in a loose f i t t i n g all glass Potter Elvehjem homogeniser. The homogenate was centrifuged at 600 g for 10 min in cold centrifuge (remi K24 swing out rotor) Remi Udyog, Bombay, India), the supernatant was again centrifuged at IO,O00 xg for 10 min (Sorvall RC 5B superspeed cold centrCfuge, SS 34 rotor, Du Pont Instruments, New Tower, CT, USA). The pellet rich in mitochondria was washed twice resuspending i t in the homogenising medium and centrifuged. The mitochondrial respiration was measured according to the method of Esterbrook (13). The mitochondrial protein content was determined by the biuret method (12). RESULTS Data gilven in Table 2 indicate that basal 02 consumption, C02 production and respilratory quots were not different between the diet restricted group and control groups. In response to NE injection, 02 consumption increased over basal value by 60-70% and the magnitude of this increase was the same in both groups. The spontaneous physical a c t i v i t y of the rats in the underfed group was higher by 54 per cent. Interscapular brown adipose tissue noss expressed as mg/IO0 body weight was also not significantly different between the two groups. Results given in Table 3 show that the levels of adenine nucleotides, ATP, ADP and AMP, expressed as ~moles/gram wet weight of tissue and the energy charge in muscle were unaltered in the energy -restricted group. In the l i v e r , ATP levels decreased by 23% and the energy charge decreased from 0.81 in control to 0.77 in diet restricted group.
1412
P.J. MOHANand B.S. NARASINGA RAO
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1413
TABLE 3 Adenine Nucleotidesand Energy charge ATP
ADP
LMP
Energy charge
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2.6•
1.00•
0.24•
0.81•
Group 11 Restricted
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0.77•
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**
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Group 11 Restricted
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Values are given as Mean • S.E.M.
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Values expressed as umoles/gram tissue
3.
Energy charge = ATP +~ADP
4.
**The liver ATP values of group II were compared with group I and was significantly different by paired ' t ' test P< 0.02
The specific ~ctivity~of ouabain sensitive ATPase expressed as umoles ADP formed mg protein- . hour- i (Table 4) increased in liver by 26% but was unaffected in muscle and kidney. The ouabain insensitive ATPase specific in kidney decreased by 21% but not in liver and muscle. The rate of state 3 respiration in liver mitochondria expressed as ug atoms oxygen, mg protein-', hour-i with both substrates, (glutamate-malate and succinate) (Table 5) decreased by 32 and 23 percent respectively. Kidney mitochondria did not show any of these changes. Tha ADP/O ratio was also unaffected. The protein content per gram of tissue was same in control and restricted group. DISCUSSION That basal 02 consumption was not affected and its implications have been reported earlier which indicates the major type of food material did not provide any further information either, between the control and diet restricted groups.
by moderate energy restriction (3). The respiration quotient being oxidased (14) in the body since i t was not different
Nerepinephrine stimulated oxygen consumption is a measure of heat production mostly from BAT (15). The role of BAT has been studied mostly in conditions of cold stress (15) and diet induced thermogenesis (7). A decreased response to norepinephrine injection and reduction in BAT mass have been shown
1414
P.J. MOHANand B.S. NARASINGARAO
TABLE 4 Ouabain sensitive and insensitive ATPase SPECIFIC ACTIVITY IN RAT LIVER, KIDNEY AND MUSCLE Group 1 Ad l i b
Group 11 restricted
Percent charge
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29.6•
29.6•
Ouabain insensitive
20.0•
15.9t1.33
-21
Liver Ouabain sensitive
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3.9•
Ouabain insensitive
2.7•
3.0•
5.5•
4.8•
17.0•
16.3•
+26
Muscle Ouabain sensitive Ouabain insensitive 1.
Values given as Mean • S.E.M.
n = 10
2.
Values expressed as specific a c t i v i t y (~moles ADP formed, mg protein -1. hr-1).
3.
*** The kidney ouabain insensitive values of group I I were compared with group 1 and was significantly different by paired 't'test P < 0.001.
4.
*** The l i v e r ouabain sensitive values of group 11 were compared with group 1 and was significantly different by paired ' t ' test P < 0.001.
in obese rat (7), and therefore, this factor has been implicated in the etiology of obesity (8). However, the role of BAT in dissipating energy as heat under normal conditions or under energy deficiency has not been studied so far. In the present study the response to norepinephrine and brown adipose tissue mass were unaltered in energy restricted rats, i t would appear that in growing young rats this parameter may not be an important process involved in energy conservation. Though alteration in physical a c t i v i t y may not constitute a physiological form of adaptation, data on physical a c t i v i t y suggests that the energy restricted rats infact do not save energy by reducing their physical movements either. I f any they appear to be more active than ad l i b fed rats in short term measurements. A long period of 24 hours a c t i v i t y measurement would have been a better indicator of the physical a c t i v i t y of the rats, but because of technical reasons, the measurement was res~cricted to only 4 hours. However, reports in l i t e r a t u r e show that moderate energy restricted rats, do have a higher (16,17) or a similar (18) physical a c t i v i t y as the ad l i b control group.
ADAPTATION TO ENERGY DEFICIENCY
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The energy charge which is given by the equation ATP + 89 is A~AMP an indicator of the thermodynamic state of the tissue cells and is known to vary over a range of 0.7 to 0.9 in biological systems (19). Since there is no significant change of these value in l i v e r and muscle, energy restricted rats, appear to be energetically as normal as control rats. There appears to be a close relationship between ATP levels, ATPase and mitochondrial respiration. This is very c l e a r l y seen in l i v e r where a decrease in ATP concentration is associated with an increase in ATP u t i l i s i n g enzyme and a decrease in the rate of ATP production as indicated by state 3 rates of l i v e r mitochondrial respiration. The reason for these biochemical changes occurring s p e c i f i c a l l y in l i v e r is not clear. I t was reported e a r l i e r that ATP levels did not change in muscle during underfeeding (20), Nat , K§ which may be expected to decrease as an adaptive mechanism to conserve energy as in obese r a t s , actually increased, in l i v e r of under fed rats. This increase in the enzyme a c t i v i t y can not be attributed to any one specific cause since t h i s is one enzyme which is influenced by a number of factors l i k e hormones (21, 22), changes in sodium level (23), potassium levels (24) changes in membrane compos i t i o n such as loss of l i p i d (25, 26) etc. The increase may probably be a non energy conserving adaptive mechanism of the cell to maintain the intra c e l l u l a r Na§ and K+ levels. The a c t i v i t y of this enzyme has been shown to increase considerably in conditions of protein and calorie malnutrition (27). Although i t is not our interest to look into the mechanism of increased energy u t i l i z a tion as a consequence of increased a c t i v i t y Na+, K+-ATPase in l i v e r may be i n s i g n i f i c a n t because i t forms a small component of total ATPases, compared to kidney and may not be involved in the energy conserving mechanisms of adaptation to energy deficiency. On the other hand, kidney which has high a c t i v i t y of ouabain insensitive component, is decreased in restricted rats, indicating a significant reduction in energy expenditure. The physiological significance of the decrease in the rate of state 3 respiration, an indicator of ATP production in l i v e r mitochondria, is d i f f i c u l t to assess. A decrease of state 3 and 4 respiration rates in rat l i v e r mitochondria, in protein deficiency has been reported (28). Ramanadhamet al (29) reported a decrease in state 3 respiration rate at 3rd week and increase at 7th week in experimental protein and calorie malnourished rats. Thus, an underfed young growing rat does not seem to conserve energy by any one of the physiological or biochemical mechanisms studied, except for small adaptive changes observed in kidney ATPases. However, other processes of wasteful energy expenditure like f u t i l e cycles of substrate cycles (30) needs to be looked into. I t may be that as reported in our earlier study (3) besides growth retardation which is important and a major response to food restriction, decreased fat content of the body and a reduction in maintenance energy expenditute may constitute the principle mechanisms by which young growing rats conserve energy when subjected to semistarvation. A decreased protein turn over may be one of the mechanisms by which energy expended for maintenance is decreased as has been observed in fasted rats (31}. This aspect is also being explored further. REFERENCES I.
Francisco Grande. Many under caloric deficiency IN: Adaptation to the environment, Hand book of physiology. D i l l DB, Adolph EF, and Fole CG., Waverley Press, Inc., BaltCmore, Maryland, 1964; pp 911-938.
2.
Apfelbaum M. Adaptation to changes in caloric intake. Sci. 1978; 2:543-559.
Prog. Food Nutr.
ADAPTATION TO ENERGYDEFICIENCY
1417
3.
MahonPF, Narasinga Rao BS. Adaptation to underfeeding in growing rats. Effect of energy r e s t r i c t i o n at two dietary protein levels on growth, feed efficiency, basal metabolism and body composition. J. Nutr. 1983; 113:79-85.
4.
Lin MH, Romsos DR, Akera T, Leveille GA. Na+, K+-ATPase enzyme units in skeletal muscle from lean and obese mice. Biochem. Biophy. Res. Commun. 1978; 80:398-404.
5.
York DA, Bray GA, Yuikimura Y. An enzymatic defect in the obese [ob/ob) mouse: loss of thyroid-induced sodium-potassium dependent adenosinetriphosphatase. Proc. Natl. Acad. Sci., USA 1978; 75:477-481.
6.
Deluise M, Blackburn GL, F lier JS. Reduceda c t i v i t y of the red cell sodium-potassium pump in human obesity. N. Engl. J. Med. 1980; 303: 1017.
7.
CawthorneMA. Metabolic aspects of obesity. Molecular aspects of medicine. BaumH, Gergely, J, Franburg BL. (eds) 1982: 352-400.
8.
Ashwell M. Brown adipose tissue-relevant to obesity. Applied Nutrition. 1983; 374:232-244.
9.
Bergmayer HU (ed) Methodsof enzymatic analysis vol 4, pp 2101-2127, Academic Press, Inc. New York, 1974.
10.
Horowitz W (ed) Vitamins and other nutrients. In: Official methods of analysis of the Association of o f f i c i a l analytical chemists. AOAC Publications 1975; pp 816-851.
11.
Scarchmidt BF, Keefe EB, Blankenship NM, Ockner RK. Validation of a recording spectrophotometric method for measurement of membrane associatedMg and Na, K-ATPase a c t i v i t y . J. Lab. Clin. Med. 1979; 93: 790-799.
12.
Gornall AG, Bardawill CJ, David MM. Determination of serum proteins by means of the Biuret reaction. J. Biol. Chem. 1947;177:751-766.
13.
Estabrook RW. Mitochondrial respiratory control and the polarographic measuremement of ADP: 0 Ratio, Methods in Enzymology (ed) Estabrook RW and Pullman HH. Vol X, Academic Press, New York 1961; 41-47.
14.
Kleiber M. The respiratory quotient. In: The f i r e of l i f e - An i n t r o duction to animal energetics, John Wiley & Sons, Inc., New York, 1961 82-93.
15.
CannonB, Johannson BW. Non shivering thermogenesis in newborn. In: Molecular aspects of Medicine, Baum H, Gergely J (eds) 1980: 119-223.
16.
Ahrens RA, Wilson JE Jr. Carbohydrate metabolism and physical a c t i v i t y in rats fed~ diets containing purified casein versus a mixture of amino acids stimulating casein. J. Nutr. 1966; 90: 63-70.
17.
Miller DS, Payne PR. Weight maintenance and food intake. 78:255-262.
18.
Frosum E, Hillman PE, Nesheim MC. Effect of energy r e s t r i c t i o n on total heat production basal metabolic rate and specific dynamic action of foods in rats. J. Nutr. 1981; 111:1691-1697.
HumanNutrition:
J. Nutr. 1962
1418
P.J. MOHANand B.S. NARASINGARAO
19.
Atkinston DE. Adenylate control and the adenylate energy charge In: Cellular energy metabolism and its regulation. Academic Press 1977; pp 85-107.
20.
Howarth RW, Baldwin RL. Concentration of selected enzymes and metabolites in rat skeletal muscle. Effects of food restriction. J. Nutr. 1971; 101:485-494.
21.
Lin MH, Akera T. Increased (Na+, K+) ATPase concentrations in various tissues of rats caused by thyroid hormones treatment. J. Biol. Chem. 1978; 253:723-726.
22.
Schmidt U, Schmid J, Schmid H, Dubach UC. Sodiumand potassium Activated ATPase. A possible target of aldosterone. J. Clin. Invest. 1975; 55:655-660.
23.
Katz AT, Epestein FH. The role of sodium potassium activated adinosine Triphosphatase in the reabsorption of sodium by the kidney. J. Clin. Invest. 1967; 46:199-2011.
24.
Silva P, Hayslett JP, Epstein H. The role of Na-K Activated Adenosine Triphosphatase in potassium adaptation. J. Clin. Invest. 1973; 52: 2665-2671.
25.
Farais RN, Bloj B, Morero RD, Sineriz F, Trucco RE. Regulation of allosteric membrane bound enzymes through changes in membrane l i p i d composition Biochem. Biophys. Acta. 1975; 414:235.
26.
Lin MH, Romsos DR, Akera T, Leveille GA. Increase in Na, K-ATPase enzyme units in l i v e r and kidney from essential fatty acid deficient rats. Experientia. 1979; 35:735-736.
27.
Pimplikar SW, Kaplay SS. Kidney, l i v e r and erythrocyte membrane Na, KAdenosine Triphosphatase in protein energy malnourished rats. Biochem. Med. 1981; 26:12-19.
28.
Tyebir RS, Kunina S, Sims NM, Dandorth EJR. Influence of diet composition on serum Tri-iodothyronine (T3) concentration, hepatic mitochondrial metabolism and shuttle system a c t i v i t y in rats. J. Nutr. 1981;111:252-259.
29.
Ramanadham M, Kaplay SS. Responseof oxidative phosphorylation in l i v e r of protein-energy malnourished rats. Nutr. Metab. 1979; 23:235-240.
30.
Newsholme EA, Crabtree B. Substrate cycles in metabolic regulation and in heat generation. In: Biochemical Adaptation to environmental charge; Smellie, RMS and Pennock JF (eds) The Biochemical Society, London, 1976 pp 61-109.
31.
McNurlan MA, Tomkins AM, Garlick PJ. The efect of starvation on the rate of protein synthesis in rat l i v e r and small intestine. Biochem. J. 1979; 178:373-379. Accepted for publication October 30, 1985.