Effect of caloric restriction on the glucose tolerance and plasma insulin of the sand rat

Effect of Caloric Restriction on the Glucose Tolerance and Plasma Insulin of the Sand Rat By DONALD

B. HACEEL, H. E. LEBOVITZ, L. A. FROHK~N,

E. MIKAT AND K. SCHMIDT-NIELSEN glucose tolerance. When these same animals were fed the synthetic diet ad libitum, they consumed about 49 calories per day, they gained weight, their plasma insulin became markedly elevated and their glucose tolerance decreased. A third group of sand rats was fed a synthetic diet in a similar manner except that the order of restricted-ad lib&urn feeding was reversed. The resulting alterations in plasma insulin and glucose tolerance in general followed the same relation to caloric intake as seen in the previous group. (Metabolism 16: No. 12, Decem-

Three groups of sand rats have been studied to determine the effects of restricted versus unrestricted caloric intake on the plasma insulin levels and glucose tolerance. One group of animals was fed throughout the experiment on an allvegetable diet of about 30 calories per day. They developed and, for the most part, maintained a low plasma insulin level and normal glucose tolerance. A second group of sand rats was fed a synthetic diet in restricted amounts so that the total caloric value averaged 30 per day. These animals lost weight, had a low plasma insulin level and a normal

ber, 1133-1139,

1967)

t has been shown that the glucose tolerance of the sand rat (Psammomys obestls) is modified by the type of food that it receives.’ Animals on an all vegetable diet remain relatively normal, while those fed Purina Laboratory Chow (or a similar synthetic diet) develop marked glucose intolerance and syndrome is reelevated plasma insulin levels.’ If this diabetes-mellitus-like lated primarily to the greater caloric content of the Chow or synthetic diet, it is likely that it can be reversed by a restriction of the available food. The present study was designed to evaluate this possibility by feeding groups of animals a standard synthetic diet either ad lib or in restricted amounts.

I

METHODS study, laboratory. From

Eight the Departments

North Carolina. Received for publication Dec. supported

nine

were

born

in seven Duke

1966. from Mary

and months L’niversity,

in this

Durham,

the U. S. Public He&h Biddle Foundation, from the U. S. Public Drs. Hackel (HE-KG-14, 188) and Schmidt-Nielsen (K6-GM-21, ,522) und a Cureer Development Awurd 954). DONALD B. HACHEL, hl.D.: Professor of Pathology, Duke University Medical Center, Durham, N.C. HAROLD E. LEUOVITZ, M.D.: Associate Professor of Medicine, Duke Uniuersit!y Medical Center, Durham, N.C. LAWRENCE A. FHOHMAN, M.D.: Assistant Professor of Medicine, State University of Medicine, Buffalo, N. Y. EILEEN M. MIKAT, A.B.: Research A&stunt, Duke University, Durham, N. C. KNUT SCHMIDT-NIELSEX, PH.D.: James B. Duke Professor of Physiology in the Department of Zoology. 1133

IIACKEL

Table 1 .-Outline

of Dietary

ET

.\L.

Groups

Weeks IL’1

one had been received 12 months before the start of the experiment; tvvo ot the laboratoryburn animals were seven months old and one was 11 months old. All animals had been maintained on a vegetable diet made up mainly of he& and beet leaves, although at timeb spinach and carrots were included. This was the regular maintenance diet of the colony. Because of its predominant beet content it vvas o f relatively high caloric value. Thus, two weeks before the first blood samples vvere tlravvn, all animals were put on a mixed vegetable diet of spinach, carrots and beet leaves ad lihitum. plrrs limitrtl amounts of beet roots. After the control blood samples were drawn the animals vvcre divided on an arbitrary hasis into three equal groups (Table 1). Group A continued to receive the control mixed vegetable diet ad lihitum. Groups B and C were both fed a synthetic diet, prepared as previously described,’ and containing de&in (5.3 per cent), casein (2.3 per cent), salt mixture (7 per cent), liver extract (3 per cent ), vitamin mixture (2.5 per cent ), corn oil (3.5 per cent ), and agur (8 pt‘r cent ). In addition, exh animal was given a daily supplement of 10 Gm. each of spinach, beets, beet greens, and carrots. For the first eight weeks of the experiment the ;mimals in Group B were restricted to 5 Cm. of the synthetic food, plus the 10 Gm. each of the different vegetables. This resulted in a daily caloric intake that rarely exceeded 31 calories. From the ninth week on, together with the limited ration of these rats were given the synthetic diet ad lihitum, mixed vegetables. The animals in Group C were given a similar dietary regime, hut iu reverse order. For the first eight weeks they were fed the synthetic diet ad Iihitum, and for the remaining 10 weeks they were restricted in their caloric intake. A glucose tolerance test (GTT) was performed on each animal prior to the start of the special dietary regimes, and at approximately two-week intervals thereafter. The tests were done from 9 A.hl. to 11 A.M., and all animals had access to food until the start of the test. Blood samples were obtained from the tail prior to and at 15, 30, 60 and 120 minute intervals after an intraperitoncnl glucose load of 1.5 Gm./Kg. Glucose was measured by a glucose oxidase method,” and plasma insulin by a rndioimmuno~~ss~ly method,’ using a sand rat pancreatic extract of insulin as it standard. In order to establish a broader baseline on which to interpret the normal range of glucost tolerance test values, a group of 19 additional normal sand rats wac evaluated. Each of these animals was fed a control mixed vegetable diet of spinach, carrots, and beets in amounts similar to that given to the Group A animals. After they had been on this diet for at least 7 weeks, a glucose tolerance test vvas done in the same manner as previously described ( Fig. 1). RESULTS At

the start of the experiment

a predominantly

beet

diet until

the sand rats, two weeks

all of which

previously,

had

had been abnormally

eating high

plasma insulin levels ( mean =261+80 ,LU/ml. ). The initial blood glucose levels were normal (mean=6823 mg. per 100 ml.), but the gIucose tolerance was

1135

EFFECT OF C.4LORIC RESTRICTIOK

I50 GLUCOSE hg

%) 100

0

30

60 TIME

so

120

(min)

Fig. l.- Glucose tolerance c(lrve. Mean values found in 19 normal sand rats. ‘The mean values are represented by the circles connected by solid lines. The shaded are:1 indicates twice the standard deviation above the nle;ul. decreased, glucose

with

the

load being

After

the

standard

mean 1212

animals

mixed

glucose

level

two

hours

in Group

vegetable

A were

regime

switched

there

drawn

2 weeks

diet still showed the plasma in Table (compare same

time,

from

(upper

mained

4. There

values

was gradual

mean

level

the limits

weight

(Table

insulin

of normal

During

this

beet

diet

decrease (Fig.

to normal

2);

the blood (low

loss of weight

increased

to slightly

50 ,.cU/ml. ). The Table

significance

(P>

lo-week

was not as great as in the first eight weeks,

above glucose

2 and Fig. .07)

period,

tolerance 1). At the

averaged

In the second

(compare latter

indicated

in Fig. which

beet)

2 to 4 weeks

of glucose

tests shown

to the

in plasma

vegetable

after another

of the experiment.

of borderline

2).

intraperitoneal

at the low concentrations

and marked

limit of normal=about

within

However,

2 with normal

weeks

plasma

there was an increase cose

insulin.

was also a return

in Table

the

to occur

to the control

levels were maintained

there the

the switch

plasma

in the first eight

10 weeks range

insulin mean

per cent

after

elevated

2 and Fig.

the

was a significant

insulin levels. It took 4 to 6 weeks for this decrease samples

after

14 mg. per 100 ml.

the

of

normal

tolerance

re-

1 ), although

in the two-hour the rate

amounting

23

period

of

glu-

10s~

to an average

of

of

7 per cent. The Group

glucose B

tolerance

changed

diet in restricted

amounts

ation of the low calorie to normal, and their caloric intake during caloric

intake

tests

and plasma

dramatically (Table synthetic

insulin

levels

of the

sand

rats

in

when they were switched to a synthetic 2 and Fig. 3). Within four weeks of the initidiet their plasma

insulin

levels had dropped

two-hour glucose tolerance became normal. The daily this period was 29.3, which is almost identical to the

of animaIs

on the

mixed

vegetable

diet,

and

the

weight

loss

1136

HACREL

VEGETABLES

200

2

4

6

ET AL.

PS-78

IO

I2

14

16

W&s

Fig. Z.-Findings in a sand rat fed a mixed vegetable diet (Croup A). The main findings at two-week intervals in one animal selected from each group are given. (See also Figs. 3 and 4.) The top curve (circles) represents the vnlues for blood glucose obtained two hours after the glucose load; 100 mg. per 100 ml. is considered the upper limit of normal. The middle group of figures (triangles) indicates the plasma insulin values from blood samples obtained prior to the glucose load. The louver set of figures (squares) gives the values for averagi body weight per week. __

Table 2.--Summary .~~~~_

of results in animals fed different diets ___ (;luco.ir

~~~

t mfr.r; )

~.

~~

\\‘eigllr

(‘ll:lll~r

EFFECT

OF CALORIC

1137

RESTRICTlOK

--

RESTR.

AD LIB

PS-87

G-

i 250-c”

Weeks

Fig. 3.-Sand rat from Croup B, fed a diet restricted in its caloric content eight weeks. followed by the same diet ad libitum. (See legend under Fig. 2.)

fog

during this time averaged 14 per cent When these same animals were switched to synthetic diet ad libitum, three of the four animals markedly increased their food intake to a mean of 49.4 calories per day. Since the fourth animal did not voluntarily increase its ingestion of checkers, and continued to lose weight, it does not properly fit into this “high calorie” group and is omitted from the mean values. It is interesting to note, however, that its insulin level remained low and its glucose tolerance stayed relatively normal the remainder of the experiment. In contrast, the other three animals in this group, whose caloric intake was high (49.4 calories a day) and who gained a large amount of weight ( +28 per cent ), showed a marked and significant rise in plasma insulin. They also showed a slight but definite decrease in glucose tolerance. It is seen by a comparison of Table 2 and Fig. 1 that the two-hour glucose level is beyond 2 times the standard deviation of the normal series. The findings in the animals in Group C were generally the same as those in Group R. although occurring at the alternate time period (Table 2 and Fig. 4). As in Group B, one of the four animals did not eat the ad libitum diet in enough volume to be considered in the “high calorie” group. This one animal, which is excluded from the mean figures, did not show the insulin elevations or abnormal glucose tolerance that was found in the rest of the group. A second animal developed a markedly abnormal glucose tolerance, its plasma insulin remained high, and it died within two weeks. The other animals showed a marked and highljr significant increase in mean plasma insulin, and markedly decreased glucose tolerance. During this period the food intake averaged 39.6 calories per day and there was a gradual increase in weight. After the eighth week, when the diet was switched to a restricted one. the mean daily caloric intake decreased to 27.9 and there was a mean weight

1138

zs!-[ 0

2

Fig. 4.--Sand rat frmn the same diet in restricted

Group

4

6

0 Weeks

IO

(1, fed ;,(Ilibitrlm

amounts.

12

for

14

eight

16

\\cel\s.

;llltl

tllf?ll

giVeI

(See legencl lulcler Fig. 2.1

loss of 16 percent. The mean plasma insulin kvel decreased significantly coinpared to the preceding period of ad libitum food intake, but the kvel did not reach the same lo~v mean as seen in Group I3 during the period of caloric restriction. The glucose tolerance reacted similarly, changing in the direction of normal, ante

but not responding

in Group

as markedly

or as lmiformly

as the glllc~e

tolcAr-

A.

A striking correlation was seen between caloric intake and plasma insulin When the synthetic diet was given ad libitum, the animals gained weight, their glucose tolerance decreased, and their plasma insulin lcvrls were clrvated. This is similar to previous observations in this ” and other z laboratories. In addition, however, these changes were reversed by wstricting the available food supply. These findings are consistent with the concept that the sand rat has evolved with its metabolic processes geared to a natural diet which is barely adecluate in amount. In this situation a diabetes-like condition would tend to conserve the available glucose, thus safeguarding the nervous system which is depe~~dent on glucose as a food. Diabetes mellitus may become manifest in hnman populations which shift from a bare subsistence level to sudden affiuencc~.” Similarly the incipient metabolic abnormality in the sand rat seems to aplwar and regress in direct relation to the caloric content of the diet. level.

It should pattern on

example, synthetic

be noted, a restricted

however, caloric

that the reversal intake was not

toward

a normal

complete.

restriction of calories after the animals had diet for nine weeks did not result in complete

metabolic

111 Grotlp

C, for

bee11 011 an ad lil)itllm return

of (lither

plasma

EFFECT

OF CALORIC

1139

RESTRICTION

inslllin or of the glucose tolerance test to the original normal level. If calories had been restricted for a longer period of time this might have been accomplished. On the other hand, it could have been a reflection of a progressively increasing insulin resistance with age, as suggested by the increase of borderline significance in plasma insulin in Group A animals (on vegetable diet throughout experiment) during the second nine weeks of the study. In keeping with this possibility are the recent findings of DeFronzo et al.’ who observed decreasing insulin responsiveness of sand rat peripheral tissues with increasing age. The finding of high plasma insulin levels in sand rats on a high calorie diet may have some similarities to observations by Karam et aLx in obese human subjects. They observed that obesity was associated with peripheral antagonism to glucose metabolism that induces compensatory overproduction of insulin. and they reasoned that carbohydrate tolerance would eventually become abnormal in such persons if the antagonism should increase beyond the ability of the islets to compensate. A similar initial increased rate of insulin synthesis in sand rats on a high calorie diet may he followed by eventual exhaustion of the islets.“,” In the human as in the sand rat. weight reduction by calorie restriction resulted in reduction of plasma inslllin levels.” REFERENCES 1. Haines,

2.

3.

4.

H. B., Hackel, D. B., and Schmidt-Nielsen, K. : Experimental diabetes mellitus induced by diet in the sand rat. Amer. J. Physiol. 208: 297. 1965. Hackel, D. B., Frohman, L., Mikat, E., Lebovitz, H. E., Schmidt-Nielsen, K., and Kinney, T. D.: Effect of diet on the glucose tolerance and plasma insulin levels of the sand rat. Diabetes

5.

6.

7.

15:105, 1966. Washko, hl. E., and Rice, E. W.: Determination of glucose by an improved enzymatic proceclurta. Clin. Chem. 7:542, 1961. Genuth, S., Frohman, L. A., and Lebovitz, H. E.: A radioimmunologic assay method for insulin using Insulin-‘1 and gel filtration. J. Clin. Endocr. 25: 1043, 1965.

8.

Miki, E., Likca, A. A., Soeldner, J S., Steinke, J., and Cahill, C. F.: Acute ketotic-type diabetic hyndromr in sand rats (Psammomys ohesus) with reference to the pancreas. special Xletaholism 15:749, 1966. Cohen, A. M.: Prevalence of diabetes among different ethnic Jewish groups in Israel. Metabolism 10:50, 1961. DeFronzo, K., hliki, E.. and Stcinkr, J.: Diabetic syndrome in sand rats. III. Observations on adiposr tissue and liver in the non-diabetic stage. Diah&)lopia :3: 140, 1967. Karam, J. H., Groclsky, G. hl., and Forsham, P. H.: The relationship of obesity and growth hormone to serum insulin levels. Ann. N\‘. Y. Acad. Sci. 131:374. 1965.