american journal of physiology 1980 239 3 r344-51

239:R344-R351, 1980. ;Am J Physiol Regul Integr Comp Physiol Y. Nishizawa and G. A. Brayparabiotic ratsEvidence for a circulating ergostatic factor: studies on

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Evidence for a circulating ergostatic factor: studies on parabiotic rats

Y. NISHIZAWA AND G. A. BRAY Harbor General Hospital- UCLA School of Medicine, Torrance, California 90509

NISHIZAWA, Y., AND G. A. BRAY. Evidence for a circulating ergostatic factor: studies on parabiotic rats. Am. J. Physiol. 239 (Regulatory Integrative Comp. Physiol. 8): R344-Rt$il, 1980.-The effect of electrolytic ventromedial hypothalamic lesions or forced overfeeding through gastric cannulas was studied in parabiotic rats. In one experiment a ventromedial hypothalamic lesion in one member of the parabiotic union was associated with an increased fatness in the lesioned rat and a decrease in fat pad weight and a rise in body density (loss of fat) of the unlesioned animal. In the other two experiments a high fat or high carbohydrate diet was tube fed to one member of a parabiotic union. The average preinfusion intake per rat was 56 kcal/day. When 90 kcal/day were given through the intragastric cannula, oral intake of calories for the two animals fell to approximately 44 kcal/day. Body fat of the non-tube-fed animal was less than in the tube-fed controls. These studies suggest the presence of an ergostatic (energy stabilizing) factor that enters and is transported in the blood with passage from one animal to another.

hypothalamic obesity; forced-feeding; weight gain; high fat diet; body density; high carbohydrate diet; hormones

IN 1959, Hervey (13) demonstrated that a ventromedial hypothalamic (VMH) lesion in one member of a parabiotic union of two rats was accompanied by weight gain and obesity in the lesioned animal and by a decrease in body fat in the unlesioned animals. This study suggested the presence of a circulating lipostatic (ergostatic) factor involved in regulation of body fat. However, doubts were cast on Hervey’s experiments because Han and his col- leagues (12) and Flemming (11) could not reproduce this observation. These latter two studies used considerably heavier animals and the studies lasted for a shorter period of time. Indeed, careful examination of Flem- ming’s data indicates that his unlesioned rats may have become leaner.

Support for a lipostatic factor has recently come from Parameswaran et al. (19), who reported that electrical stimulation of the lateral hypothalamus in one member of two parabiotic rats produced increased feeding in that animal. The fat pads in the stimulated animals were heavier, and there was a reduction in the weight of the fat pad in the other animal. The concentrations of glucose, glucagon, and insulin were also lower in the unstim- ulated animal, eliminating them as contenders for the “lipostatic” factor. Because all of the experiments described above utilized rats in which hypothalamic func- tion had been disturbed, we undertook a study to evalu-

ate the effects of forced feeding using a similar experimental design with parabiotic animals.

MATERIALS AND METHODS

AnimaZs. The animals used in these studies were fe- male albino rats purchased from Charles River Labora- tories (Wilmington, MA).

Parabiosis. A lateral incision was made from the fore- leg to the gluteal muscle along adjacent sides of each animal. The adjoining muscles on the lateral side of the peritoneum of two animals were then divided and the abdominal muscles on the right side of one animal were sutured to the abdominal muscles on the left side of the other animal such that peritoneal surfaces were con- nected. Ligatures were placed around the right and left clavicles, and a strong ligature was placed between the gluteal muscles so that the animals could not pull them- selves apart. They then were sutured skin-to-skin with ventral and dorsal skin opposed. Following surgery, which was performed on animals weighing less than 100 g each, the animals were allowed to recover and gain weight. Pairs were used when they reached 440-550 g.

Diets. Both before surgery and postoperatively, the animals were given ad libitum access l to Purina labora tory chow (Ralston Purina, St. Louis, MO). This diet was continued for the animals with electrolytic lesions. For tube feeding, two formula diets with the following com- position were used.

Caloric Content High Fat High Car- bohydrate

Dried milk powder (Carnation) Lipid emulsion (Lipomul), ml Corn syrup (Ka.d, g begs, g Vitamins (Polyvisol) , ml Diluted to k&/ml

g 3.5 k&g 6.0 kcal/ml 3.0 kWg 4.2 kWg

28.8 21.0

1.0 180 ml

1.26

28.8 1.5

19.2 14.2

1.0 180 ml

1.26

One of these diets, or an equivalent volume of 0.15 M NaCl, was fed through the stomach tube five times a day.

Experimental procedures. Pairs of rats weighing between 440 and 550 g were used for these studies. Hypo- thalamic lesions were produced in one member *of the parabiotic union by passing an anodal current of 2 mA for 15 s, using coordinates and techniques described previously (14, 18).

R344 0363-6119/80/0000-0000$01.25 Copyright 0 1980 the American Physiological Society

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PARABIOTIC RATS R345

Cross circulation was determined by the intravenous injection of Evans Blue dye (T-1824). The concentration of dye was measured in each animal at 30.min intervals between 30 and 120 min after injection (19).

(Fig. 1) (h/2 = 115 min). This is comparable to a transfer rate of 0.723 h-’ and suggest that approximately 10 vol of blood are exchanged per day (19).

Intragastric intubation of nutrients was performed in two experiments. In the first, the Silastic catheter was inserted into the stomach and sutured in place. It was then led out through the back muscles and passed sub- cutaneously to a needle located on the back of the skull and mounted with dental cement, which was held in place by screws in the skull. With this procedure, a syringe load of liquid diet could be injected into the stomach through the needle on the back of the skull.

For the second experiment with intragastric intubation of nutrients, the Silastic tubing was notched in four places and passed into the stomach and then through the pylorus into the duodenum such that the tip of the catheter rested in the duodenum. The more proximal notches in the tube allowed passage of liquid diet into the stomach and the more distal ones into the intestine.

At the end of the second experiment, with intragastric intubation of nutrients, an acute load of liquid diet was given by intragastric injection into both the saline- treated animals and into the chronically treated rats. Half the saline-treated rats received a 30.kcal load of the high carbohydrate liquid diet and the other half the high fat liquid diet. The chronically fed rats received a 30.kcal load of the diet with which they had been fed previously.

Experiment 2. Ventromedial hypothalamic lesions in one member of six parabiotic pairs were followed by an increase in the combined weight of the animals and by a rise in food intake that occurred primarily during the daylight hours (6,15) (Fig. 2). During the 9 days following the hypothalamic lesion, there was a weight gain of approximately 60 g. Organ weights for these animals are summarized in Table 1. The combined weight of the lesioned animals was 121 g (or 29%) above that of the control animals. The fat pad weights were identical in the sham-operated pair (Fig. 3). In the lesioned animal, on the other hand, the mean fat pad weight was 3.5 times heavier than in the sham-operated control animals. In the unlesioned member of the experimental pair, the fat pad was significantly lighter than in the control pairs (P < 0.01). The salivary glands of the VMH-lesioned animals were significantly smaller (P < 0.05) than in the unlesioned parabiotic mate, but were similar in the sham- lesioned group (16).

Samples of blood were obtained through polyethylene catheters inserted into the jugular vein on each animal for the 5 h following the intragastric tube feeding.

Body weight was measured weekly until the time of the experiment and then three times weekly for the remainder of the study. At death, the body weight of both animals of each parabiosed pair was obtained before and after separation. The weights of the perimetrial and retroperitoneal fat pad, the liver, salivary glands, uterus, and pituitary were recorded. Body fat was determined by use of the Lee index (1)

Experiment 3. In this experiment, the intragastric liquid diet entered the stomach through a single opening in the end of a Silastic catheter. Two pairs of animals that died were found to have large distended stomachs. For this reason, the experiment was terminated after 10 days. The weight of the fat pads are shown in Fig. 4. The weight of the fat pads in animals infused with either diet was significantly heavier than in the saline-treated controls. The weight of fat pads from the saline-intubated member of each parabiotic union was similar to those in non-tube-fed partners. The fat pad from the rats intu-

where BW = body weight in g, and Z = nasal-anal length in cm, or from measurements of body density (10). The gut and lungs were removed from the decapitated carcass. Hair was removed with a depilatory (Nair). The carcass was then weighed in and out of water. The density was used to obtain body fat from a regression of density and fat (W. T. Dahms, unpublished data). Glu- cose was determined by the glucose oxidase method and free fatty acids by titration (9). Insulin was determined by double antibody radioimmunoassay (17) using rat insulin standards; triglycerides were determined by a commercial kit (Dade and Co.) and glycerol by the glycerol dehydrogenase method of Davidson and Karjala (8). Evans Blue dye was measured spectrophotometricalIy on alkalinized deproteinized serum (19).

RESULTS

100 i I I

0 30 60 90 120 TIME (Minutes)

I I

180

Experiment 1. After injection of Evans Blue dye into one member of two parabiotic rats, equilibrium between the parabiotic animals was nearly complete by 120 min

FIG. 1. Transfer between parabiotic rats. Evans Blue dye was injected into 1 animal and concentration in serum of each animal measured for 2 h. Although equilibrium had not quite been reached, estimate of t1,2 is 115 min.

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R346 Y. NISHIZAWA AND G. A. BRAY

520 0 Doytlme

q Nlghtime

-0OD (gm)

40

1 FIG. 2. Weight gain after VMH lesion in parabiotic rata. One animal of each pair was lesioned bilaterally in ventromedial hypothalamus after recording food intake daily for 8 days. After lesion, rate of weight increased for pair, and this was result of food intake primarily during day.

420

12 TIME (Day)

TABLE 1. Effect of a VMH lesion in one member of a parabiotic pair of rats

Experimental

VMH Sham VMH

Control

Sham

Combined wt, g Density Fat tissue

w g % Body wt

Liver, g Salivary glands, g Uterus, g Pituitary, mg Glucose, mg/dl FFA. ueo/l

(n = 6) 539 + 11

1.0569 + 0.0084 1.0980 * 0.0027

9.7 f 1.91 1.80 f 0.50 7.18 + 0.50 0.40 + 0.018 0.59 + 0.08 10.5 -e 0.51

147.8 + 11.3 409*59

1.08 zk 0.18 0.20 + 0.03 7.20 + 0.90 0.46 + 0.018 0.76 1- 0.06 10.8 + 0.27

133.1 f 15.8 457 + 42

(n = 4) 418 + 19

1.0866 + 0.0018 1.0822 f 0.0018

2.75 + 0.17 0.65 f 0.26 5.99 + 0.61 0.38 + 0.01 0.49 f 0.02 11.8 + 1.0

126.0 + 13.6 502 + 50

2.78 +- 0.26 0.66 + 0.21 5.69 f 0.32 0.41 f 0.02 0.54 f 0.10 10.5 f 0.53

123.1 -+: 13.3 520 f 22

1.0800-

1.0600-

[ SHAM SHAM VMH SHAM

FIG. 3. Fat pads and body density after VMH lesions. Perimetrial fat pads were significantly lighter in unlesioned sham-operated animal after VMH lesion in other animal. Body density increased, indicating that total fat had declined.

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6-- cl TUBE FED

Ezl NON-FED PARTNER

Fat Carb Saline FIG. 4. Retroperitoneal fat pad weights of tube-fed rats. Effects of

overfeeding 1 animal on fat pad weights of the overfed and ad lib. fed animal are shown. Both high carbohydrate and high fat diet seemed to lower weight of fat pads in unfed animal in expt 3.

FOOD INTAKE

3c

bated with the high carbohydrate diet and the high fat diet weighed significantly less than in the animals intubated with saline. The data also suggest a greater reduction in fat pad weight in the animals intubated with the high fat diet.

Experiment 4. In the final experiment, the intragastric tube was passed into the duodenum and had perforations along the section that passes through the stomach. Food intake was combined for each pair and expressed as grams per day for each dietary group (Fig. 5). Prior to initiation of tube feeding, the parabiotic animals ate 27- 30 g of solid food (approx 115 kcal/day) per pair of animals. During intragastric intubation of either the high carbohydrate or high fat diet, there was a reduction in combined oral intake of solid food, which by the end of the 2nd day almost completely matched the quantity of calories given by stomach tube to one animal. In contrast, the animals receiving intragastric saline showed no sig- nificant changes in their intake of solid food, which remained essentially constant throughout. When caloric intake by stomach tube was increased to 90 kcal/day the combined intake of solid food by the parabiotic animals fell further and ranged between 10 and 13 g/day or approximately 40-56 kcal/day. On many days, it was significantly below half that of the animals intubated with saline. Both diets produced a comparable reduction in food intake. By the end of the experiment, the pairs of rats were ingesting a total caloric intake that was ap-

FIG. 5. Food intake of rats in expt 4. Tube feeding saline had no animal. When total energy fed to 1 animal was 90 kcal/day, combined effect on food intake of parabiotic pair. However, both high carbohy- intake rose to 42 g/day (128 kca.l/day). drate and high fat diets produced a decline in food intake of ad lib. fed

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R348

BW

g

Forced Tubed Feeding

CONTROL STAGE ad lib tube- feeding / \.I /\ /\

Y. NISHIZAWA AND G. A. BRAY

,

0

0

I

0

-e - -

f

f

, ; ; I - :

/

f

/

f

. - l I

f

/ * /

0-c 8’ / 0’ t

0 / 0 . ”

.’

. L.

0’ 8.

4

T T T

A I .

0 1 2 3 4 5 6 wks

FIG. 6. Weight gain of tube-fed rats. When tube feeding surpassed rate of gain in carbohydrate fed animals (HF, high fat; HC, high 6 kcal/day, body weight of pair of animals rose with a slightly higher carbohydrate).

El FED SIDE

El i$i NON- FED SIDE

HFD HCD HFD

3 BWt ------+X1( NA- length

HCD CONTROL

. 250

HFD HCD CONTROL

FIG. 7. Fat pad weight and Lee index of animals in expt 4. At end of smaller than in saline-fed control animals whose fat pads had same 14 days of overfeeding, fat pads of overfed rats were significantly weight. Changes in Lee index of animals showed same changes (HFD, heavier and those of nonfed rat in parabiotic union were significantly high fat diet; HCD, high carbohydrate diet).

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proximately 15 kcal/day higher than the total intake when they were allowed to eat ad libitum at the beginning of the experiment. This was accompanied by an increase in body weight of .5 g in 4 days for the rats fed the high carbohydrate liquid diet and 5 g in 6 days for rats given the high fat liquid diet (Fig. 6). Both diets produced an increase in the weight of the fat pads in the tube-fed animals and a reduction in the weight of the fat pads in the unfed animals relative to the saline-fed controls (Fig. 7). In the animals receiving intragastric saline, fat pad weights were just under 3 g. In the animals tube fed the liquid diets, the decline in weight of the fat pads was greater with the high fat diet and was similar to the one noted in the previous experiment (Fig. 4).

On the final day of the experim .ent, the fed partners received an intragastric me lal of the sam .e type and quantity that they had been fed chronically. The control (saline-fed) animals received intragastrically either the high carbohydrate or high fat diet in an amount equivalent to that given to the experimental animals. The changes in glucose in the fed and unfed animals are summarized in Figs. 8 and 9, and the saline-fed animals that received an acute caloric gavage for this study (Fig. 9) differed from the animals chronically gavaged with diet (Fig. 8). The basal glucose was higher and the rise smaller than in the chronically fed animals. Thirty minutes after tube feeding, the glucose level in rats given the high fat diet intragastrically was significantly lower in the fed animal. Only at 180 min after the acutely admin- istered high carbohydrate diet did the fed animals have significantly higher glucose values. In the chronically fed

animals, basal glucose was th .e same in both members of the carbohydrate fed group9 but different in the pairs chronically fed the high fat diet. After the acute load of the high fat diet, a sustained rise in glucose occurred in the fed animal with little change in the nonfed partner. In contrast, a peak occurred at 30-40 min in the carbohydrate-fed rat.

DISCUSSION

The present data with a VMH-lesioned rat parabiosed to a lean animal are consistent with those of Hervey (13) in showing that the VMH-lesioned animals had a higher body fat content (lower density) than the unlesioned animals, which were also less fat than the sham-operated controls. Thus, within 9 days after the VMH lesion, body density rose and the weight of the fat pads was reduced in the u nlesioned animal. The VMH-lesioned member of each pair also had smaller salivary glands, confirming earlier observations (16) that have been interpreted as indicating that VMH-lesioned rats have reduced activity of the sympathetic nervous system (5, 18).

The tube feeding experiments also support the concept of a circulating ergostatic or lipostatic factor. When caloric intake was provided to one animal by stomach tube, the food intake of the pair of animals was suppressed to less than 50% of the base-line level of food intake. Fat pad weights were smaller in the non -tube-fed animals, an indication that they were eating fewer calories than needed for maintenance of body fat stores. This effect occurred with both high carbohydrate and high fat diets. In these studies, i .n contrast to those using electrical

1 HF GROUP (fed-side)

FIG. 8. Changes in glucose in fed and nonfed animal after an acute load of diet.

Afikr 2 wk on M k&/day a shgle t&e- fed meal equal to % of total daily intake was given and blood was sampled from both animals for 180 min. There was a slow rise in glucose in nonfed animal. Glucose in high carbohydrate fed animals showed a rapid rise and return to base line. Glucose in high fat fed rats was

e-- 4 HC GROUP (fed-side) initially higher and continued to rise for

HC GROUP (non-fed side) 180 min.

e-4 HF GROUP (non-fed side)

1 I

0

I I I I I I 1 I I I I 1

60 120 180 TIME (minutes)

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R350 Y. NISHIZAWA AND G. A. BRAY

HC (fed)

I ! I 1 I I I 1 I I I I 1 I

0 60 120 180 200

1

TIME (minutes)

1 : I I I I I I I I I 1. I 1

0 60 120 180 TIME (minutes)

FIG. 9. Charges in glucose after an acute load of high carbohydrate (HC) or high fat (HF) diet in rats previously tube-fed saline. Glucose rose higher in HC fed rats than in the HF fed rats. These acute effects are in contrast to acute response in animals previously adapted to these diets (Fig. 8).

factor. The first is from the expanding fat depot. It is conceivable that the adipose tissue releases a factor(s) that modulates feeding (11). If this were the case, we would expect a gradual decline in food intake in the unfed-parabiosed animal. However, food intake dropped within the first 2 days and showed no further decline as the fat depots filled. This makes the fat organ less likely than the gastrointestinal tract or liver as a site for the ergostatic factor.

There are numerous gastrointestinal, hepatic, or pancreatic candidates for such an ergostatic factor (22). Because all of the extra calories provided in the experiments on tube- fed rats entered -the body through the gastrointestinal tract a gastrointestinal hormone is high on the list. Pancreatic factors released as a result of food ingestion are also possibilities. However, the low levels of insulin and glucagon in the unfed animals reported by Parameswaran et al. (19), and the effects of acute and chronic loading in the present experiments suggest nei- ther glucose nor these two pancreatic hormones are the circulating factor. Glycerol (5) would be a possibility since Wirtshafter and Davis (21) have recently shown that injections of glycerol can lower body weight in rats. Several other authors thave also suggested the possibility of circulating factors modulating feeding behavior, but as yet there is no unequivocal evidence for any of them. Smith and Gibbs (20) have proposed that cholecystokinin, a gastrointestinal hormone, might play this role. Other candidates might include bombesin (7), pancreatic polypeptide (3), glicentin (3), or neurotensin (2). The rate of transfer across the parabiotic union is about 1% of cardiac output or about 10 vol of blood per day. This means that the factor produced in the fed animals must have a long enough half-life to accumulate in the other animal. With a short half-life, for most peptides, none of them would seem to be candidates. Although the precise nature of this circulating ergostatic or lipostatic factor is unknown, its identification will add significantly to the understanding of the regulation of body fat.

stimulation or VMH lesions, no injury to the hypothalamus was produced. We thus concur with Hervey (13) and This research was supported in part by National Institutes of Health

Parameswaran et al. (19) that overfeeding one animal Grant AY-1516? leads to depression of food intake in the other via a

Address reprint requests to: G. A. Bray, Harbor-UCLA Medical c en t er, 1000 W. Carson St., Torrance, CA 90509.

humoral or ergostatic (energy stabilizing) mechanism. There are two likely sites for the origin of an ergostatic Received 13 August 1979; accepted in final form 10 March 1980.

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2. BLOOM, S. R. Gut hormones. Proc. Nutr. Sot. 37: 259-271,1978. 3. BRAY, G. A. Endocrine factors in the modulation of food intake.

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12. HAN, P. W., A.-C. LIU, AND S. LEPKOVSKY. Food intake of parabiotic rats. Am. J. Physiol. 205: 1139-1143, 1963.

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14. INOUE, S., AND G. A. BRAY. The effect of subdiaphragrnatic vagot- omy in rats with ventromedial hypothalamic obesity. EndocrinoZ- ogy 100: 10%114,1977.

15. INOUE, S., G. A. BRAY, AND Y. MULLEN. Transplantation of pancreatic beta-cells prevents the development of hypothalamic obe-

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RUITER. The involvement of a humoral factor in the regulation of body weight in parabiotic rats. Am. J. Physiol. 232 (Regulatory Integrative Comp. PhysioZ. 1): R150-157, 1977.

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21. WIRTSHAFTER, D., AND J. D. DAVIS. Body weight: reduction by long-term glycerol treatment. Science 195: 1271-1274, 1977.

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