Metabolic effects of hypothalamic hyperphagia

Metabolic

Effects

R. G. MacKenzie,

of Hypothalamic

R. Luboshitzky,

J. K. Goldman,

Hyperphagia and L. L. Bernardis

In order to test the hypothesis that the enhanced gluconeogenesis of hypothalamic obesity remains responsive to changes in food intake, we have measured gluconeogenesis in two models of hypothalamic obesity under both hyperphagic and normophagic conditions. The results show that hyperphagia partially decreases gluconeogenesis and fully restores liver glycogen in both models. The discussion section relates our present findings to the enhanced glucose utilization previously noted after VMH destruction and to the recent hypothesis that hyperphagia is a response to body protein depletion.

A

RECENT HYPOTHESIS states that the hyperphagia exhibited by certain rodent models of obesity can be viewed as a response to body protein depletion secondary to metabolic disturbances which divert nutrients to fat depots and away from protein synthetic pathways.’ Although this view has received experimental support only in the genetically obese Zucker rat, it has also been proposed to account for hypothalamic hyperphagia.’ Experiments from this laboratory have demonstrated increased in vivo and in vitro diversion of glucose into the fat pads of VMH-damaged rats.* In addition, we have recently demonstrated increased lipogenesis within 2 hr post-lesion followed by increased gluconeogenesis at 4-6 hr post-lesion.3 These data suggest that enhanced gluconeogenesis is required to maintain normoglycemia in the presence of accelerated glucose conversion to lipid. A chronic elevation in gluconeogenesis could serve as a sink for amino acids and eventually lower the level of tissue protein. In this regard, weanling male VMH rats exhibit decreased body protein content and increased urea production indicative of accelerated amino acid deamination.4,5 In the present work we studied two models of hypothalamic obesity in adult female rats under hyperphagic and normophagic conditions. Carcass and liver composition and gluconeogenic activity were measured to evaluate the effects of the two food intake levels. Two models were used in order to ascertain that the changes noted were not specific to one mode of hypothalamic manipulation. Our hypothesis was that gluconeogenesis is enhanced in hypothalamic obesity, but that it remains at least partially responsive to food intake and, therefore, can be attenuated or eliminated by hyperphagia with attendant increases in exogenous glucose supply.

MATERIALS

AND

METHODS

Animals Female

Sprague-Dawley

rats (Madison,

Wisconsin)

weighing

231 + 2.7 (mean + SE) grams were housed in a temperaturecontrolled room (23O C) with a 12 hr/day light cycle. The rats were habituated for one wk to the environment, and a powdered diet (Ralston Purina Lab Chow No. 5001) was available in a feeder

Metabolism, Vol. 30, No. 5 (May), 198 1

designed to keep spillage at a maximum of 0. I g/day.” Food intake and body weight measurements were begun one wk prior to surgery. Surgery was performed under sodium hexobarbital anesthesia (14 mg/lOO g body weight) and parasagittal knife cuts were stereotaxitally placed in 20 rats using a retractable wire knife as described by Gold et al.’ In control rats the needle of the microsyringe was lowered to 8.0 mm below the cortex but the knife was not extended. Following the stereotaxic atlas of Koenig and Klippel’ the cuts were bilaterally placed 1.O mm from the midline, extending from A6.0 to A3.0 in the anterior-posterior plane and from the base of the brain dorsally for 3.0 mm. Knife-cut placement was verified by microscopic examination of cresyl-violet stained brain section sliced at 40 microns. All rats ate ad libitum during the first post-operative wk, and at the end of this period 14 of the knife-cut rats exhibited postoperative weight gains (41.2 2 5.5 g) and food intake (31.6 i- 1.5 g/day) which greatly differed from control values (body weight gain, 2.4 + 4.9 g; food intake, 17.4 f 1.I g/day). From this point, seven of the knife cut rats were fed 21 g/day then

(normal

for the remaining

for the next week and

of the experiment intake obese). This regimen was employed to normalize the

18 g/day

two weeks

food intake of this group. and possible “meal-feeding”

effects were

minimized by feeding ‘/j of the daily portion every 8 hr.’ In the second

experiment,

electrolytic

lesions of the VMH

were

placed according to previously described methods’” in seven female

Sprague-Dawley rats weighing approximately 230 grams. In five control rats of similar weight the electrodes were lowered just dorsal to the VMH without current Row. Lesion placement was verified as above. Body weight and food (powdered chow) intake were monitored on post-operative days l-14 and 21-28. At the end of the first two wk. four VMH rats exhibited body weight gains (45.25 + 9.0) and food intake (26.5 f 2.6 g/day) that greatly exceeded control values (body weight gains. 8.6 + 5.4; food intake. 18.3 + 0.5 g/day).

Gluconeogenesis We studied gluconeogenesis by injecting rats with NaH’%XI;and measuring the incorporation

incorporation

of 14C into glucose and glycogen. The

occurs as CO, is fixed in the conversion of pyruvate to

From the Departments of Medicine and Surgery, Veterans Administration Medical Center, Buffalo. New York; State University of New York at Buflalo. Buffalo. New York. Received for publication August 5. 1980.

Supported by Veterans Administration Merit Review Research Funds. National Science Foundation Grant #PCM76 84381 and National Institutes of Health Grant #I F32 NSO6Ol Y. Address reprint requests to Dr. Jack K. Goldman. VA Medical Center, 3495 Bailey Avenue, Buffalo, New York 14215. G 1981 by Grune & Stratton. Inc. 0026&O495/8I/3005-0014$01.00/0 493

MACKENZIE ET AL.

494

OP

phosphoenolpyruvate. The intraperitoneal injections consisted of 9 pCi/lOO g body weight of NaH14C0, (New England Nuclear, Boston, MA; 6.2 mCi/mole) dissolved in Krebs Ringer phosphate buffer (60 NCi/ml). All rats were without food for at least three hr prior to the injections to assure that glucose absorption from the gut was minimal and steady state conditions existed. Blood was collected 30 min later in chilled, capped tubes containing sodium fluoride and heparin and spun at 4O C to obtain plasma. The specific activities of plasma bicarbonate, glucose and liver glycogen were determined as previously described.’ Blood samples were obtained from the tip of the tail when gluconeogenesis was measured in VMH rats during the hyperphagic phase and all other samples were from the trunk of rats decapitated 30 minutes after NaH?OI injections.

Carcass analysis In this study, “carcass” was prepared by removal of the head, paws, tail, skin, liver, and gut. The omental fat was stripped from the intestines and included with the carcass. The carcasses were weighed, chopped into several pieces, autoclaved for one hour and brought to a final volume of 400 ml by the addition of H,O containing 0. I% detergent (Manoxol OT, BDG, Poole, England) and 0.2% antifoam agent (Antifoam E, Leverkusen-Bayerwerke, Germany). The carcasses so treated were then homogenized in a Waring blender. Lipids were Folch-extracted” from a 2.0 ml aliquot of the homogenate and measured gravimetrically and carcass protein was determined by the Lowry technique.”

RESULTS

Knife Cut Experiment Figure 1 depicts the rapid weight gain due to increased post-operative food intake and the gradual body weight normalization of the normal-intake-obese group after hyperphagia was prevented. Table 1 shows that the carcass adiposity of knife cut rats on this regimen was greater than control rats but less than knife cut rats fed ad libitum. There were no group differences in either carcass proteins or liver lipid or protein percents. However, hyperphagia did result in

A

B

300 E E .cr, g 260 _ ~ c c% 22oL

1 14 18 22

I 1 048

; -7

Fig. 1. Food intake pair-fed-obese (ml end SE; OP = operation; A B = pair-fed group put

, J 26

and body weight of normophagic-lean (0 1, hyperphagic-obese (0) rats. Bars indicate = pair-fed group put on 21 g/day regimen: on 18 g/day regimen.

increased liver size and, therefore, greater amounts of hepatic lipids and protein on a per organ basis. Figure 2 demonstrates that the hyperphagia of knife-cut obese rats attenuated changes in gluconeogenesis and normalized hepatic glycogen levels. Gluconeogenesis, as measured by the incorporation of CO, into plasma glucose, increased 114% in the normal-intake-obese group compared to only 36% in the hyperphagic-obese group. Gluconeogenesis, as measured by the incorporation of CO2 in liver glyco-

Table 1. Carcass and Liver Composition

of Knife-cut

and Control Rats Group Differences

Control (A) Wet

carcass

weight

(g)

g Lipids/carcass

Pair-fed obese 181

* 3.8

148.8

7.8 i 0.7

11.9

148.7

+ 2.5

Hyperphagic-obese

164.3

(C)

F value

+ 5.6

31.6 88.5 26.8

A vs C

B vs C

p<.o1

p < .Ol

p < .Ol

p<.Ol

p < .Ol

p < .05

p < .Ol

p < .05 NS

NS

1.2

16.8

k

Carcass, % lipid

5.25

+ 0.4

8.0

+ 0.8

10.2

+ 0.9

g Protein/carcass

17.4 + 0.4

17.4

k 0.6

8.52

+ 1.4

0.8

NS

NS

Carcass, % protein

11.7 2 0.4

11.7

k 0.6

11.3

+

1.0

0.3

NS

NS

7.5 k 0.3

6.7

+ 0.2

8.5

+ 0.4

15.6

NS

p < .05

pi.01

638

+ 42

6.8

NS

p < .05

p < .05

9.5

+ 0.6

9.6

0.4

NS

NS

k 73

1,749

11.8

NS

Wet liver weight (g) mg lipids/liver Liver, % lipid mg protein/liver Liver, % protein

700 k 55 9.3 k 0.8 1,442

+ 86

19.2 + 1.1

1,406 21.0

t

*

Values mean k SE. NS not statistically significant. F values determined by one-way ANOVA. Differences between means determined by Neuman-Keuls Test. n 7 rats per group.

1.2

1.9

A vs B

816+51

20.8

+ 0.5 t

80

k 0.9

1.75

NS

p i

.05 NS

NS

NS p < .05 NS

GLUCONEOGENESIS

IN HYPOTHALAMIC

ug glycogen/mg -NW 0000

OBESITY

liver

495

2 Wks POST OP

glycemia mM V

0

P

4 Wks POST OP

0

NS

PEq CO, incorporated per pg liver glycogen rUul4 oulocn

Fig.

2.

Gluconeogenesis

in controls

(stippled

n

pair-fad-obese (vertical-lined from

dividing

plasma indicate

CO,.

column. DPM/pg Each

differences

Neuman-Keuls

column. n =

7) rats.

glucose

column

r1

NEq CO2 incorporated per flmole plasma glucose

=

column.

groups

the

of CO,

by specific means

as

n

=

=7

hyperphagic-obese

Incorporation

or glycogen

represents

between

(open

7) and

T

? SE.

determined

derived

activity

of

Brackets by

the

test.

500% in normal-intake-obese rats gen. increased versus 108% in hyperphagic-obese rats. The 60% decrement of hepatic glycogen levels in the normalintake group was not due to an imposed fast brought about by the timing of the feeding schedule. In the period 16-8 hr prior to food removal, normophagiclean rats ate 8.7 + 0.9 grams and normal-intake-obese rats ate 5.85 I 0.1 grams. In the subsequent 8 hr period the former group consumed 5.7 k 1.O grams and the latter group again ate 5.85 + 0.1 grams.

Fig. 3. VMN phagic the liver

VMH-lesion

Experiment

Figure 3 indicates that 2 wk after the operation the VM l-l rats exhibited hyperphagia, accelerated body weight gain and a 32% increase in gluconeogenesis as measured by the incorporation of 14C into plasma glucose. Four wk after lesioning the same rats were obese as shown in Table 2. Figure 3 shows that when the hyperphagia had abated at 4 wk gluconeogenesis was more accelerated as measured by the incorporation of 14C into plasma glucose (14 1%). Liver glycogen labelling, measured only at 4 wk, was increased 60%.

rats

Gluconeogenesis [stippled

post-operative

incorporation glycogen

Columns between

which

means

(open

n = 4) during

phases.

of “C

represent

in control

column.

Gluconeogenesis

from are

means

as determined

NaH”CO;

isolated +

SE.

column,

hyperphagic into

as

described

Brackets

by Students

was plasma indicate

n = 5) and and normomeasured glucose in

by and

Methods. differences

t tests.

DISCUSSION

In the present studies restriction of food intake to control rat levels causes a 50% decrease in liver glycogen of adult female rats with knife-cut hypothalamic obesity whereas hyperphagia permits these rats to maintain normal liver glycogen. Similarly, our results show that these rats exhibit enhanced gluconeogenesis in the presence of normophagia but not where hyperphagia is permitted to occur. The present

496

MACKENZIE ET AL.

Table 2.

Carcass Composition

and Substrate

Levels in VMH and

Control Rats Four Wk Postoperatively VMH (4)

Control(5) Wet carcass wt (g) Carcass, % lipid

146.7

+ 4.7

152.9

5.3 + 0.2

+ 9.8

7.7 t 1.0’

Carcass, % protein

7.7 * 0.7

7.9 + 0.3

Glycemia, mM

6.8 + 0.1

6.8 * 0.1

pg glycogen/mg liver

16.0 + 2.3

18.4 f

1.1

Values mean ? SEM. Differences between means determined by Student t test. *Significant difference between means, p < .Ol; all other comparisons between means do not attain statistical significance.

data also show that adult female rats with electrolytic VMH lesions have increased gluconeogenesis even in the presence of hyperphagia and that this process is even more accelerated after 2 wk of spontaneous normophagia. These findings are compatible with increased rates of glucose utilization in these rats with a consequent need for accelerated gluconeogenesis to maintain normoglycemia as had been previously postulated in weanling male rats with electrolytic VMH lesions.3 We assume that the increased glucose utilization is due primarily to the hyperinsulinemia of VMHdamaged ratsI since chronic hyperinsulinemia stimulates adipocyte transport and metabolism of glucose.‘4 We interpret the present findings to support the hypothesis that hypothalamic obesity is associated with increased gluconeogenesis and that this accelera-

tion can be partially or completely reversed by hyperphagia. The data do not explain why the hyperphagia is transient or why it is not even more marked so as to fully block the need for increased gluconeogenesis after electrolytic lesions. Perhaps this method of lesion production produces other more primary enhancing effects on gluconeogenesis.‘5 Unlike Zucker rats’ and weanling male VMH ratsi the adult female rats in the present study do not exhibit decreased carcass protein after VMH damage. This discrepancy may be due to sex, age, or strain differences. Regardless of the reason though, the present data do not support the hypothesis that hypothalamic hyperphagia is solely a response to body protein depletion. Finally, we have observed that hyperphagia normalizes liver glycogen content in knife-cut rats, and in electrolytic-lesioned rats normophagia is seen with normal liver glycogen levels. However, further studies will be needed to determine whether hepatic glycogen levels contribute to the level of food intake in hypothalamic obesity. ACKNOWLEDGMENT We would like to thank Marjorie Kodis and Geraldine McEwen for excellent technical assistance and Renee Chase who typed the manuscript. We would like to thank Dr. Mark Kristal, Department of Psychology, for the use of a stereotaxic instrument.

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on adipose tissue metabolism in the rat. Can J Physiol Pharmacol 43:857-868, 1972 IO. Frohman LA, Bernardis LL: Growth hormone and insulin levels in weanling rats with ventromedial hypothalamic lesions. Endocrinol82: I 125-l 132, 1968 I I. Folch J, Lees M, Sloane-Stanley GH: A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem 226:497-509, 1957 12. Lowry OH, Rosebrough NJ, Farr AL, et al: Protein measurement with the Folin phenol reagent. J Biol Chem 193:265-275, 1951 13. Han PW, Frohman LA: Hyperinsulinemia in tube-fed hypophysectomized rats bearing hypothalamic lesions. Am J Physiol 219:1632-1636, 1970 14. Kobayashi M, Olefsky JM: Effect of experimental hyperinsulinemia on intracellular glucose metabolism of isolated adipocytes. Diabetologia 17:111-I 16. 1979 15. Shimazu T: Nervous control of peripheral metabolism. Acta Physiol Polon 3O:Suppl. 18:1I18, 1979 16. Goldman JK, Bernardis LL: Protein metabolism in weanling rats with hypothalamic obesity. Proc Sot Exp Biol Med 15 1: 155159, 1976