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N-cp (corpulent) rat
Physiology&Behavior,Vol. 36, pp. 127-131. Copyright©Pergamon Press Ltd.. 1986. Printed in the U.S.A.
0031-9384/86 $3.00 + .00
Nonshivering Thermogenesis in the Diabetic SHR/N-cp (Corpulent) Rat O R I E N L . T U L P , 1 C A R L T. H A N S E N
AND OTHO E. MICHAELIS,
IV
Department of Nutrition and Food Sciences, Drexel University, Philadelphia, PA 19104 Division of Small Animal Genetics, National Institute of Health, Bethesda, MD 20205 Carbohydrate Nutrition Laboratory, US Department of Agriculture, Beltsville, MD 20705 R e c e i v e d 26 N o v e m b e r 1984 TULP, O. L., C. T. HANSEN AND O. E. MICHAELIS, IV. Nonshivering thermogenesis in the diabetic SHR/N-cp (corpulent) rat. PHYSIOL BEHAV 36(1) 127-131, 1986.--The effects of isoenergetic sucrose and starch-based diets on thermogenesis were investigated in young adult, male, lean and corpulent SHR/N-cp rats. Corpulent rats gained weight 1.5 times more rapidly than lean, and sucrose diets resulted in more rapid weight gains in both phenotypes. Rates of resting and of norepinephrine-stimulated oxygen consumption were similar in both groups of lean rats and in sucrose-fed corpulent rats, but were decreased in starch-fed corpulent rats. The thermic response to injected norepinephrine occurred normally in all groups. Colonic and rectal temperatures were greater in lean than in corpulent rats. Acute cold exposure (5°C) resulted in decreases in rectal but not colonic temperature in lean rats fed both diets, but resulted in lower temperatures at both sites in corpulent rats, with the greatest decreases being observed in the starch fed corpulent rats. Fifty percent of the corpulent but none of the lean rats succumbed within 24--48 hours following cold exposure. Urinary vanilmandelic acid (VMA) excretion was greater in lean than in corpulent rats, and the sucrose diet induced a greater increment in urinary VMA excretion in lean rats than in corpulent rats. These results are consistent with an impaired activation of sympatheticallymediated thermogenesis via nutritional or environmental stimuli in the corpulent genotype of the SHR/N-cp rat in concert with an economy in energy expenditure which may be contributing factors in the causation of their obese state. Thermogenesis
Catecholamines
Obesity
Diabetes
Rat comes visably apparent by the animal's greater weight and exterior dimensions by about 5 weeks of age [22]. Additionally, the obese members of this strain exhibit hypertriglyceridemia, hyperinsulinemia, hyperglycemia, and glycosuria by 9 weeks of age [11,12]. The diabetic characteristics persist well into adulthood [11,28]. In contrast, lean animals of this strain demonstrate moderate hypertension presumably due to the influence of the trait for hypertension derived from the SHR (okamoto derived) component of the background stock [12]. Although the basic characteristics of obesity in this new strain are similar to those of other obese rodent strains including the closely related LA/N-cp (LA-corpulent) rat [8,9], the cause of obesity has not been fully established. The closely related L A / N - c p (LAcorpulent) rat has been shown to have an impairment in the activation of non-shivering thermogenesis following both environmental and nutritional stimuli [20,25]. Rothwell et al. [16] have determined in chemically-diabetic rats that insulin action is an essential prerequisite for the development and expression of thermogenesis, presumably via enhancing cellular glucose uptake and metabolism, or by modulating the activity of critical enzymes. The purpose of the present study was to determine if the obesity in the corpulent phenotype of the SHR/N-cp strain was associated with an impairment in the activation of expression o f the sympathetic component of non-shivering thermogenesis. The capacity for expression of thermogenesis is of particular inter-
P R O C E S S E S of adaptive thermogenesis represent an important mechanism of heat production and of modulation of energy expenditure during periods of adjustment to nutritional or environmental stress [1]. Himms-Hagen [4] has recently proposed that biochemical defects in non-shivering thermogenesis may represent a possible causation of obesity in several genetically obese rodent strains and may also represent a causative factor of obesity in man, and Schutz and coworkers have recently demonstrated diminished dietaryinduced thermogenesis in obese women [17]. The principle mechanism of adaptive thermogenesis in the rat would appear to be via brown adipose tissue, which is under the regulatory control of the sympathetic nervous system [15, 16, 18, 26]. Recent work by Himms-Hagen et al. [4,6] indicate that in some genetically obese rodent strains such as the fa/fa rat, the biochemical mechanism of non-shivering thermogenesis may be impaired centrally (i.e., at the level of neuroactivation), while in others such as the obese ob/ob mouse biochemical impairments in thermogenesis may exist in the effector tissue (i.e., brown adipose tissue). The SHR/N-cp rat represents a newly developed, cogenic strain, where the obesity develops as the consequence of an autosomal recessive trait, and appears in one-fourth of the offspring of breeding pairs that are heterozygous for the trait [3,11]. The obesity is characterized by increased adipose mass, adipocyte size, and adipocyte number in retroperitoneal, subcutaneous and other fat depots, and it be-
~Requests for reprints should be addressed to Dr. Often L. Tulp, Dept. of Nutrition and Food Science, Nesbitt Hall, Drexel University, 32nd and Chestnut Sts., Philadelphia, PA 19104.
127
128
TULP, H A N S E N A N D M I C H A E L I S BODY WEIGHT OF SHR-CORPULENT RATS
THERMOGENESIS IN DIABETIC SHR-CORPULENT RATS
600
CORP-SU j-O
CORP-ST
_....~ . . . . . . "~
LEAN-SU LEAN-ST
~
400
z:r'"
GRAMS
..'J
LEAN
CORPULENT
STARCH SUCROSE
STARCH SUCROSE
z 20
d m
200
0
2 4 6 8 WEEKS OF DIET TREATMENT
10
[]
- - n o r e p i n e p h r i n e , l O O u g / k g BW,sc
FIG. 1. Body weights of SHR-corpulent rats. Rats were fed the starch (ST) or sucrose (SU) based diets from weaning for 10 weeks, and body weights recorded at weekly intervals. ©=lean, ST; @=lean, SU; []=corp ST; II=corp SU. n=6 rats/treatment group. BW corpulent > lean after 2 weeks (0=<0.05); SU>ST for both phenotypes from 6 weeks (0=<0.05).
FIG. 2. Thermogenesis in diabetic SHR-corpulent rats. Measurements ofthermogenesis obtained midway in dark phase of light cycle after a normal overweight feed. Copr SU RMA< all other groups (0 = <0.05). Thermogenic response to norepinephrine is similar in all groups, n=6 rat/treatment group.
est due to the prominent insulin resistance and diabetic state that is apparent in the corpulent phenotype of this strain of rat [11, 12, 28], and the reported requirement for insulin for several components of NST [2,16].
of the capacity to activate thermogenesis via endogenous mechanisms, and was determined by placing animals acutely into pre-chilled hanging steel cages identical to those to which the animals were accustomed to. Rectal and colonic temperatures were measured immediately before and at hourly intervals after being placed in the cold environment by inserting flexible fast response rectal thermisters to distances of 3 and 12 cm (measured from center of thermister sensor tip) respectively into the anal canal (Yellow Springs Instruments, Yellow Springs, OH) as outlined elsewhere [25]. Distances of less than 3 cm resulted in highly variable and inconsitant readings in this and other studies [20,25]. Animals were removed from the cold environment and placed under a warming lamp to recover if colonic temperature dropped to 26°C or below. Body temperatures of all animals were allowed to return to their pretreatment temperatures in the laboratory before returning them to their usual environment in the animal quarters. No animals succumbed during the immediate recovery period. Survival data was computed on the number of animals that survived cold exposure 24-48 hours post-treatment. All animals that succumbed during this post-treatment period were subjected to a post-mortem examination in the animal pathology laboratories of the National Institutes of Health, Bethesda, MD 20205. Data were analyzed by analysis of variance with application of the Student-Newman-Keuls procedure for identification of mutually exclusive subgroups [13].
METHOD Twelve lean (genotype approximately 2/3 + / - and 1 / 3 - / for corpulent trait) and twelve corpulent (genotype + / + for cp/cp) male rats were obtained from the laboratory of one of us (C.T.H.), and maintained individually in hanging steel wirebottomed cages at 2 2 +- l°C in a laminar flow hood and with a reverse light cycle (12 hr light: 12 hr dark, light period commencing at 1800 hr daily) in the U S D A laboratories from 4 until 12 weeks of age. Rats were fed isoenergetic diets containing 54% carbohydrate as cooked cornstarch or sucrose, 20% protein, 16% fat, plus 6.5% vitamins, minerals, and non-nutritive fiber [10] continuously from 4 weeks of age. Measurements of body weight were obtained weekly with an Ohaus animal balance (Fisher Scientific, Boston, MA). During the tenth week of the study, animals were placed in metabolic cages, and 24 hour urine samples collected in 1.0 ml 6 M HCI for determination of vanilmandelic acid excretion using the UV spectrophotometric procedure outlined by Pisano et al. [14]. Urine samples were stored at -20°C until assayed. The presence of glycosuria was determined qualitatively with the Clinistix method (Ames Division of Miles Laboratories, Elkhart, IN). Measurements of resting and of norepinephrinestimulated (100 p~g/kg body weight, SC) oxygen consumption were measured by indirect calorimetry during the dark phase of the light cycle at 29.5°C (thermal neutrality) in a small animal respirometer apparatus essentially as outlined by Stock [19]. This level of norepinephrine administration represents an approximate 1/2 maximal dose in normally-fed lean rodents and in preliminary trials was found to be safe in the lean hypertensive animals of this study. These data are expressed relative to body weight (0.75) as outlined previously [21,23]. The animals' capacity to maintain body temperature in a cold 5°C environment was taken as an indicator
RESULTS
Weight gain of rats is shown in Fig. 1, and shows that body weights of corpulent rats increased 1.5 to 1.8 times more rapidly in corpulent than in lean animals, and that the rate of weight gain was consistently more rapid in sucrosefed than in starch-fed rats of either phenotype. Additionally, the sucrose diet resulted in a greater absolute increment in body weight in the corpulent than in the lean animals. Measures of thermogenesis (Fig. 2) show that rates of resting and of norepinephrine-stimulated oxygen consump-
T H E R M O G E N E S I S IN C O R P U L E N T RATS
129
TABLE 1 URINE VOLUME, GLUCOSE, AND VANILMANDELICACID CONTENT OF SHR/N-cp RATS Group
n
Volume, ml
lean ST* lean SU corpulent ST corpulent SU ANOVA (2x2) Phenotype diet
5 6 6 5
15.0 ± 23.3 ± 14.2 _ 50.0 ±
1.0 2.7 2.5 15.1
Glycosuria No No Yes Yes
COLONIC
40 1 36 ]
VMA, /~g/24hr 92 154 57 91
± 29 ___44 ± 12 ± 12
A
LEAN RATS
oc
=
=,
TEMPERATURE
=
=
ST-LEAN
=
= SU-LEAN
32 RECTAL T E M P E R A T U R E 38 SU-LEAN ST-LEAN
32
NS <0.05
---
•°t
<0.05 <0.05
28
0
*ST=starch diet; SU=sucrose diet. tion were similar in both groups of lean rats and in the sucrose-fed corpulent rats. Resting oxygen consumption was decreased by 17% in the starch-fed corpulent animals, but the increase following norepinephrine stimulation averaged 40%, similar to that observed in the other three groups. Measures of urinary volume, qualitative glucose content, and of 24 hour vanilmandelic acid excretion are shown in Table 1. VMA excretion was greater in starch-fed lean than in starch-fed corpulent rats. Although 24 hour VMA excretion was greater in both lean and corpulent rats consuming the sucrose than the starch diets, the absolute increment due to diet in the lean rats was nearly twice that observed in the sucrose-fed corpulent rats (mean increase=0.63 vs. 0.34/xg VMA/24 hours, respectively). All corpulent animals had a positive urinary glucose determination, while none of the lean animals exhibited glycosuria. Urine volumes were greatest in the sucrose-fed corpulent, intermediate in the sucrose-fed lean animals, and lowest in the starch-fed animals of either phenotype. All animals of both phenotypes were observed to shiver soon after placement in the cold environment, and at periodic intervals during the cold exposure. Colonic temperatures were greater than rectal temperatures in both phenotypes, irrespective of antecedent diet (Fig. 3A and 3B), and continued to remain so through the period of cold treatment. After one hour of cold exposure, rectal temperatures of both lean and corpulent rats were significantly decreased below their initial temperatures. Colonic temperatures were unchanged in lean animals fed either diet at this time, but were decreased in both groups of corpulent animals as the period of cold exposure progressed, with the greatest decrements observed in the starch-fed corpulent animals (Fig. 3B). These differences in colonic and rectal temperatures persisted throughout the 4 hours of cold exposure. The period of cold exposure was terminated after 4 hours because of the apparent difficulty of the corpulent animals to maintain adequate body temperatures, despite visable evidence of continued shivering. Recovery from cold exposure was uneventful among lean animals fed either diet, but 50% of the corpulent animals of both dietary groups succumbed during the 24-48 hours post-treatment, despite the immediate post-treatment reacclimitization period, and reestablishment of normal rectal and colonic temperatures before returning the animals to the animal quarters. Postmortem examinations of these animals were inconclusive for a cause of death, and were negative for significant coronary artery disease involvement.
i
2
3
4
H O U R S OF C O L D E X P O S U R E
CORPULENT
B
RATS COLONIC
TEMPERATURE
40
_
36
;
..........
ST-CORP
32
oc RECTAL TEMPEF~TURE
3e
'°t
"-.
j
32
•
"'g" . . . . .
28
6
i
~ .....
2
~ .....
3
z SU-CORP --~ S T - C O R P
4
H O U R S OF C O L D E X P O S U R E
FIG. 3. Colonic and rectal temperatures of cold-exposed rats. Measurements were recorded with a fast response flexible thermister insected to constant depths of 3 cm for rectal and 12 cm for colonic temperatures after stable readings obtained (about 10 see). n=6 rats/treatment group. A=lean rats; B=corpulent rats. Symbols as for Fig. 1. DISCUSSION The results of this study indicate that the corpulent members of the SHR/N-cp rat strain exhibit an impairment in environmentally-induced alterations in non-shivering thermogenesis, in addition to a less pronounced decrease in their capacity to respond to nutritionally-induced alterations in thermogenesis. In contrast, the expression of NST via environmental and nutritional factors occurred normally in the lean animals of this strain. In starch fed corpulent animals, resting metabolic rate, daily vanilmandelic acid excretion, and the capacity to maintain normal body temperatures in a cold environment were decreased compared to similarly-fed lean animals. Although the more rapid decrease in rectal temperature observed in corpulent than in lean rats might be consistent with an impairment in shivering-induced heat production or in an impairment in the control of heat loss, this is not likely to represent the predominate causative factor. Corpulent animals were observed to continue to shiver after 4 hr of cold exposure, and their greater adiposity would offer a greater insulative effect than was present in lean animals. Although the absolute difference between the colonic and rectal temperatures in the two dietary groups were
130
TULP, HANSEN AND MICHAELIS
of similar magnitude, the temperatures in the starch fed corpulent rats at both sites were significantly lower than those in all other groups throughout the trial. Feeding of sucrose-rich diets, which normally result in greater sympathetic nervous activity and increased N S T and sometimes in greater energy intake in other strains of rats [27], resulted in increases in V M A excretion that were quantitatively nearly only half as great in the corpulent as occurred in similarly-fed lean littermates. Significant diet-induced differences in food intake were not observed in either phenotype. These increases in VMA excretion are reflective of greater net catecholamine metabolism, and were associated with moderate increases in resting thermogenesis and in an improved capacity to maintain body temperatures in a cold environment in the lean animals, while the same parameters did not increase further in the corpulent rats. It is recognized that the greater carcass fat content presumed to be present in the sucrose-fed lean rats and in the corpulent rats fed either diet may result in an inaccurate estimate of resting metabolic rate because of an inability to accurately access the fat-free or true respiring mass of the animals. When this limitation was addressed by relating thermogenesis to lean body mass, thermogenic responses to diet become more clear-cut [21]. Despite this limitation, resting thermogenesis was significantly decreased in the starch-fed corpulent rat. The studies were conducted during the dark phase of the light cycle, at a time when the animals would be in a normally active post prandial state, and when normal thermogenic responses would be most likely to be optimal. Measurements of thermogenesis were obtained under conditions of thermal neutrality, in order to minimize artefacts attributable to environmentally-induced heat loss secondary to low ambient temperatures. Because of the reported predisposition and later development of coronary artery disease in the Koletsky rat from which the corpulent trait was derived [7], animals of this study were limited to an approximate 1/2 maximal dose of norepinephrine in an effort to avert or minimize the risk of a
potential sympathomimetic mishap or cardiovascular failure from occurring. Nonetheless, the thermogenic responses to a submaximal dose of norepinephrine occurred normally in both lean and corpulent animals regardless of antecedent diet, and averaged a 40% increase in all groups. There were no differences in the chronology or magnitude of response to norepinephrine in any group. These observations are consistent with a normal thermogenic response to noradrenergic stimuli in peripheral tissues, but an impairment in the normal activation process for NST in the corpulent phenotype of this strain. Centrally-mediated sympathomimetic processes, in concert with thyroidal, adrenocortical, and insulindependent functions, are presumed to be principal components of this response [2, 4, 5, 15, 21, 23-26]. In normally-fed and in cafeteria-fed rats sympathetically-mediated brown adipose tissue is thought to be the major effector of N S T [15,21], and its capacity to respond may be impaired in several obese rodent models [4]. The extent to which insulin resistance might contribute to impairments in NST in obese animals could not be determined, but is likely to be a contributing factor because of the requirement for insulin action in expression of N S T [2]. Regardless of the nature of the impairment in the animals of the present study, the corpulent animals exhibited demonstrable impairments in nutritionally and environmently-stimulated parameters of thermogenesis which are consistent with an economy of energy expenditure, and in all likelihood, are a significant contributor to the development of their obese state.
ACKNOWLEDGEMENTS The authors wish to thank Drs. Sheldon Reiser, CNL, USDA; Dr. Wayne Smith, Colby College, and Dr. Stanley Segall, Drexel University, for use of laboratory facilities, and Mary Lou Vendryes and Brenda Jones of Drexel University for administrative assistance in the preparation of this manuscript. Supported by Colby A-23089 and a Drexel University Minigrant to O.L.T.
REFERENCES 1. Danforth, E., Jr. Hormonal control of thermogenesis. Life Sci 28: 1821-1827, 1981. 2. Gavin, L. A., F. A. McMahon and M. Moeller. The mechanism of impaired T3 production from T4 in diabetes. Diabetes 30: 694--699, 1981. 3. Hansen, C. T. Two new congenic rat strains for nutrition and obesity research. Fed Proc 42: 537, 1983. 4. Himms-Hagen, J. Obesity may be due to a malfunctioning of brown fat. Can Med Assoc J 121: 1361-1364, 1980. 5. Himms-Hagen, J. and M. Desaultals. A mitochondrial defect in brown adipose tissue of the obese (ob/ob) mouse: failure to respond to cold by an increase in binding. Biochem Biophys Res Commun 83: 628--636, 1978. 6. Himms-Hagen, J. Role of thermogenesis in brown adipose tissue in the regulation of energy balance. In: The Adipocyte and Obesity; Cellular and Molecular Mechanisms, edited by A. Angel et al. New York: Raven Press, 1983, pp. 259-270. 7. Koletsky, S. New type of spontaneously hypertensive rat with hyperlipidemia and endocrine gland defects. In: Spontaneous Hypertension, its Pathogenesis and Complications, edited by K. Okamoto, Tokyo: Igaky Shoin Ltd., 1972, pp. 194--197. 8. Michaelis, O. E., IV, K. C. Ellwood, J. Hallfrisch and C. T. Hansen. Effect of dietary sucrose and genotype on metabolic parameters of a new strain of genetically obese rat: LA/N corpulent. Nutr Res 3: 217-228, 1983.
9. Michaelis, O. E., IV, K. C. Ellwood, O. L. Tulp and M. R. C. Greenwood. Effect of feeding sucrose or starch diets on parameters of glucose tolerance in the LA/N corpulent rat. Nutr Res, in press, 1986. 10. Michaelis, O. E., IV, R. E. Martin, L. B. Gardner and K. C. Ellwood. Effect of simple and complex carbohydrate on lipogenic parameters of spontaneously hypertenisve rats. Nutr Rep lnt 24: 313-321, 1981. 11. Michaelis, O. E., IV, K. C. Ellwood, J. M. Judge, N. W. Schoene and C. T. Hansen. Effect of dietary sucrose on the SHR/N coruplent rat: a new model for insulin independent diabetes. Am J Clin Nutr 39: 612-618, 1981. 12. Michaelis, O. E., IV, K. C. Ellwood, J. M. Judge, N. W. Schoene and C. T. Hansen. The SHR/N-corpulent rat: A new model for maturity-onset diabetes. Fed Proc 42: 674, 1983. 13. Nie, N., C. H. Hull, J. G. Jenkins, K. Steinbrenner and D. H. Bart. Statistical Package for the Social Sciences, chapter 2. New York: McGraw-Hill, 1975, pp. 426-428. 14. Pisano, J. J., J. R. Crout and D. Abraham. Determination of 3-methoxy-4-hydroxymandelic acid in urine. Clin Chem Acta 7: 285-290, 1962. 15. Rothwell, N. J. and M. J. Stock. A role for brown adipose tissue in diet induced thermogenesis. Nature 281: 31-35, 1979. 16. Rothwell, N. J., M. J. Stock and B. P. Warwick. Investigation into the role of insulin in diet-induced thermogenesis in the rat. Proc Nutr Soc 81: 349A, 1981.
THERMOGENESIS
IN CORPULENT
RATS
17. Schutz, Y., T. Bessard and E. Jequier. Diet induced thermogenesis measured over a whole day in obese and non-obese women. Am J Clin Nutr 40: 542-552, 1984. 18. Smith, R. E. and B. A. Horowitz. Brown fat and thermogenesis. Physiol Rev 49: 330--425, 1969. 19. Stock, M. J. An automatic closed circuit oxygen consumption apparatus for small animals. J Appl Physiol 39: 849-850, 1975. 20. Tulp, O. L. Impaired activation of thermogenesis in the corpulent rat. Life Sci 35: 1699-1704, 1984. 21. Tulp, O. L., R. Frink and E. Danforth, Jr. Effect of cafeteria feeding on brown and white adipose tissue cellularity, thermogenesis, and body composition in rats. J Nutr 112: 22502260, 1982. 22. Tulp, O. L., M. H. Gregory, C. Maggio, M. R. C. Greenwood and O. E. Michaelis, IV. Effect of diet and genotype on growth and adipose cellularity in the corpulent (corp) rat: A new model of obesity. Fed Proc 41: 458A, 1982.
131
23. Tulp, O. L. and P. P. Krupp. Thermogenesis in thyroidectomized protein-malnourished rats. J Nutr 114: 2365-2372, 1984. 24. Tulp, O. L., P. P. Krupp and E. S. Horton. Characteristics of thyroid function in experimental protein malnutrition. J Nutr 109: 1321-1332, 1979. 25. Tulp, O. L. and S. J. Shields. Thermogenesis in cafeteria fed LA/N-cp (corpulent) rats. Nutr Res 4: 325-332, 1984. 26. York, D. A. Energy balance, diet induced thermogenesis and brown adipose tissue in lean and obese (fa/fa) Zucker rats after adrenalectomy. J Nutr 113: 1395--1402, 1983. 27. Young, J. B. and L. Landsberg. Stimulation of the sympathetic nervous system during sucrose feeding. Nature 269: 615-617, 1977. 28. Zador, C. M., O. L. Tulp and E. L. Lein. Insulin independent diabetes in adult female SHR-corpulent rats. Chem Abstr, in press, 1985.
0031-9384/86 $3.00 + .00
Nonshivering Thermogenesis in the Diabetic SHR/N-cp (Corpulent) Rat O R I E N L . T U L P , 1 C A R L T. H A N S E N
AND OTHO E. MICHAELIS,
IV
Department of Nutrition and Food Sciences, Drexel University, Philadelphia, PA 19104 Division of Small Animal Genetics, National Institute of Health, Bethesda, MD 20205 Carbohydrate Nutrition Laboratory, US Department of Agriculture, Beltsville, MD 20705 R e c e i v e d 26 N o v e m b e r 1984 TULP, O. L., C. T. HANSEN AND O. E. MICHAELIS, IV. Nonshivering thermogenesis in the diabetic SHR/N-cp (corpulent) rat. PHYSIOL BEHAV 36(1) 127-131, 1986.--The effects of isoenergetic sucrose and starch-based diets on thermogenesis were investigated in young adult, male, lean and corpulent SHR/N-cp rats. Corpulent rats gained weight 1.5 times more rapidly than lean, and sucrose diets resulted in more rapid weight gains in both phenotypes. Rates of resting and of norepinephrine-stimulated oxygen consumption were similar in both groups of lean rats and in sucrose-fed corpulent rats, but were decreased in starch-fed corpulent rats. The thermic response to injected norepinephrine occurred normally in all groups. Colonic and rectal temperatures were greater in lean than in corpulent rats. Acute cold exposure (5°C) resulted in decreases in rectal but not colonic temperature in lean rats fed both diets, but resulted in lower temperatures at both sites in corpulent rats, with the greatest decreases being observed in the starch fed corpulent rats. Fifty percent of the corpulent but none of the lean rats succumbed within 24--48 hours following cold exposure. Urinary vanilmandelic acid (VMA) excretion was greater in lean than in corpulent rats, and the sucrose diet induced a greater increment in urinary VMA excretion in lean rats than in corpulent rats. These results are consistent with an impaired activation of sympatheticallymediated thermogenesis via nutritional or environmental stimuli in the corpulent genotype of the SHR/N-cp rat in concert with an economy in energy expenditure which may be contributing factors in the causation of their obese state. Thermogenesis
Catecholamines
Obesity
Diabetes
Rat comes visably apparent by the animal's greater weight and exterior dimensions by about 5 weeks of age [22]. Additionally, the obese members of this strain exhibit hypertriglyceridemia, hyperinsulinemia, hyperglycemia, and glycosuria by 9 weeks of age [11,12]. The diabetic characteristics persist well into adulthood [11,28]. In contrast, lean animals of this strain demonstrate moderate hypertension presumably due to the influence of the trait for hypertension derived from the SHR (okamoto derived) component of the background stock [12]. Although the basic characteristics of obesity in this new strain are similar to those of other obese rodent strains including the closely related LA/N-cp (LA-corpulent) rat [8,9], the cause of obesity has not been fully established. The closely related L A / N - c p (LAcorpulent) rat has been shown to have an impairment in the activation of non-shivering thermogenesis following both environmental and nutritional stimuli [20,25]. Rothwell et al. [16] have determined in chemically-diabetic rats that insulin action is an essential prerequisite for the development and expression of thermogenesis, presumably via enhancing cellular glucose uptake and metabolism, or by modulating the activity of critical enzymes. The purpose of the present study was to determine if the obesity in the corpulent phenotype of the SHR/N-cp strain was associated with an impairment in the activation of expression o f the sympathetic component of non-shivering thermogenesis. The capacity for expression of thermogenesis is of particular inter-
P R O C E S S E S of adaptive thermogenesis represent an important mechanism of heat production and of modulation of energy expenditure during periods of adjustment to nutritional or environmental stress [1]. Himms-Hagen [4] has recently proposed that biochemical defects in non-shivering thermogenesis may represent a possible causation of obesity in several genetically obese rodent strains and may also represent a causative factor of obesity in man, and Schutz and coworkers have recently demonstrated diminished dietaryinduced thermogenesis in obese women [17]. The principle mechanism of adaptive thermogenesis in the rat would appear to be via brown adipose tissue, which is under the regulatory control of the sympathetic nervous system [15, 16, 18, 26]. Recent work by Himms-Hagen et al. [4,6] indicate that in some genetically obese rodent strains such as the fa/fa rat, the biochemical mechanism of non-shivering thermogenesis may be impaired centrally (i.e., at the level of neuroactivation), while in others such as the obese ob/ob mouse biochemical impairments in thermogenesis may exist in the effector tissue (i.e., brown adipose tissue). The SHR/N-cp rat represents a newly developed, cogenic strain, where the obesity develops as the consequence of an autosomal recessive trait, and appears in one-fourth of the offspring of breeding pairs that are heterozygous for the trait [3,11]. The obesity is characterized by increased adipose mass, adipocyte size, and adipocyte number in retroperitoneal, subcutaneous and other fat depots, and it be-
~Requests for reprints should be addressed to Dr. Often L. Tulp, Dept. of Nutrition and Food Science, Nesbitt Hall, Drexel University, 32nd and Chestnut Sts., Philadelphia, PA 19104.
127
128
TULP, H A N S E N A N D M I C H A E L I S BODY WEIGHT OF SHR-CORPULENT RATS
THERMOGENESIS IN DIABETIC SHR-CORPULENT RATS
600
CORP-SU j-O
CORP-ST
_....~ . . . . . . "~
LEAN-SU LEAN-ST
~
400
z:r'"
GRAMS
..'J
LEAN
CORPULENT
STARCH SUCROSE
STARCH SUCROSE
z 20
d m
200
0
2 4 6 8 WEEKS OF DIET TREATMENT
10
[]
- - n o r e p i n e p h r i n e , l O O u g / k g BW,sc
FIG. 1. Body weights of SHR-corpulent rats. Rats were fed the starch (ST) or sucrose (SU) based diets from weaning for 10 weeks, and body weights recorded at weekly intervals. ©=lean, ST; @=lean, SU; []=corp ST; II=corp SU. n=6 rats/treatment group. BW corpulent > lean after 2 weeks (0=<0.05); SU>ST for both phenotypes from 6 weeks (0=<0.05).
FIG. 2. Thermogenesis in diabetic SHR-corpulent rats. Measurements ofthermogenesis obtained midway in dark phase of light cycle after a normal overweight feed. Copr SU RMA< all other groups (0 = <0.05). Thermogenic response to norepinephrine is similar in all groups, n=6 rat/treatment group.
est due to the prominent insulin resistance and diabetic state that is apparent in the corpulent phenotype of this strain of rat [11, 12, 28], and the reported requirement for insulin for several components of NST [2,16].
of the capacity to activate thermogenesis via endogenous mechanisms, and was determined by placing animals acutely into pre-chilled hanging steel cages identical to those to which the animals were accustomed to. Rectal and colonic temperatures were measured immediately before and at hourly intervals after being placed in the cold environment by inserting flexible fast response rectal thermisters to distances of 3 and 12 cm (measured from center of thermister sensor tip) respectively into the anal canal (Yellow Springs Instruments, Yellow Springs, OH) as outlined elsewhere [25]. Distances of less than 3 cm resulted in highly variable and inconsitant readings in this and other studies [20,25]. Animals were removed from the cold environment and placed under a warming lamp to recover if colonic temperature dropped to 26°C or below. Body temperatures of all animals were allowed to return to their pretreatment temperatures in the laboratory before returning them to their usual environment in the animal quarters. No animals succumbed during the immediate recovery period. Survival data was computed on the number of animals that survived cold exposure 24-48 hours post-treatment. All animals that succumbed during this post-treatment period were subjected to a post-mortem examination in the animal pathology laboratories of the National Institutes of Health, Bethesda, MD 20205. Data were analyzed by analysis of variance with application of the Student-Newman-Keuls procedure for identification of mutually exclusive subgroups [13].
METHOD Twelve lean (genotype approximately 2/3 + / - and 1 / 3 - / for corpulent trait) and twelve corpulent (genotype + / + for cp/cp) male rats were obtained from the laboratory of one of us (C.T.H.), and maintained individually in hanging steel wirebottomed cages at 2 2 +- l°C in a laminar flow hood and with a reverse light cycle (12 hr light: 12 hr dark, light period commencing at 1800 hr daily) in the U S D A laboratories from 4 until 12 weeks of age. Rats were fed isoenergetic diets containing 54% carbohydrate as cooked cornstarch or sucrose, 20% protein, 16% fat, plus 6.5% vitamins, minerals, and non-nutritive fiber [10] continuously from 4 weeks of age. Measurements of body weight were obtained weekly with an Ohaus animal balance (Fisher Scientific, Boston, MA). During the tenth week of the study, animals were placed in metabolic cages, and 24 hour urine samples collected in 1.0 ml 6 M HCI for determination of vanilmandelic acid excretion using the UV spectrophotometric procedure outlined by Pisano et al. [14]. Urine samples were stored at -20°C until assayed. The presence of glycosuria was determined qualitatively with the Clinistix method (Ames Division of Miles Laboratories, Elkhart, IN). Measurements of resting and of norepinephrinestimulated (100 p~g/kg body weight, SC) oxygen consumption were measured by indirect calorimetry during the dark phase of the light cycle at 29.5°C (thermal neutrality) in a small animal respirometer apparatus essentially as outlined by Stock [19]. This level of norepinephrine administration represents an approximate 1/2 maximal dose in normally-fed lean rodents and in preliminary trials was found to be safe in the lean hypertensive animals of this study. These data are expressed relative to body weight (0.75) as outlined previously [21,23]. The animals' capacity to maintain body temperature in a cold 5°C environment was taken as an indicator
RESULTS
Weight gain of rats is shown in Fig. 1, and shows that body weights of corpulent rats increased 1.5 to 1.8 times more rapidly in corpulent than in lean animals, and that the rate of weight gain was consistently more rapid in sucrosefed than in starch-fed rats of either phenotype. Additionally, the sucrose diet resulted in a greater absolute increment in body weight in the corpulent than in the lean animals. Measures of thermogenesis (Fig. 2) show that rates of resting and of norepinephrine-stimulated oxygen consump-
T H E R M O G E N E S I S IN C O R P U L E N T RATS
129
TABLE 1 URINE VOLUME, GLUCOSE, AND VANILMANDELICACID CONTENT OF SHR/N-cp RATS Group
n
Volume, ml
lean ST* lean SU corpulent ST corpulent SU ANOVA (2x2) Phenotype diet
5 6 6 5
15.0 ± 23.3 ± 14.2 _ 50.0 ±
1.0 2.7 2.5 15.1
Glycosuria No No Yes Yes
COLONIC
40 1 36 ]
VMA, /~g/24hr 92 154 57 91
± 29 ___44 ± 12 ± 12
A
LEAN RATS
oc
=
=,
TEMPERATURE
=
=
ST-LEAN
=
= SU-LEAN
32 RECTAL T E M P E R A T U R E 38 SU-LEAN ST-LEAN
32
NS <0.05
---
•°t
<0.05 <0.05
28
0
*ST=starch diet; SU=sucrose diet. tion were similar in both groups of lean rats and in the sucrose-fed corpulent rats. Resting oxygen consumption was decreased by 17% in the starch-fed corpulent animals, but the increase following norepinephrine stimulation averaged 40%, similar to that observed in the other three groups. Measures of urinary volume, qualitative glucose content, and of 24 hour vanilmandelic acid excretion are shown in Table 1. VMA excretion was greater in starch-fed lean than in starch-fed corpulent rats. Although 24 hour VMA excretion was greater in both lean and corpulent rats consuming the sucrose than the starch diets, the absolute increment due to diet in the lean rats was nearly twice that observed in the sucrose-fed corpulent rats (mean increase=0.63 vs. 0.34/xg VMA/24 hours, respectively). All corpulent animals had a positive urinary glucose determination, while none of the lean animals exhibited glycosuria. Urine volumes were greatest in the sucrose-fed corpulent, intermediate in the sucrose-fed lean animals, and lowest in the starch-fed animals of either phenotype. All animals of both phenotypes were observed to shiver soon after placement in the cold environment, and at periodic intervals during the cold exposure. Colonic temperatures were greater than rectal temperatures in both phenotypes, irrespective of antecedent diet (Fig. 3A and 3B), and continued to remain so through the period of cold treatment. After one hour of cold exposure, rectal temperatures of both lean and corpulent rats were significantly decreased below their initial temperatures. Colonic temperatures were unchanged in lean animals fed either diet at this time, but were decreased in both groups of corpulent animals as the period of cold exposure progressed, with the greatest decrements observed in the starch-fed corpulent animals (Fig. 3B). These differences in colonic and rectal temperatures persisted throughout the 4 hours of cold exposure. The period of cold exposure was terminated after 4 hours because of the apparent difficulty of the corpulent animals to maintain adequate body temperatures, despite visable evidence of continued shivering. Recovery from cold exposure was uneventful among lean animals fed either diet, but 50% of the corpulent animals of both dietary groups succumbed during the 24-48 hours post-treatment, despite the immediate post-treatment reacclimitization period, and reestablishment of normal rectal and colonic temperatures before returning the animals to the animal quarters. Postmortem examinations of these animals were inconclusive for a cause of death, and were negative for significant coronary artery disease involvement.
i
2
3
4
H O U R S OF C O L D E X P O S U R E
CORPULENT
B
RATS COLONIC
TEMPERATURE
40
_
36
;
..........
ST-CORP
32
oc RECTAL TEMPEF~TURE
3e
'°t
"-.
j
32
•
"'g" . . . . .
28
6
i
~ .....
2
~ .....
3
z SU-CORP --~ S T - C O R P
4
H O U R S OF C O L D E X P O S U R E
FIG. 3. Colonic and rectal temperatures of cold-exposed rats. Measurements were recorded with a fast response flexible thermister insected to constant depths of 3 cm for rectal and 12 cm for colonic temperatures after stable readings obtained (about 10 see). n=6 rats/treatment group. A=lean rats; B=corpulent rats. Symbols as for Fig. 1. DISCUSSION The results of this study indicate that the corpulent members of the SHR/N-cp rat strain exhibit an impairment in environmentally-induced alterations in non-shivering thermogenesis, in addition to a less pronounced decrease in their capacity to respond to nutritionally-induced alterations in thermogenesis. In contrast, the expression of NST via environmental and nutritional factors occurred normally in the lean animals of this strain. In starch fed corpulent animals, resting metabolic rate, daily vanilmandelic acid excretion, and the capacity to maintain normal body temperatures in a cold environment were decreased compared to similarly-fed lean animals. Although the more rapid decrease in rectal temperature observed in corpulent than in lean rats might be consistent with an impairment in shivering-induced heat production or in an impairment in the control of heat loss, this is not likely to represent the predominate causative factor. Corpulent animals were observed to continue to shiver after 4 hr of cold exposure, and their greater adiposity would offer a greater insulative effect than was present in lean animals. Although the absolute difference between the colonic and rectal temperatures in the two dietary groups were
130
TULP, HANSEN AND MICHAELIS
of similar magnitude, the temperatures in the starch fed corpulent rats at both sites were significantly lower than those in all other groups throughout the trial. Feeding of sucrose-rich diets, which normally result in greater sympathetic nervous activity and increased N S T and sometimes in greater energy intake in other strains of rats [27], resulted in increases in V M A excretion that were quantitatively nearly only half as great in the corpulent as occurred in similarly-fed lean littermates. Significant diet-induced differences in food intake were not observed in either phenotype. These increases in VMA excretion are reflective of greater net catecholamine metabolism, and were associated with moderate increases in resting thermogenesis and in an improved capacity to maintain body temperatures in a cold environment in the lean animals, while the same parameters did not increase further in the corpulent rats. It is recognized that the greater carcass fat content presumed to be present in the sucrose-fed lean rats and in the corpulent rats fed either diet may result in an inaccurate estimate of resting metabolic rate because of an inability to accurately access the fat-free or true respiring mass of the animals. When this limitation was addressed by relating thermogenesis to lean body mass, thermogenic responses to diet become more clear-cut [21]. Despite this limitation, resting thermogenesis was significantly decreased in the starch-fed corpulent rat. The studies were conducted during the dark phase of the light cycle, at a time when the animals would be in a normally active post prandial state, and when normal thermogenic responses would be most likely to be optimal. Measurements of thermogenesis were obtained under conditions of thermal neutrality, in order to minimize artefacts attributable to environmentally-induced heat loss secondary to low ambient temperatures. Because of the reported predisposition and later development of coronary artery disease in the Koletsky rat from which the corpulent trait was derived [7], animals of this study were limited to an approximate 1/2 maximal dose of norepinephrine in an effort to avert or minimize the risk of a
potential sympathomimetic mishap or cardiovascular failure from occurring. Nonetheless, the thermogenic responses to a submaximal dose of norepinephrine occurred normally in both lean and corpulent animals regardless of antecedent diet, and averaged a 40% increase in all groups. There were no differences in the chronology or magnitude of response to norepinephrine in any group. These observations are consistent with a normal thermogenic response to noradrenergic stimuli in peripheral tissues, but an impairment in the normal activation process for NST in the corpulent phenotype of this strain. Centrally-mediated sympathomimetic processes, in concert with thyroidal, adrenocortical, and insulindependent functions, are presumed to be principal components of this response [2, 4, 5, 15, 21, 23-26]. In normally-fed and in cafeteria-fed rats sympathetically-mediated brown adipose tissue is thought to be the major effector of N S T [15,21], and its capacity to respond may be impaired in several obese rodent models [4]. The extent to which insulin resistance might contribute to impairments in NST in obese animals could not be determined, but is likely to be a contributing factor because of the requirement for insulin action in expression of N S T [2]. Regardless of the nature of the impairment in the animals of the present study, the corpulent animals exhibited demonstrable impairments in nutritionally and environmently-stimulated parameters of thermogenesis which are consistent with an economy of energy expenditure, and in all likelihood, are a significant contributor to the development of their obese state.
ACKNOWLEDGEMENTS The authors wish to thank Drs. Sheldon Reiser, CNL, USDA; Dr. Wayne Smith, Colby College, and Dr. Stanley Segall, Drexel University, for use of laboratory facilities, and Mary Lou Vendryes and Brenda Jones of Drexel University for administrative assistance in the preparation of this manuscript. Supported by Colby A-23089 and a Drexel University Minigrant to O.L.T.
REFERENCES 1. Danforth, E., Jr. Hormonal control of thermogenesis. Life Sci 28: 1821-1827, 1981. 2. Gavin, L. A., F. A. McMahon and M. Moeller. The mechanism of impaired T3 production from T4 in diabetes. Diabetes 30: 694--699, 1981. 3. Hansen, C. T. Two new congenic rat strains for nutrition and obesity research. Fed Proc 42: 537, 1983. 4. Himms-Hagen, J. Obesity may be due to a malfunctioning of brown fat. Can Med Assoc J 121: 1361-1364, 1980. 5. Himms-Hagen, J. and M. Desaultals. A mitochondrial defect in brown adipose tissue of the obese (ob/ob) mouse: failure to respond to cold by an increase in binding. Biochem Biophys Res Commun 83: 628--636, 1978. 6. Himms-Hagen, J. Role of thermogenesis in brown adipose tissue in the regulation of energy balance. In: The Adipocyte and Obesity; Cellular and Molecular Mechanisms, edited by A. Angel et al. New York: Raven Press, 1983, pp. 259-270. 7. Koletsky, S. New type of spontaneously hypertensive rat with hyperlipidemia and endocrine gland defects. In: Spontaneous Hypertension, its Pathogenesis and Complications, edited by K. Okamoto, Tokyo: Igaky Shoin Ltd., 1972, pp. 194--197. 8. Michaelis, O. E., IV, K. C. Ellwood, J. Hallfrisch and C. T. Hansen. Effect of dietary sucrose and genotype on metabolic parameters of a new strain of genetically obese rat: LA/N corpulent. Nutr Res 3: 217-228, 1983.
9. Michaelis, O. E., IV, K. C. Ellwood, O. L. Tulp and M. R. C. Greenwood. Effect of feeding sucrose or starch diets on parameters of glucose tolerance in the LA/N corpulent rat. Nutr Res, in press, 1986. 10. Michaelis, O. E., IV, R. E. Martin, L. B. Gardner and K. C. Ellwood. Effect of simple and complex carbohydrate on lipogenic parameters of spontaneously hypertenisve rats. Nutr Rep lnt 24: 313-321, 1981. 11. Michaelis, O. E., IV, K. C. Ellwood, J. M. Judge, N. W. Schoene and C. T. Hansen. Effect of dietary sucrose on the SHR/N coruplent rat: a new model for insulin independent diabetes. Am J Clin Nutr 39: 612-618, 1981. 12. Michaelis, O. E., IV, K. C. Ellwood, J. M. Judge, N. W. Schoene and C. T. Hansen. The SHR/N-corpulent rat: A new model for maturity-onset diabetes. Fed Proc 42: 674, 1983. 13. Nie, N., C. H. Hull, J. G. Jenkins, K. Steinbrenner and D. H. Bart. Statistical Package for the Social Sciences, chapter 2. New York: McGraw-Hill, 1975, pp. 426-428. 14. Pisano, J. J., J. R. Crout and D. Abraham. Determination of 3-methoxy-4-hydroxymandelic acid in urine. Clin Chem Acta 7: 285-290, 1962. 15. Rothwell, N. J. and M. J. Stock. A role for brown adipose tissue in diet induced thermogenesis. Nature 281: 31-35, 1979. 16. Rothwell, N. J., M. J. Stock and B. P. Warwick. Investigation into the role of insulin in diet-induced thermogenesis in the rat. Proc Nutr Soc 81: 349A, 1981.
THERMOGENESIS
IN CORPULENT
RATS
17. Schutz, Y., T. Bessard and E. Jequier. Diet induced thermogenesis measured over a whole day in obese and non-obese women. Am J Clin Nutr 40: 542-552, 1984. 18. Smith, R. E. and B. A. Horowitz. Brown fat and thermogenesis. Physiol Rev 49: 330--425, 1969. 19. Stock, M. J. An automatic closed circuit oxygen consumption apparatus for small animals. J Appl Physiol 39: 849-850, 1975. 20. Tulp, O. L. Impaired activation of thermogenesis in the corpulent rat. Life Sci 35: 1699-1704, 1984. 21. Tulp, O. L., R. Frink and E. Danforth, Jr. Effect of cafeteria feeding on brown and white adipose tissue cellularity, thermogenesis, and body composition in rats. J Nutr 112: 22502260, 1982. 22. Tulp, O. L., M. H. Gregory, C. Maggio, M. R. C. Greenwood and O. E. Michaelis, IV. Effect of diet and genotype on growth and adipose cellularity in the corpulent (corp) rat: A new model of obesity. Fed Proc 41: 458A, 1982.
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23. Tulp, O. L. and P. P. Krupp. Thermogenesis in thyroidectomized protein-malnourished rats. J Nutr 114: 2365-2372, 1984. 24. Tulp, O. L., P. P. Krupp and E. S. Horton. Characteristics of thyroid function in experimental protein malnutrition. J Nutr 109: 1321-1332, 1979. 25. Tulp, O. L. and S. J. Shields. Thermogenesis in cafeteria fed LA/N-cp (corpulent) rats. Nutr Res 4: 325-332, 1984. 26. York, D. A. Energy balance, diet induced thermogenesis and brown adipose tissue in lean and obese (fa/fa) Zucker rats after adrenalectomy. J Nutr 113: 1395--1402, 1983. 27. Young, J. B. and L. Landsberg. Stimulation of the sympathetic nervous system during sucrose feeding. Nature 269: 615-617, 1977. 28. Zador, C. M., O. L. Tulp and E. L. Lein. Insulin independent diabetes in adult female SHR-corpulent rats. Chem Abstr, in press, 1985.