- Email: [email protected]
Adipose tissue metabolism of weanling rats after destruction of ventromedial hypothalamic nuclei: Effect of hypophysectomy and growth hormone
Adipose Tissue Metabolism Destruction
of Ventromedial
Effect of Hypophysectomy By
JACK
Rats After
Hypothalamic
Nuclei:
and Growth Hormone
K. GOLDMAN, J. DAVID SCHNATZ, LEE L. BERNARDIS AND
LAWRENCE A. FROHMAN
This report describes the effects of hypophysectomy and growth hormone treatment on the endocrine-metabolic alterations associated with destruction of the ventromedii hypotbaiamic nuclei (VMN) in weanihrg rats. In addition to previously noted characteristics of the syndrome (increased plasma insulin and triglyceride levels without hyperphagia, normal plasma glucose, and decreased plasma growth hormone levels with impaired linear growth), changes in in vitro adipose tissue metabolism of glucose-U-
D
of Weanling
Cl4 and paimitate-l-C14 were observed. Glucose oxidation and incorporation into lipid were markediy elevated, whereas paimitate osidation was decreased in adipose tissue of VMN rats. Although hypophysectomy and growth hormone treatment alone or in combination produced effects in rats with or without VMN lesions, neither signiticantly modified the characteristic changes observed in untreated rats with VMN lesions. (Metabolism 19: No. 11, November, 995 1005, 1970)
ESTRUCTION
OF THE VENTROMEDIAL NUCLEI in weanling rats produces increased body fat content, hyperinsulinemia and hypertriglyceridemia with normoglycemia and normal food intake. Growth hormone deficiency and decreased linear growth have also been noted.l-O The hormonal alterations and the lack of hyperphagia suggest that metabolic factors are implicated in the fat accumulation. The present communication describes a study of in vitro carbohydrate and lipid metabolism by adipose tissue of weanling rats subjected to bilateral destruction of the ventromedial nuclei. The role of growth hormone deficiency in the entire syndrome has also been investigated using rats additionally subjected to hypophysectomy and/or growth hormone therapy.
From the Department of Medicine, State University of New York and the Veterans Administration Hospital, Buffalo, N.Y. This work was supported in part by NIH Grants AM-11456, HD-03331 and AM-11746; American Heart Association and Mid-Hudson Heart Association Grants: USPHS General Research Support Grant 5-SOI-FR-054000-07; United Health Foundation of Western New York Grant CG-67-UB-8; and a Veterans Administration Clinical Investigatorship. Received for publication July 2, 1970. JACK K. GOLDMAN, M.D.: Assistant Professor of Medicine, State University of New York at Buffalo; Clinical Investigator, Buffalo Veterans Administration Hospital, Buffalo, N.Y. J. DAVD SCNNATZ, M.D.: Associate Professor of Medicine, State University of New York at Buffalo. LEE L. BERNARDIS,PH.D.: Assistant Clinical Professor of Medicine, State University of New York at Buffalo. LAWRENCE A. FROHMAN, M.D.: Associate Professor of Medicine, State University of New York at Buflalo. METABOLISM, VOL. 19, No.
11 (NOVEMBER), 1970
995
996
GOLDMAN ET AL. MATERIALS AND METHODS
Animals and operative Procedures Weanling male Holtzman rats were individually caged and maintained on lab chow (Teklad) and tap water ad libitum. This diet contained 3.4 calories per gram distributed as 24.7 per cent protein, 6.5 per cent fat, 55.7 per cent carbohydrate, 3.3 per cent fiber and 6.1 per cent minerals. At the age of 27 days (31 days in Experiment 2), they were anesthetized with ether and body weight and length measured. On the following day, electrolytic lesions were produced in the ventromedial hypothalamic nuclei using a HorsleyClarke stereotaxic instrument to position 0.25-mm. stainless steel electrodes coated with spar varnish and bared at the tip. The coordinates were those previously described.’ In one group of rats (VMN) a direct anodal current of 1.50 mA was passed for 10 seconds to produce lesions, and in a second group (Sham) no current was allowed to flow. The incisions were closed with stainless steel clips. In all experiments food intake was measured twice weekly, and on the 15th postoperative day body weight and length were again measured. The following day sacrifice was accomplished by decapitation, and blood and tissues collected for the subsequent determination of plasma and pituitary growth hormone,3 and plasma insulin,3 glucose (Autoanalyzer) and triglycerides.* After removal of tissues for metabolic studies, the decapitated carcasses were dried and extracted for gravimetric lipid and dry fat-free residue determinations.
Incubations and C140p Measurement The thin distal portion of one epididymal fat pad (SO-150 mg.) was incubated with 16.6 &moles glucose U-C14 (0.5 PC) in 2 ml. Krebs-Ringer bicarbonate buffer (pH 7.4) equilibrated with 95 per cent O,-5 per cent CO,,9 and a similar portion of the other fat pad with 250 mpmoles ammonium palmitate-1-C 14 (0.25 pC) in 2 ml. oxygenated KrebsRinger phosphate buffer (pH 7.4).9 Blank flasks were incubated without tissue. Flasks were sealed with serum stoppers holding hanging plastic cups and the Krebs-Ringer bicarbonate flasks gassed for five minutes with the O,-CO, gas mixture. After 3 hours of incubation with shaking at 37’C, 0.2 ml. Hyamine was injected tnrough the stopper into the hanging cups, and 0.3 ml. 4N H,SO, was injected into the medium. To collect the evolved Cr40,, incubation was continued for one hour, after which the Hyamine containing cups were removed, wiped with methanol and placed in scintillation vials containing 10 ml. of toluene with 3 Gm. PPO (2,5 diphenyloxazole) and 0.6 Gm. POPOP (p-bis 2 (5-phenyloxazolyl) benzene) per liter. Counting was performed in a Nuclear-Chicago scintillation counter.
Incorporation of Cl” Glucose into Tissue Lipids Tissues were removed from the incubation flasks, rinsed three times in saline and homogenized in 20 ml. chloroform-methanol (2: 1). The residue was separated by centrifugation and immediately dissolved in 35 per cent KOH for protein determination.10 Lipids in the supernatant were extracted, washed by the Folch procedure11 and dried at 40°C under nitrogen. Dried lipids were weighed and redissolved in chloroform-methanol, and aliquots were counted.
Verification of Lesions The brain of each rat was fixed in formalin and serially sectioned for localization of the lesion.ls Lesions were read on a coded basis and animals were excluded from statistical analysis if their lesions were asymmetrical or not primarily confined to the ventromedial nuclei. Figure 1 illustrates a typical lesion. Table 1 defines the treatment protocol and number of rats accepted for statistical analysis by Student’s t test13 in each of the four types of experiments run. RESULTS
In normal weanling rats subjected to VMN destruction (Table 2) previously described changes in body composition and plasma insulin and triglyceride were
ADIPOSE TISSUE
997
METABOLISM
section through hypothalamus of representative rat showing size, Fig. l.-Coronal location and symmetry of destructive lesion in ventromedial nuclei. X 15. observed. Adipose tissue of these rats demonstrated decreased protein content, increased glucose oxidation and incorporation into lipid and decreased oxidation of palmitic acid. Results of growth hormone treatment of VMN and Sham rats are also shown in Table 2. In both Sham and VMN rats growth hormone decreased adipose tissue lipid and increased adipose tissue protein, and in VMN rats it increased linear growth slightly but not significantly. However, none of the VMN induced alterations was reversed by growth hormone therapy. Table L-Protocol for Three Separate Experiments: Treatment Groups and Number of Animals per Treatment Group in Individual Experiments .__~
Ehp;;rey
Treatment
t
Untreated
1. VMN Sham 2. VMN Sham 3. VMN Sham
7* 5 6
8
GHP
HYPOX t
Hypox + GH I
___.
6 5 8
7 6 6 8 7 .--__ verified lesions on which data in subse-
:* Numbers refer to animals with histologically quent tables are based. i Intact animals treated with daily subcutaneous injections of 1 mg. ovine growth hormone (NIH GH-S8) after hypothalamic manipulation. $ Animals hypophysectomized at 24 days of age and injected with corticosterone (0.5 mg.) and thyroxine (2.5 pg.) daily after hypothalamic manipulation at 32 days of age. $ Animals hypophysectomized at 21 days of age. Hypothalamic manipulation at 28 days of age. Thereafter same as Hypox but with addition of growth hormone treatment as for GH.
* Not statistically
significant
(p > .05).
23 k 1 27 + 3 166 + 3 80 -c 9 162 + 9 22 + 2 30 f 2 19 -c 0.5 26 k 2 160 k 11 163 -t- 14 122 + 16 27 + 7 121 * 12 69 2 2 7-+1 22 k 2 23 k 3 4+2
21 f 2 46 + 3 182 k 5 96 k 9 173 -c 14 5 r 0.4 8&l 24 f 0.5 33 -c 3 169 k 3 100 k 6 35 5 5 82 ? 25 246 k 33 66 + 3 12 + 1 6?1 6rl 30 -r- 4 22 f 2 42 Ifr 2 179 + 2 93 * 7 167 k 9 6 r 0.6 8+-l 24 + 0.3 32 _e 2 168 -c 2 alI rt 5 37 +- 4 151 k 14 58 f 1 16 + 1 5kl 4 -c 0.2 30 + 3
III GH-Sham
k 1 k 1 + 2 +- 5 2 4 -c 1 f 1 + 0.2 + 1 c 5 k 7 + 30 93 -c 6 56 + 3 11 + 1 21 2 3 19 r 3 322
23 33 171 103 181 18 27 20 31 154 138 136
cd&N
in VMN and Sham Rats (Experiment
SalinSMN
Hormone
I Saline-Sham
of Growth
Food intake (Gm./day) F.I. Naso-anal growth (mm.) A NAL Naso-anal length (mm.) NAL Body weight gain (Gm.) A BW Final body weight (Gm.) BW Carcass fat (% wet weight) % CF Carcass fat (Gm.) CF Dry fat-free residue (% wet weight) % DFF Dry fat-free residue (Gm.) DFF Plasma glucose (mg./lOO ml.) PL. GLUC. Plasma triglyceride (mg./ 100 ml.) PL. TG. Plasma insulin ($_J./ml.) PL. INS. Plasma growth hormone (ng./ml.) PL. GH. Pituitary growth hormone bg./gland) PIT. GH. Adipose tissue lipid (% wet weight) A. T. LIP. Adipose tissue protein (mg./Gm. wet weight) A. T. PR. Glucose oxidation (dpm/rg. protein) GL. OX. Glucose to lipid (dpm/&g. protein) GL. LIP. Pahnitate oxidation (dpm/mg. tissue) P. A. OX.
Variable
Table Z.-Effects
1)
.Ol .Ol NS NS .OOl .OOl .OOl NS NS .Ol .OOl .05 .Ol NS .Ol .OOl .OOl .OOl
NS*
I-II -
NS NS NS NS NS NS NS .05 .05 .02 NS .05 NS
.OOl .OOl .OOl NS .05 .OOl .Ol .Ol NS .Ol .Ol .OOl .OOl
I-III
NS NS NS NS NS
P-c
NS .Ol .05 NS NS
III-IV
NS
NS NS NS .Ol .Ol NS NS
NS
NS
NS
NS
NS
NS NS NS NS NS
II-IV
n
I Intact Sham
3.-Effects
F.I. 21 -c 1 P, NAL 37 _’ 3 NAL 185 f 2 ABW 46 -c 9 BW 178 _’ 10 % CF 5 k 0.5 CF 6-cl % DFF 28 k 1 DFF 3.5 k 2 PL. GLUC. 152 i 3 PL. TG. 84 + 3 PL. INS. 33 -c 3 PL. GH. 42 + 2 PIT. GH. 588 k 73 A. T. LIP. 65 k 2 A. T. PR. 22 * 3 GL. OX. 4+-l GL. LIP. 4e1 P. A. OX. 58 r 10 __~~___ ___ * Abbreviations as in Table 2. i Not statistically significant (p > .0.5).
Variable’
Table
11 -r- 1 I?1 143 -r- 1 -19+-2 69 k 1 7 r 0.3 4 +- 0.1 27 ? 1 13 ? 0.3 136 2 3 116 r 13 29 -I- 4 5.6 -c 1.5 68 r 2 18 -r- 1 6-r-1 5rfil 56 ?z 11
rt 3 k 3 -’ 2 -c 17 ? 18 f 3 -e 5 f 1 * 2 -c 3 ? 8 -c 30 k 1 2 35 _c 4 c 1 -c 1 s 1 k 9
” 1 r 1 -c 2 2 8 -c 8 2 2 + 2 r 1 -c 1 -r- 4 -c 23 r 41 -c 1.4 68 r 1 12 -c 2 25 k 3 25 f 2 25 ” 10
16 2.5 148 24 114 22 20 22 19 156 206 140 2.8
HyD2klN ._
and Hypophysectomized
23 16 178 46 208 20 34 21 28 157 114 123 10 258 66 10 15 16 28
in Intact
__ III HYDOXSham
.____.~.
Destruction
IntacL4N
of VMN
NSt .OOl NS NS NS .OOl .OOl .Ol .OS NS .Ol .Ol .05 .Ol NS .Ol .OOl .OOl .05
I-II
.Ol NS NS .OOl .02 .OOl .OOl .Ol .OS .Ol .Ol .02 NS NS .Ol .OOl .OOl .05
III-rV
Rats (Experiment
2) V<
.OOl .OOl .OOl .OOl .OOl .OOl NS NS .Ol .Ol .OS NS .02 NS NS NS NS NS
I-III
.05 .OOl .OOl NS .OOl NS .OOl NS .Ol NS .Ol NS .Ol NS NS .02 .Ol NS
II-IV
1000
GOLDMAN ET AL.
Table 3 compares the results of VMN destruction in normal and in previously hypophysectomized rats maintained on corticosterone and thyroxine. Prior hypophysectomy sharply reduced food intake in both Sham and VMN rats, but less so in the latter. Hypox-Sham animals lost weight whereas Hypox-VMN animals gained weight possibly because of greater food intake. On the other hand, HypoxVMN rats ate less and gained less than did intact-VMN. Hypophysectomy stopped linear growth and thus obscured any effects of VMN destruction on this variable. The decreased plasma glucose of Hypox-Sham rats was not seen in Hypox-VMN rats who exhibited better food intake. Hypophysectomy increased plasma triglycerides in both Sham and VMN animals and potentiated the increased glucose utilization of VMN rats. Hypophysectomy did not affect the changes seen in intact VMN rats regarding per cent carcass composition, plasma insulin, adipose tissue protein, or palmitate oxidation. Table 4 (Experiment 3) present results of studies utilizing hypophysectomized rats maintained on corticosterone and thyroxine with and without growth hormone replacement. The rise in plasma glucose after VMN destruction in hypophysectomized rats was again noted and persisted with growth hormone treatment. Although food intake did not increase in Hypox-VMN rats as it did in Experiment 2 (Table 3), weight gain was again increased by VMN destruction. Hyperinsulinemia and hypertriglyceridemia appeared after VMN destruction with or without growth hormone replacement. Growth hormone, however, produced a rise in insulin but a fall in triglyceride in both Sham and VMN rats. The change in insulin levels was not significant in VMN animals, however, due to large individual variation. Growth hormone treatment did not eliminate any of the changes produced by VMN destruction in hypophysectomized animals. DISCUSSION
Marked alterations in adipose tissue composition and metabolism have been demonstrated in addition to previously described changes associated with VMN destruction. Although fat cell counts have not been performed, the decreased adipose tissue protein content presumably is a reflection of increased cell size and hence decreased cell number per gram of tissue. Such changes have been demonstrated in other forms of obesityI and recently in mature (hyperphagic) rats with hypothalamic lesions. l5 The increased glucose oxidation and incorporation into lipid is compatible with known effects of insulin on adipose tissuelS and the hyperinsulinemia seen in VMN rats may explain these findings. The data do not support the hypothesis that resistance to insulin by adipose tissue accounts for the concomitant findings of hyperinsulinemia with normoglycemia in VMN rats. Normoglycemia in VMN rats with increased adipose tissue glucose utilization suggests either increased hepatic glucose production or decreased glucose utilization associated with insulin resistance in tissues other than fat. Interpretation of the data on palmitate utilization is difficult since multiple tissue pools of free fatty acid exist.17 However, if the data are accepted at face value, they suggest a decreased lipid utilization for energy production and are compatible with the adipose tissue accumulation exhibited by VMN rats. Growth hormone has been shown to affect adipose tissue metabolism of carbohydrate,l* lipidlQ and proteinzQ However, the changes in adipose tissue metabolism associated with VMN destruction
100’
100’ 100’ 100’
IO’
ZO’ SO’
IO’
SN SN SN SN SN
100’ 100’
SN SN SN SN SN SN
SN
SN
100’ IO’ ZO’ IO’ IO’ SN SN
SN
SN
SO SO’ ZO’
_~ __ IO’ 100’ SN SN 100’ ZO’ 100’ 100’ 100’ 100’ ZO’
ZO’
_ _ _I_.
9 L I I
? + 7 T
ZEI 99 LSI EE
E + 59 zz T LZI 81 7 ZSI s i 821
IT8
OP L 7 LP P ? Lfi
sT
9 + EL E + @II z T 601 z T PP I TPSI T T PE
E 7 zz
1 T 69
E 7 OE PT It so + L
LE T OZI
__II 3 6P P 7 zs E 7 ES ITL P T 89 II TP8 fP 7 zzz z 7 ZZI E T 901 P 7 OP I T WI Z? IZ
I?L z T OL 178 6 T PO1 E T 901 z 7 IL ITS I T9EI I i SI
ZS 7 861 P T 9z t + LZ
‘d1-I Tlf) ‘X0 ‘-ED ‘Xd ‘.I, ‘V ‘dI? ‘.L ‘V ‘SNI ‘-Id ‘5X ‘?d ‘Xl?t) ‘?d M8 M8v 1VN 1VN v
‘X0 ‘V ‘d
1002
GOLDMAN
ET AL.
in these experiments were not appreciably altered by hypophysectomy or growth hormone treatment alone or in combination. This indicates that these changes are not related to altered pituitary function and cannot be attributed to changes in growth hormone dynamics. Our data regarding effects of growth hormone (GH) on food intake, weight gain and linear growth in VMN rats are in accord with Han’~.~l GH produces hyperphagia and only partially corrects impaired linear growth in intact VMN rats, suggesting that GH deficiency is not the only factor responsible for these findings. On the other hand, in hypophysectomized VMN rats GH does not produce mild hyperphagia and corrects the impairment of linear growth suggesting that some other pituitary factor inhibits these effects of GH in the intact rat. The possibility that VMN destruction renders rats resistant to growth hormone is untenable since Hypox-VMN rats grew as well as Hypox-Sham animals when both were treated with growth hormone. Total grams of carcass fat and dry-fat-free residue were not affected by growth hormone treatment, but hypophysectomy reduced both variables in VMN rats and the latter in Sham rats. Comparison of these data to Han’s is not appropriate in view of differences in experimental design. Our findings on per cent carcass composition indicate that obesity (defined as increased per cent carcass fat) is not dependent on hyperphagia, growth hormone deficiency or impaired linear growth in agreement with Han’s thesis.21 The hypertriglyceridemia is likewise not a function of altered pituitary function since it appeared after VMN destruction in hypophysectomized rats, growthhormone treated rats, and hypophysectomized-growth-hormone treated rats. The hypertriglyceridemic effect of hypophysectomy alone (Table 3) and its reversal by growth hormone administration (Table 4) have not been previously reported although case reports of hypertriglyceridemia associated with hypopituitarism have been published. 22-25The mechanism responsible for this finding is uncertain at present. Plasma insulin levels in the fed state were not affected by hypophysectomy or growth hormone alone, but in hypophysectomized rats they were raised by growth hormone administration. The lack of concurrent hypoglycemia may be related to increased food intake or growth hormone mediated insulin resistance. In Hypox-VMN animals growth hormone produced a 50 per cent rise in plasma insulin which failed of significance because of large individual variation. The hyperinsulinemia of VMN rats is, however, clearly independent of pituitary function since it is equally apparent in all treatment protocols. The variation in plasma insuhn levels apparent between Experiments 2 and 3 may be the result of seasonal factors which are known26 to influence insulin levels. Obesity in laboratory animals can be genetically transmitted as in the obesehyperglycemic mouse, or it can be induced experimentally by force feeding, by administration of insulin or gold-thioglucose, or by electrolytic destruction of the ventromedial hypothalamic nuclei.27 Electrolytic VMN destruction produces obesity with characteristics of both types. The predominant pattern seems to genetic28-33 and hyperphagic Z8.30,31,34 vary with age.2 Lesions in mature rats produce obesity, hyperphagia and decreased rate of weight loss with starvation. 2,36On a metabolic level these animals
ADIPOSE
TISSUE
1003
METABOLISM
demonstrate increased lipogenesis from acetate in the fed but not the fasted state,30r34decreased lipolytic response to epinephrine in vitro,35 and a decreased adipose tissue ratio of linoleic to palmitic acid.33 All of these changes can be ascribed to hyperinsulinemia which in turn has been attributed to hyperphagia. The metabolic pattern observed in VMN rats resembles a persistence of the immediate postprandial phase characterized by hyperinsulinemia, decreased growth hormone and increased storage of calories as fat.36 In weanling VMN rats, however, this pattern is not dependent on hyperphagia, hyperglycemia or decreased growth hormone. It persists with fasting and dietary alteration.37 Han has recently shown that adult rats with hypothalamic lesions become obese even if hypophysectomized and placed in paired activity and feeding status with their respective controls,38~3HHe concluded from this that these findings are independent of hyperphagia and are instead related to the diminished oxygen consumption exhibited by these animals. This fall in oxygen consumption could be explained by a shift from fatty acid to carbohydrate oxidation and by a stimulation of lipogenesis from glucose both of which we have described in the present paper. Adipose tissue carbohydrate and lipid metabolism are altered in VMN rats, and the alterations are conducive to the accumulation of adipose tissue. The origin and development of these changes remain subjects for speculation. However, they may well involve alterations of hypothalamic function with subsequent effects on peripheral metabolism mediated both directly through neural pathways and indirectly through hormonal alterations. This study expands the description of abnormalities seen in weanling VMN rats by demonstrating alterations in adipose tissue metabolism. The role of growth hormone deficiency has been investigated and found to be minimal since the syndrome can be equally well produced in hypophysectomized or growth hormone treated animals. ACKNOWLEDGMENTS The authors wish to express their gratitude to Miss Jane Asmus, Miss Diana Hojnicki, Mrs. Marjorie Kodis, Miss Maureen Mills, Miss Elizabeth Rybak, Miss Patricia Zuber and Mr. Michael Bohorsky for valuable technical assistance. REFERENCES 1. Bemardis, L. L.: Development of hyperphagia in female rats with ventromedial hypothalamic lesions placed at four different ages. Experientia 22:593, 1966. 2. -, and Skelton, F. R.: Growth and obesity following ventromedial hypothalamic lesions placed in female rats at four different ages. Neuroendocrinology 1: 265, 1965166. 3. Frohman, L. A., and Bernardis, L. L.: Growth hormone and insulin levels in weanling rats with ventromedial hypothalamic lesions. Endocrinology 82: 1125, 1968. 4. -, Bernardis, L. L., Schnatz, J. D., and Burek, L.: Effect of ventromedial hypothalamic nucleus (VMN) destruction on
carbohydrate and lipid metabolism in weanling rats. Amer. J. Physiol. 216:1496, 1969. 5. Han, P. W., Lin, C. H., Chu, K. C.. Mu, J. Y., and Liu, A. C.: Hypothalamic obesity in weanling rats. Amer. J. Physiol. 209:627, 1964. 6. -, and Liu, A. C.: Obesity and impaired growth of rats force-fed 40 days after hypothalamic lesions. Amer. J. Physiol. 211:229, 1966. 7. Bernardis, L. L., and Skelton, F. R.: Stereotaxic localization of the supraoptic, ventromedial and mamillary nuclei in the hypothalamus of weanling to mature rats. Amer. J. Anat. 116:69, 1965.
1004 8. Van Handel, E., and Zilversmit, D. B.: Micromethod for the direct determination of serum triglycerides. J. Lab. Clin. Med. 50: 152, 1957. 9. Umbreit, W. W., Burris, R. H., and Stauffer, J. F.: Manometric Techniques (ed. 3). Minneapolis, Minn., Burgess, 1957, p. 149. 10. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J.: Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265, 1951. 11. Folch, J., Lees, M., and Sloane-Stanley, G. H.: A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226:497, 1957. 12. DeGroot, J.: The rat forebrain in stereotaxic coordinates. Verh. Koninl. Ned. Akad. Wetenschap. afd. Natureuk. 4: 1, 1959. 13. Snedecor, G. W., and Cochran, W. G.: Statistical Methods (ed. 6). Ames, Iowa, Iowa State University Press, 1967. 14. Salans, L. B., Knittle, J. L., and Hirsch, J.: The role of adipose cell enlargement in the carbohydrate intolerance of human obesity. J. Clin. Invest. 46: 1112, 1967. 15. Hirsch, J., and Han, P. W.: Cellularity of rat adipose tissue: Effects of growth, starvation, and obesity. J. Lipid Res. 10:77, 1969. 16. Winegrad, A. I., and Renold, A. E.: Studies on rat adipose tissue in vitro. I. Effects of insulin on the metabolism of glucose, pyruvate and acetate. J. Biol. Chem. 233~267, 1958. 17. Vaughan, M., Steinberg, D.. and Pittman, R.: On the interpretation of studies measuring uptake and esterification of (l-l%) palmitic acid by rat adipose tissue in vitro. Biochem. Biophys. Acta 84: 154, 1964. 18. Goodman, H. M.: A comparative study of the effects of insulin and growth hormone on hexose metabolism in adipose tissue. Endocrinology 80:45, 1967. 19. Goldman, J. K., and Bressler, R.: Growth hormone stimulation of fatty acid utilization by adipose tissue. Endocrinology 81: 1306, 1967. 20. Swislocki, N. I.: Growth hormone effects on histidine incorporation into protein by adipose tissue from hypophysectomized rats. Biochem. Biophys. Acta 169:556, 1968.
GOLDMAN ET AL. 21. Han, P. W.: Hypothalamic obesity in rats without hyperphagia. Trans. N.Y. Acad. Sci. 30:229, 1967. 22. Summers, V. K., Hipkin, L. J., and Davis, J. C.: Serum lipids in diseases of the pituitary. Metabolism 16: 1106, 1967. 23. Tabatznik, B., and Rabinowitz, D.: Hyperglyceridemia and lipemia retinalis in hypopituitarism. Bull. J. Hopkins Hosp. 107: 175, 1960. 24. DeMedeiros-Neto, G. A., Wajchenberg, B. L., Schneider, J., DeAssis, L. M., Kieffer, J., Pieroni, R. R., and Centra, A. B. U.: Hyperlipemia and panhypopituitarism: Report of 2 cases. Metabolism 12:659, 1963. 25. Jacobs, D. R., Krieger, 0. T., and Charles, R. N.: Late appearance of hyperlipemia in hypopituitarism. Ann. Intern. Med. 55:640, 1961. 26. Howland, R. J., and Nowell, N. W.: Seasonal changes of plasma insulin concentration in the rat. J. Endocr. 4O:vi, 1968. 27. Mayer, J.: Some aspects of the problem of regulation of food intake and obesity. New Eng. J. Med. 274:662, 1966. 28. Bates, M. W., Zomzely, C., and Mayer, J.: Fat metabolism in three forms of experimental obesity; instantaneous rates of lipogenesis in vivo. Amer. J. Physiol. 181: 187, 1955. 29. Shigeta, Y., and Shreeve, W. W.: Fatty acid synthesis from glucose-l-H3 and glucose-104 in obese-hyperglycemic mice. Amer. J. Physiol. 206:1085, 1964. 30. Christophe, J., Jeanrenaud, B., Mayer, J., and Renold, A. E.: Metabolism in vitro of adipose tissue in obese-hyperglycemic and gold-thioglucose-treated mice. I. Biol. Chem. 236:642, 1961. 31. LeBoeuf, B., Lochaya, S., LeBoeuf, N., Wood, F. C., Jr., Mayer, J., and Cahill, G. F., Jr.: Glucose metabolism and mobilization of fatty acids by adipose tissue from obese mice. Amer. J. Physiol. 201:19, 1961. 32. Lochaya, S., LeBoeuf, N., Mayer, J., and LeBoeuf, B.: Adipose tissue metabolism of obese mice on standard and high-fat diets. Amer. J. Physiol. 201:23, 1961. 33. Hellman, B., and Westman, S.: Palmitate utilization in obese hyperglycemic mice. Acta Physiol. Stand. 61:65, 1964. 34. Bates, M. W., Mayer, J., and Nauss, S. F.: Fat metabolism in three forms of experimental obesity; acetate incorporation. Amer. J. Physiol. 180:304, 1955.
ADIPOSE
TISSUE
METABOLISM
35. Haessler, H. A., and Crawford, J. D.: Fatty acid composition and metabolic activity of depot fat in experimental obesity. Amer. I. Physiol. 213:255, 1967. 36. Rabinowitz, D., and Zierler, K. L.: A metabolic regulating device based on the actions of human growth hormone and of insulin, singly and together, on the human forearm. Nature 199:913, 1963. 37. Frohman, L. A., Goldman, J. K., Schnatz, J. D., and Bernardis, L. L.: Hypothalamic obesity in the weanling rat: Effect
1005
of dietary alteration and starvation. Abstract 226, Program of 5’1st Meeting of the Endocrine Society 1969, p. 143. 38. Han, P. W.: Obesity in force-fed, hypophysectomized rats bearing hypothalamic lesions. Proc. Sot. Exp. Biol. Med. 127: 10.57, 1968. 39. -: Energy metabolism of tube-fed hypophysectomized rats bearing hypothalamic lesions. Amer. J. Physiol. 215:1343, 1968.
of Ventromedial
Effect of Hypophysectomy By
JACK
Rats After
Hypothalamic
Nuclei:
and Growth Hormone
K. GOLDMAN, J. DAVID SCHNATZ, LEE L. BERNARDIS AND
LAWRENCE A. FROHMAN
This report describes the effects of hypophysectomy and growth hormone treatment on the endocrine-metabolic alterations associated with destruction of the ventromedii hypotbaiamic nuclei (VMN) in weanihrg rats. In addition to previously noted characteristics of the syndrome (increased plasma insulin and triglyceride levels without hyperphagia, normal plasma glucose, and decreased plasma growth hormone levels with impaired linear growth), changes in in vitro adipose tissue metabolism of glucose-U-
D
of Weanling
Cl4 and paimitate-l-C14 were observed. Glucose oxidation and incorporation into lipid were markediy elevated, whereas paimitate osidation was decreased in adipose tissue of VMN rats. Although hypophysectomy and growth hormone treatment alone or in combination produced effects in rats with or without VMN lesions, neither signiticantly modified the characteristic changes observed in untreated rats with VMN lesions. (Metabolism 19: No. 11, November, 995 1005, 1970)
ESTRUCTION
OF THE VENTROMEDIAL NUCLEI in weanling rats produces increased body fat content, hyperinsulinemia and hypertriglyceridemia with normoglycemia and normal food intake. Growth hormone deficiency and decreased linear growth have also been noted.l-O The hormonal alterations and the lack of hyperphagia suggest that metabolic factors are implicated in the fat accumulation. The present communication describes a study of in vitro carbohydrate and lipid metabolism by adipose tissue of weanling rats subjected to bilateral destruction of the ventromedial nuclei. The role of growth hormone deficiency in the entire syndrome has also been investigated using rats additionally subjected to hypophysectomy and/or growth hormone therapy.
From the Department of Medicine, State University of New York and the Veterans Administration Hospital, Buffalo, N.Y. This work was supported in part by NIH Grants AM-11456, HD-03331 and AM-11746; American Heart Association and Mid-Hudson Heart Association Grants: USPHS General Research Support Grant 5-SOI-FR-054000-07; United Health Foundation of Western New York Grant CG-67-UB-8; and a Veterans Administration Clinical Investigatorship. Received for publication July 2, 1970. JACK K. GOLDMAN, M.D.: Assistant Professor of Medicine, State University of New York at Buffalo; Clinical Investigator, Buffalo Veterans Administration Hospital, Buffalo, N.Y. J. DAVD SCNNATZ, M.D.: Associate Professor of Medicine, State University of New York at Buffalo. LEE L. BERNARDIS,PH.D.: Assistant Clinical Professor of Medicine, State University of New York at Buffalo. LAWRENCE A. FROHMAN, M.D.: Associate Professor of Medicine, State University of New York at Buflalo. METABOLISM, VOL. 19, No.
11 (NOVEMBER), 1970
995
996
GOLDMAN ET AL. MATERIALS AND METHODS
Animals and operative Procedures Weanling male Holtzman rats were individually caged and maintained on lab chow (Teklad) and tap water ad libitum. This diet contained 3.4 calories per gram distributed as 24.7 per cent protein, 6.5 per cent fat, 55.7 per cent carbohydrate, 3.3 per cent fiber and 6.1 per cent minerals. At the age of 27 days (31 days in Experiment 2), they were anesthetized with ether and body weight and length measured. On the following day, electrolytic lesions were produced in the ventromedial hypothalamic nuclei using a HorsleyClarke stereotaxic instrument to position 0.25-mm. stainless steel electrodes coated with spar varnish and bared at the tip. The coordinates were those previously described.’ In one group of rats (VMN) a direct anodal current of 1.50 mA was passed for 10 seconds to produce lesions, and in a second group (Sham) no current was allowed to flow. The incisions were closed with stainless steel clips. In all experiments food intake was measured twice weekly, and on the 15th postoperative day body weight and length were again measured. The following day sacrifice was accomplished by decapitation, and blood and tissues collected for the subsequent determination of plasma and pituitary growth hormone,3 and plasma insulin,3 glucose (Autoanalyzer) and triglycerides.* After removal of tissues for metabolic studies, the decapitated carcasses were dried and extracted for gravimetric lipid and dry fat-free residue determinations.
Incubations and C140p Measurement The thin distal portion of one epididymal fat pad (SO-150 mg.) was incubated with 16.6 &moles glucose U-C14 (0.5 PC) in 2 ml. Krebs-Ringer bicarbonate buffer (pH 7.4) equilibrated with 95 per cent O,-5 per cent CO,,9 and a similar portion of the other fat pad with 250 mpmoles ammonium palmitate-1-C 14 (0.25 pC) in 2 ml. oxygenated KrebsRinger phosphate buffer (pH 7.4).9 Blank flasks were incubated without tissue. Flasks were sealed with serum stoppers holding hanging plastic cups and the Krebs-Ringer bicarbonate flasks gassed for five minutes with the O,-CO, gas mixture. After 3 hours of incubation with shaking at 37’C, 0.2 ml. Hyamine was injected tnrough the stopper into the hanging cups, and 0.3 ml. 4N H,SO, was injected into the medium. To collect the evolved Cr40,, incubation was continued for one hour, after which the Hyamine containing cups were removed, wiped with methanol and placed in scintillation vials containing 10 ml. of toluene with 3 Gm. PPO (2,5 diphenyloxazole) and 0.6 Gm. POPOP (p-bis 2 (5-phenyloxazolyl) benzene) per liter. Counting was performed in a Nuclear-Chicago scintillation counter.
Incorporation of Cl” Glucose into Tissue Lipids Tissues were removed from the incubation flasks, rinsed three times in saline and homogenized in 20 ml. chloroform-methanol (2: 1). The residue was separated by centrifugation and immediately dissolved in 35 per cent KOH for protein determination.10 Lipids in the supernatant were extracted, washed by the Folch procedure11 and dried at 40°C under nitrogen. Dried lipids were weighed and redissolved in chloroform-methanol, and aliquots were counted.
Verification of Lesions The brain of each rat was fixed in formalin and serially sectioned for localization of the lesion.ls Lesions were read on a coded basis and animals were excluded from statistical analysis if their lesions were asymmetrical or not primarily confined to the ventromedial nuclei. Figure 1 illustrates a typical lesion. Table 1 defines the treatment protocol and number of rats accepted for statistical analysis by Student’s t test13 in each of the four types of experiments run. RESULTS
In normal weanling rats subjected to VMN destruction (Table 2) previously described changes in body composition and plasma insulin and triglyceride were
ADIPOSE TISSUE
997
METABOLISM
section through hypothalamus of representative rat showing size, Fig. l.-Coronal location and symmetry of destructive lesion in ventromedial nuclei. X 15. observed. Adipose tissue of these rats demonstrated decreased protein content, increased glucose oxidation and incorporation into lipid and decreased oxidation of palmitic acid. Results of growth hormone treatment of VMN and Sham rats are also shown in Table 2. In both Sham and VMN rats growth hormone decreased adipose tissue lipid and increased adipose tissue protein, and in VMN rats it increased linear growth slightly but not significantly. However, none of the VMN induced alterations was reversed by growth hormone therapy. Table L-Protocol for Three Separate Experiments: Treatment Groups and Number of Animals per Treatment Group in Individual Experiments .__~
Ehp;;rey
Treatment
t
Untreated
1. VMN Sham 2. VMN Sham 3. VMN Sham
7* 5 6
8
GHP
HYPOX t
Hypox + GH I
___.
6 5 8
7 6 6 8 7 .--__ verified lesions on which data in subse-
:* Numbers refer to animals with histologically quent tables are based. i Intact animals treated with daily subcutaneous injections of 1 mg. ovine growth hormone (NIH GH-S8) after hypothalamic manipulation. $ Animals hypophysectomized at 24 days of age and injected with corticosterone (0.5 mg.) and thyroxine (2.5 pg.) daily after hypothalamic manipulation at 32 days of age. $ Animals hypophysectomized at 21 days of age. Hypothalamic manipulation at 28 days of age. Thereafter same as Hypox but with addition of growth hormone treatment as for GH.
* Not statistically
significant
(p > .05).
23 k 1 27 + 3 166 + 3 80 -c 9 162 + 9 22 + 2 30 f 2 19 -c 0.5 26 k 2 160 k 11 163 -t- 14 122 + 16 27 + 7 121 * 12 69 2 2 7-+1 22 k 2 23 k 3 4+2
21 f 2 46 + 3 182 k 5 96 k 9 173 -c 14 5 r 0.4 8&l 24 f 0.5 33 -c 3 169 k 3 100 k 6 35 5 5 82 ? 25 246 k 33 66 + 3 12 + 1 6?1 6rl 30 -r- 4 22 f 2 42 Ifr 2 179 + 2 93 * 7 167 k 9 6 r 0.6 8+-l 24 + 0.3 32 _e 2 168 -c 2 alI rt 5 37 +- 4 151 k 14 58 f 1 16 + 1 5kl 4 -c 0.2 30 + 3
III GH-Sham
k 1 k 1 + 2 +- 5 2 4 -c 1 f 1 + 0.2 + 1 c 5 k 7 + 30 93 -c 6 56 + 3 11 + 1 21 2 3 19 r 3 322
23 33 171 103 181 18 27 20 31 154 138 136
cd&N
in VMN and Sham Rats (Experiment
SalinSMN
Hormone
I Saline-Sham
of Growth
Food intake (Gm./day) F.I. Naso-anal growth (mm.) A NAL Naso-anal length (mm.) NAL Body weight gain (Gm.) A BW Final body weight (Gm.) BW Carcass fat (% wet weight) % CF Carcass fat (Gm.) CF Dry fat-free residue (% wet weight) % DFF Dry fat-free residue (Gm.) DFF Plasma glucose (mg./lOO ml.) PL. GLUC. Plasma triglyceride (mg./ 100 ml.) PL. TG. Plasma insulin ($_J./ml.) PL. INS. Plasma growth hormone (ng./ml.) PL. GH. Pituitary growth hormone bg./gland) PIT. GH. Adipose tissue lipid (% wet weight) A. T. LIP. Adipose tissue protein (mg./Gm. wet weight) A. T. PR. Glucose oxidation (dpm/rg. protein) GL. OX. Glucose to lipid (dpm/&g. protein) GL. LIP. Pahnitate oxidation (dpm/mg. tissue) P. A. OX.
Variable
Table Z.-Effects
1)
.Ol .Ol NS NS .OOl .OOl .OOl NS NS .Ol .OOl .05 .Ol NS .Ol .OOl .OOl .OOl
NS*
I-II -
NS NS NS NS NS NS NS .05 .05 .02 NS .05 NS
.OOl .OOl .OOl NS .05 .OOl .Ol .Ol NS .Ol .Ol .OOl .OOl
I-III
NS NS NS NS NS
P-c
NS .Ol .05 NS NS
III-IV
NS
NS NS NS .Ol .Ol NS NS
NS
NS
NS
NS
NS
NS NS NS NS NS
II-IV
n
I Intact Sham
3.-Effects
F.I. 21 -c 1 P, NAL 37 _’ 3 NAL 185 f 2 ABW 46 -c 9 BW 178 _’ 10 % CF 5 k 0.5 CF 6-cl % DFF 28 k 1 DFF 3.5 k 2 PL. GLUC. 152 i 3 PL. TG. 84 + 3 PL. INS. 33 -c 3 PL. GH. 42 + 2 PIT. GH. 588 k 73 A. T. LIP. 65 k 2 A. T. PR. 22 * 3 GL. OX. 4+-l GL. LIP. 4e1 P. A. OX. 58 r 10 __~~___ ___ * Abbreviations as in Table 2. i Not statistically significant (p > .0.5).
Variable’
Table
11 -r- 1 I?1 143 -r- 1 -19+-2 69 k 1 7 r 0.3 4 +- 0.1 27 ? 1 13 ? 0.3 136 2 3 116 r 13 29 -I- 4 5.6 -c 1.5 68 r 2 18 -r- 1 6-r-1 5rfil 56 ?z 11
rt 3 k 3 -’ 2 -c 17 ? 18 f 3 -e 5 f 1 * 2 -c 3 ? 8 -c 30 k 1 2 35 _c 4 c 1 -c 1 s 1 k 9
” 1 r 1 -c 2 2 8 -c 8 2 2 + 2 r 1 -c 1 -r- 4 -c 23 r 41 -c 1.4 68 r 1 12 -c 2 25 k 3 25 f 2 25 ” 10
16 2.5 148 24 114 22 20 22 19 156 206 140 2.8
HyD2klN ._
and Hypophysectomized
23 16 178 46 208 20 34 21 28 157 114 123 10 258 66 10 15 16 28
in Intact
__ III HYDOXSham
.____.~.
Destruction
IntacL4N
of VMN
NSt .OOl NS NS NS .OOl .OOl .Ol .OS NS .Ol .Ol .05 .Ol NS .Ol .OOl .OOl .05
I-II
.Ol NS NS .OOl .02 .OOl .OOl .Ol .OS .Ol .Ol .02 NS NS .Ol .OOl .OOl .05
III-rV
Rats (Experiment
2) V<
.OOl .OOl .OOl .OOl .OOl .OOl NS NS .Ol .Ol .OS NS .02 NS NS NS NS NS
I-III
.05 .OOl .OOl NS .OOl NS .OOl NS .Ol NS .Ol NS .Ol NS NS .02 .Ol NS
II-IV
1000
GOLDMAN ET AL.
Table 3 compares the results of VMN destruction in normal and in previously hypophysectomized rats maintained on corticosterone and thyroxine. Prior hypophysectomy sharply reduced food intake in both Sham and VMN rats, but less so in the latter. Hypox-Sham animals lost weight whereas Hypox-VMN animals gained weight possibly because of greater food intake. On the other hand, HypoxVMN rats ate less and gained less than did intact-VMN. Hypophysectomy stopped linear growth and thus obscured any effects of VMN destruction on this variable. The decreased plasma glucose of Hypox-Sham rats was not seen in Hypox-VMN rats who exhibited better food intake. Hypophysectomy increased plasma triglycerides in both Sham and VMN animals and potentiated the increased glucose utilization of VMN rats. Hypophysectomy did not affect the changes seen in intact VMN rats regarding per cent carcass composition, plasma insulin, adipose tissue protein, or palmitate oxidation. Table 4 (Experiment 3) present results of studies utilizing hypophysectomized rats maintained on corticosterone and thyroxine with and without growth hormone replacement. The rise in plasma glucose after VMN destruction in hypophysectomized rats was again noted and persisted with growth hormone treatment. Although food intake did not increase in Hypox-VMN rats as it did in Experiment 2 (Table 3), weight gain was again increased by VMN destruction. Hyperinsulinemia and hypertriglyceridemia appeared after VMN destruction with or without growth hormone replacement. Growth hormone, however, produced a rise in insulin but a fall in triglyceride in both Sham and VMN rats. The change in insulin levels was not significant in VMN animals, however, due to large individual variation. Growth hormone treatment did not eliminate any of the changes produced by VMN destruction in hypophysectomized animals. DISCUSSION
Marked alterations in adipose tissue composition and metabolism have been demonstrated in addition to previously described changes associated with VMN destruction. Although fat cell counts have not been performed, the decreased adipose tissue protein content presumably is a reflection of increased cell size and hence decreased cell number per gram of tissue. Such changes have been demonstrated in other forms of obesityI and recently in mature (hyperphagic) rats with hypothalamic lesions. l5 The increased glucose oxidation and incorporation into lipid is compatible with known effects of insulin on adipose tissuelS and the hyperinsulinemia seen in VMN rats may explain these findings. The data do not support the hypothesis that resistance to insulin by adipose tissue accounts for the concomitant findings of hyperinsulinemia with normoglycemia in VMN rats. Normoglycemia in VMN rats with increased adipose tissue glucose utilization suggests either increased hepatic glucose production or decreased glucose utilization associated with insulin resistance in tissues other than fat. Interpretation of the data on palmitate utilization is difficult since multiple tissue pools of free fatty acid exist.17 However, if the data are accepted at face value, they suggest a decreased lipid utilization for energy production and are compatible with the adipose tissue accumulation exhibited by VMN rats. Growth hormone has been shown to affect adipose tissue metabolism of carbohydrate,l* lipidlQ and proteinzQ However, the changes in adipose tissue metabolism associated with VMN destruction
100’
100’ 100’ 100’
IO’
ZO’ SO’
IO’
SN SN SN SN SN
100’ 100’
SN SN SN SN SN SN
SN
SN
100’ IO’ ZO’ IO’ IO’ SN SN
SN
SN
SO SO’ ZO’
_~ __ IO’ 100’ SN SN 100’ ZO’ 100’ 100’ 100’ 100’ ZO’
ZO’
_ _ _I_.
9 L I I
? + 7 T
ZEI 99 LSI EE
E + 59 zz T LZI 81 7 ZSI s i 821
IT8
OP L 7 LP P ? Lfi
sT
9 + EL E + @II z T 601 z T PP I TPSI T T PE
E 7 zz
1 T 69
E 7 OE PT It so + L
LE T OZI
__II 3 6P P 7 zs E 7 ES ITL P T 89 II TP8 fP 7 zzz z 7 ZZI E T 901 P 7 OP I T WI Z? IZ
I?L z T OL 178 6 T PO1 E T 901 z 7 IL ITS I T9EI I i SI
ZS 7 861 P T 9z t + LZ
‘d1-I Tlf) ‘X0 ‘-ED ‘Xd ‘.I, ‘V ‘dI? ‘.L ‘V ‘SNI ‘-Id ‘5X ‘?d ‘Xl?t) ‘?d M8 M8v 1VN 1VN v
‘X0 ‘V ‘d
1002
GOLDMAN
ET AL.
in these experiments were not appreciably altered by hypophysectomy or growth hormone treatment alone or in combination. This indicates that these changes are not related to altered pituitary function and cannot be attributed to changes in growth hormone dynamics. Our data regarding effects of growth hormone (GH) on food intake, weight gain and linear growth in VMN rats are in accord with Han’~.~l GH produces hyperphagia and only partially corrects impaired linear growth in intact VMN rats, suggesting that GH deficiency is not the only factor responsible for these findings. On the other hand, in hypophysectomized VMN rats GH does not produce mild hyperphagia and corrects the impairment of linear growth suggesting that some other pituitary factor inhibits these effects of GH in the intact rat. The possibility that VMN destruction renders rats resistant to growth hormone is untenable since Hypox-VMN rats grew as well as Hypox-Sham animals when both were treated with growth hormone. Total grams of carcass fat and dry-fat-free residue were not affected by growth hormone treatment, but hypophysectomy reduced both variables in VMN rats and the latter in Sham rats. Comparison of these data to Han’s is not appropriate in view of differences in experimental design. Our findings on per cent carcass composition indicate that obesity (defined as increased per cent carcass fat) is not dependent on hyperphagia, growth hormone deficiency or impaired linear growth in agreement with Han’s thesis.21 The hypertriglyceridemia is likewise not a function of altered pituitary function since it appeared after VMN destruction in hypophysectomized rats, growthhormone treated rats, and hypophysectomized-growth-hormone treated rats. The hypertriglyceridemic effect of hypophysectomy alone (Table 3) and its reversal by growth hormone administration (Table 4) have not been previously reported although case reports of hypertriglyceridemia associated with hypopituitarism have been published. 22-25The mechanism responsible for this finding is uncertain at present. Plasma insulin levels in the fed state were not affected by hypophysectomy or growth hormone alone, but in hypophysectomized rats they were raised by growth hormone administration. The lack of concurrent hypoglycemia may be related to increased food intake or growth hormone mediated insulin resistance. In Hypox-VMN animals growth hormone produced a 50 per cent rise in plasma insulin which failed of significance because of large individual variation. The hyperinsulinemia of VMN rats is, however, clearly independent of pituitary function since it is equally apparent in all treatment protocols. The variation in plasma insuhn levels apparent between Experiments 2 and 3 may be the result of seasonal factors which are known26 to influence insulin levels. Obesity in laboratory animals can be genetically transmitted as in the obesehyperglycemic mouse, or it can be induced experimentally by force feeding, by administration of insulin or gold-thioglucose, or by electrolytic destruction of the ventromedial hypothalamic nuclei.27 Electrolytic VMN destruction produces obesity with characteristics of both types. The predominant pattern seems to genetic28-33 and hyperphagic Z8.30,31,34 vary with age.2 Lesions in mature rats produce obesity, hyperphagia and decreased rate of weight loss with starvation. 2,36On a metabolic level these animals
ADIPOSE
TISSUE
1003
METABOLISM
demonstrate increased lipogenesis from acetate in the fed but not the fasted state,30r34decreased lipolytic response to epinephrine in vitro,35 and a decreased adipose tissue ratio of linoleic to palmitic acid.33 All of these changes can be ascribed to hyperinsulinemia which in turn has been attributed to hyperphagia. The metabolic pattern observed in VMN rats resembles a persistence of the immediate postprandial phase characterized by hyperinsulinemia, decreased growth hormone and increased storage of calories as fat.36 In weanling VMN rats, however, this pattern is not dependent on hyperphagia, hyperglycemia or decreased growth hormone. It persists with fasting and dietary alteration.37 Han has recently shown that adult rats with hypothalamic lesions become obese even if hypophysectomized and placed in paired activity and feeding status with their respective controls,38~3HHe concluded from this that these findings are independent of hyperphagia and are instead related to the diminished oxygen consumption exhibited by these animals. This fall in oxygen consumption could be explained by a shift from fatty acid to carbohydrate oxidation and by a stimulation of lipogenesis from glucose both of which we have described in the present paper. Adipose tissue carbohydrate and lipid metabolism are altered in VMN rats, and the alterations are conducive to the accumulation of adipose tissue. The origin and development of these changes remain subjects for speculation. However, they may well involve alterations of hypothalamic function with subsequent effects on peripheral metabolism mediated both directly through neural pathways and indirectly through hormonal alterations. This study expands the description of abnormalities seen in weanling VMN rats by demonstrating alterations in adipose tissue metabolism. The role of growth hormone deficiency has been investigated and found to be minimal since the syndrome can be equally well produced in hypophysectomized or growth hormone treated animals. ACKNOWLEDGMENTS The authors wish to express their gratitude to Miss Jane Asmus, Miss Diana Hojnicki, Mrs. Marjorie Kodis, Miss Maureen Mills, Miss Elizabeth Rybak, Miss Patricia Zuber and Mr. Michael Bohorsky for valuable technical assistance. REFERENCES 1. Bemardis, L. L.: Development of hyperphagia in female rats with ventromedial hypothalamic lesions placed at four different ages. Experientia 22:593, 1966. 2. -, and Skelton, F. R.: Growth and obesity following ventromedial hypothalamic lesions placed in female rats at four different ages. Neuroendocrinology 1: 265, 1965166. 3. Frohman, L. A., and Bernardis, L. L.: Growth hormone and insulin levels in weanling rats with ventromedial hypothalamic lesions. Endocrinology 82: 1125, 1968. 4. -, Bernardis, L. L., Schnatz, J. D., and Burek, L.: Effect of ventromedial hypothalamic nucleus (VMN) destruction on
carbohydrate and lipid metabolism in weanling rats. Amer. J. Physiol. 216:1496, 1969. 5. Han, P. W., Lin, C. H., Chu, K. C.. Mu, J. Y., and Liu, A. C.: Hypothalamic obesity in weanling rats. Amer. J. Physiol. 209:627, 1964. 6. -, and Liu, A. C.: Obesity and impaired growth of rats force-fed 40 days after hypothalamic lesions. Amer. J. Physiol. 211:229, 1966. 7. Bernardis, L. L., and Skelton, F. R.: Stereotaxic localization of the supraoptic, ventromedial and mamillary nuclei in the hypothalamus of weanling to mature rats. Amer. J. Anat. 116:69, 1965.
1004 8. Van Handel, E., and Zilversmit, D. B.: Micromethod for the direct determination of serum triglycerides. J. Lab. Clin. Med. 50: 152, 1957. 9. Umbreit, W. W., Burris, R. H., and Stauffer, J. F.: Manometric Techniques (ed. 3). Minneapolis, Minn., Burgess, 1957, p. 149. 10. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J.: Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265, 1951. 11. Folch, J., Lees, M., and Sloane-Stanley, G. H.: A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226:497, 1957. 12. DeGroot, J.: The rat forebrain in stereotaxic coordinates. Verh. Koninl. Ned. Akad. Wetenschap. afd. Natureuk. 4: 1, 1959. 13. Snedecor, G. W., and Cochran, W. G.: Statistical Methods (ed. 6). Ames, Iowa, Iowa State University Press, 1967. 14. Salans, L. B., Knittle, J. L., and Hirsch, J.: The role of adipose cell enlargement in the carbohydrate intolerance of human obesity. J. Clin. Invest. 46: 1112, 1967. 15. Hirsch, J., and Han, P. W.: Cellularity of rat adipose tissue: Effects of growth, starvation, and obesity. J. Lipid Res. 10:77, 1969. 16. Winegrad, A. I., and Renold, A. E.: Studies on rat adipose tissue in vitro. I. Effects of insulin on the metabolism of glucose, pyruvate and acetate. J. Biol. Chem. 233~267, 1958. 17. Vaughan, M., Steinberg, D.. and Pittman, R.: On the interpretation of studies measuring uptake and esterification of (l-l%) palmitic acid by rat adipose tissue in vitro. Biochem. Biophys. Acta 84: 154, 1964. 18. Goodman, H. M.: A comparative study of the effects of insulin and growth hormone on hexose metabolism in adipose tissue. Endocrinology 80:45, 1967. 19. Goldman, J. K., and Bressler, R.: Growth hormone stimulation of fatty acid utilization by adipose tissue. Endocrinology 81: 1306, 1967. 20. Swislocki, N. I.: Growth hormone effects on histidine incorporation into protein by adipose tissue from hypophysectomized rats. Biochem. Biophys. Acta 169:556, 1968.
GOLDMAN ET AL. 21. Han, P. W.: Hypothalamic obesity in rats without hyperphagia. Trans. N.Y. Acad. Sci. 30:229, 1967. 22. Summers, V. K., Hipkin, L. J., and Davis, J. C.: Serum lipids in diseases of the pituitary. Metabolism 16: 1106, 1967. 23. Tabatznik, B., and Rabinowitz, D.: Hyperglyceridemia and lipemia retinalis in hypopituitarism. Bull. J. Hopkins Hosp. 107: 175, 1960. 24. DeMedeiros-Neto, G. A., Wajchenberg, B. L., Schneider, J., DeAssis, L. M., Kieffer, J., Pieroni, R. R., and Centra, A. B. U.: Hyperlipemia and panhypopituitarism: Report of 2 cases. Metabolism 12:659, 1963. 25. Jacobs, D. R., Krieger, 0. T., and Charles, R. N.: Late appearance of hyperlipemia in hypopituitarism. Ann. Intern. Med. 55:640, 1961. 26. Howland, R. J., and Nowell, N. W.: Seasonal changes of plasma insulin concentration in the rat. J. Endocr. 4O:vi, 1968. 27. Mayer, J.: Some aspects of the problem of regulation of food intake and obesity. New Eng. J. Med. 274:662, 1966. 28. Bates, M. W., Zomzely, C., and Mayer, J.: Fat metabolism in three forms of experimental obesity; instantaneous rates of lipogenesis in vivo. Amer. J. Physiol. 181: 187, 1955. 29. Shigeta, Y., and Shreeve, W. W.: Fatty acid synthesis from glucose-l-H3 and glucose-104 in obese-hyperglycemic mice. Amer. J. Physiol. 206:1085, 1964. 30. Christophe, J., Jeanrenaud, B., Mayer, J., and Renold, A. E.: Metabolism in vitro of adipose tissue in obese-hyperglycemic and gold-thioglucose-treated mice. I. Biol. Chem. 236:642, 1961. 31. LeBoeuf, B., Lochaya, S., LeBoeuf, N., Wood, F. C., Jr., Mayer, J., and Cahill, G. F., Jr.: Glucose metabolism and mobilization of fatty acids by adipose tissue from obese mice. Amer. J. Physiol. 201:19, 1961. 32. Lochaya, S., LeBoeuf, N., Mayer, J., and LeBoeuf, B.: Adipose tissue metabolism of obese mice on standard and high-fat diets. Amer. J. Physiol. 201:23, 1961. 33. Hellman, B., and Westman, S.: Palmitate utilization in obese hyperglycemic mice. Acta Physiol. Stand. 61:65, 1964. 34. Bates, M. W., Mayer, J., and Nauss, S. F.: Fat metabolism in three forms of experimental obesity; acetate incorporation. Amer. J. Physiol. 180:304, 1955.
ADIPOSE
TISSUE
METABOLISM
35. Haessler, H. A., and Crawford, J. D.: Fatty acid composition and metabolic activity of depot fat in experimental obesity. Amer. I. Physiol. 213:255, 1967. 36. Rabinowitz, D., and Zierler, K. L.: A metabolic regulating device based on the actions of human growth hormone and of insulin, singly and together, on the human forearm. Nature 199:913, 1963. 37. Frohman, L. A., Goldman, J. K., Schnatz, J. D., and Bernardis, L. L.: Hypothalamic obesity in the weanling rat: Effect
1005
of dietary alteration and starvation. Abstract 226, Program of 5’1st Meeting of the Endocrine Society 1969, p. 143. 38. Han, P. W.: Obesity in force-fed, hypophysectomized rats bearing hypothalamic lesions. Proc. Sot. Exp. Biol. Med. 127: 10.57, 1968. 39. -: Energy metabolism of tube-fed hypophysectomized rats bearing hypothalamic lesions. Amer. J. Physiol. 215:1343, 1968.