Actions of Growth Hormone: Enhancement of Insulin Utilization with Inhibition of Insulin Effect on Blood Glucose in Dogs By JAMES CAMPBELL AND The disappearance of labeled immunore- active insulin (L-IIU) from blood serum was measured in dogs, after a trace dose of 1311-insulin, given intravenously, had intermixed in the body fluids. The degree of labeling of insulin was low, and the same preparation was used in all tests. The disappearance of L-IIU followed an exponential course, the mean value of the fractional rate constant (k) was 1.86 + 0.20 per cent min.-l. An integral method of analysis of the data gave similar values. Growth hormone (GH, 2 mg./Kg. of body weight/day) injected for 4 days, did not alter this fractional rate appreciably. However, the mass rate of irreversible loss of insulin was greatly increased (19- fold), due chiefly to elevation of en- dogenous immunoreactive insulin in “postabsorptive” serum. The enhanced rate of utilization of insulin, induced by GH, was thus dependent on, and coupled with increased secretion of insulin. Dur- KRISHNA SUDHA RASTOGI ing GH administration, serum gbrcose rose, but to a lesser degree than insulin: the IIWglucose ratio (23 microunits/mg. under the control conditions) rose, about lo-fold. Serum free fatty acid, and esterified fatty acid levels were both in- creased by GH. The responsiveness to insulin, within 2 hours of intravenous injection of 0.1 unit/Kg., was tested in these dogs. During GH treatment, the reduction in serum glucose was less rapid, but the sharp fall in free fatty acids was not prevented. Insulin did not affect esterified fatty acid levels, under both the control and GH treatment con- ditions, however. A hypothesis is out- lined for the effect of GH in diverting preferentially the utilization of the large amounts of insulin from processes of carbohydrate metabolism to those in- volved in protein synthesis. (Metabolism 18: No. 11, November, 930-944, 1969) T HE HYPERINSULINEMIA produced by the administration of growth hormone in the dog1J is associated with enhanced secretion of insulin.3 The action of growth hormone on insulin utilization in depancreatized and Houssay dogs,4 indicated that growth hormone could be expected to increase greatly the flux of insulin in intact animals. Extension of the investigation to intact dogs, made feasible by determination of the immunoreactive labeled insulin independently of endogenous insulin, re- vealed that growth hormone treatment increased the mass rate of transfer of insulin from the blood, without altering the fractional rate of disappearance, as reported briefly.” The results of this study and a hypothesis for the mode of From the Departnzetzt of P/zysiology, University of Toronto, Toronto, CanudLz. Supported by the Medical Research Council of Catzada and the J.P. Bickell Foundation. Received for publication April 30, 1969. JAMES CAMPBELL, PH.D.: Professor, Departmerzt of Physiology, University of Toronto, Tororzto, Canada. KRISHNA SUDHA RASTOGI, PH.D.: Researclz Associate, Departmerzt of Physiology, University of Toronto, Toronto. Canada. 930 METABOLISM,VOL. 18,No. 11 (NOVEMBER), 1969
Transcript
Actions of Growth Hormone: Enhancement of Insulin
Utilization with Inhibition of Insulin Effect on
Blood Glucose in Dogs
By JAMES CAMPBELL AND
The disappearance of labeled immunore- active insulin (L-IIU) from blood serum was measured in dogs, after a trace dose of 1311-insulin, given intravenously, had intermixed in the body fluids. The degree of labeling of insulin was low, and the same preparation was used in all tests. The disappearance of L-IIU followed an exponential course, the mean value of the fractional rate constant (k) was 1.86 + 0.20 per cent min.-l. An integral method of analysis of the data gave similar values. Growth hormone (GH, 2 mg./Kg. of body weight/day) injected for 4 days, did not alter this fractional rate appreciably. However, the mass rate of irreversible loss of insulin was greatly increased (19- fold), due chiefly to elevation of en- dogenous immunoreactive insulin in “postabsorptive” serum. The enhanced rate of utilization of insulin, induced by GH, was thus dependent on, and coupled with increased secretion of insulin. Dur-
KRISHNA SUDHA RASTOGI
ing GH administration, serum gbrcose rose, but to a lesser degree than insulin: the IIWglucose ratio (23 microunits/mg. under the control conditions) rose, about lo-fold. Serum free fatty acid, and esterified fatty acid levels were both in- creased by GH. The responsiveness to insulin, within 2 hours of intravenous injection of 0.1 unit/Kg., was tested in these dogs. During GH treatment, the reduction in serum glucose was less rapid, but the sharp fall in free fatty acids was not prevented. Insulin did not affect esterified fatty acid levels, under both the control and GH treatment con- ditions, however. A hypothesis is out- lined for the effect of GH in diverting preferentially the utilization of the large amounts of insulin from processes of carbohydrate metabolism to those in- volved in protein synthesis. (Metabolism 18: No. 11, November, 930-944, 1969)
T HE HYPERINSULINEMIA produced by the administration of growth hormone in the dog1J is associated with enhanced secretion of insulin.3
The action of growth hormone on insulin utilization in depancreatized and Houssay dogs,4 indicated that growth hormone could be expected to increase greatly the flux of insulin in intact animals.
Extension of the investigation to intact dogs, made feasible by determination of the immunoreactive labeled insulin independently of endogenous insulin, re- vealed that growth hormone treatment increased the mass rate of transfer of insulin from the blood, without altering the fractional rate of disappearance, as reported briefly.” The results of this study and a hypothesis for the mode of
From the Departnzetzt of P/zysiology, University of Toronto, Toronto, CanudLz. Supported by the Medical Research Council of Catzada and the J.P. Bickell Foundation. Received for publication April 30, 1969. JAMES CAMPBELL, PH.D.: Professor, Departmerzt of Physiology, University of Toronto,
Tororzto, Canada. KRISHNA SUDHA RASTOGI, PH.D.: Researclz Associate, Departmerzt of Physiology, University of Toronto, Toronto. Canada.
930 METABOLISM, VOL. 18, No. 11 (NOVEMBER), 1969
ACTIONS OF GROWTH HORMONE 9.31
action of growth hormone in directing the utilization of the great amounts of insulin made available in the body are presented.
MATERIALS AND METHODS
Normal adult male dogs were accustomed for about 1 month to laboratory conditions.1 and brought to a state of nutritional repletion (body weight 12.6-16.9 Kg.). For 3 weeks prior to and during the experimental period, the daily diet consisted of cooked lean meats (Jubilee Animal Ration, Canada Packers, Toronto) 32 Gm./Kg., and chow (Ralston Purina Co.) 8 Gm./Kg., divided into two meals, given at 10 a.m. and -1 p.m. The food was eaten completely, within 10 minutes.
Insulir~ Disappearance Test
Two initial samples of venous blood were taken about 18 hours after a meal. A trace dose of labeled insulin (ls*I-insulin, bovine, specific activity 4 mc./mg., Abbott Laboratories, NO. 6797) was injected intravenously, equivalent to 2 PC., in 0.4 ml., per kilogram of body weight. The solution was prepared fresh in 0.04 M phosphate buffer, pH 7.4, containing 1 mg./ml. of bovine plasma albumin (Armour Laboratories) and 9 mg./ ml. of sodium chloride. For each test in each dog, a “phantom” was prepared, by addition of labeled insulin to the serum in vitro. The same preparation of rsiI-insulin was used for tests under both the control and growth hormone treatment conditions.
Antibody-bound isotopic insulin was determined by the method of Wallish,u which is based on that of Herbert et al.7 To 0.4 ml. of serum, in duplicate, was added 0.4 ml. of diluted guinea pig anti-bovine-insulin serum (l/1000 in saline containing albumin, 3 mg./lOO ml.). The insulin-binding capacity of the antiinsulin serum added was about 8-fold in excess of the maximum amount of insulin expected. Duplicate controls con- tained 0.4 ml. serum and 0.4 ml. of the saline-albumin diluent. After incubation at 37O C for 2 hours, the solutions were cooled in an ice-bath and 1 ml. of cold, dextran-coated charcoal suspension (dextran 0.50/c, Norit A charcoal 5% ) was added with mixing (Vortex Mixer). The tubes were centrifuged (3000 rpm, O-4” C) and the radioactivities of the supernatants were determined in a gamma-scintillation counter (Nuclear-Chicago).
The concentration (denoted by square brackets) of injected, labeled immunoreactive insulin [I.-IRIl in serum was calculated:
[L-IRII = 2.5 (RA, - RA,,), cpm/ml. (1)
where RA, and RA, are the radioactivities of the antibody-containing and the control tubes. respectively. The amount of labeled, immunoreactive insulin injected (Q, cpm: Kg. of body weight) was obtained from determinations on the phantom. The L-IRI concentration could then be expressed as the fraction of the amount injected per kilogram of hody weight per unit volume (milliliter or liter) of serum:
In addition to the above procedure, the amount of isotope precipitable by trichloro- acetic acid (TCA) was determined in each sample of serum and in the phantom. To 0.4 ml. of serum, in duplicate, was added 1 ml. of cold 20 per cent TCA. After centri- fugation, the precipitate was washed with 1.5 ml. of 5 per cent TCA, and the radio- activity of the precipitate was determined.
The data obtained by both methods were plotted on semilogarithmic coordinates (Fig. 2). The rate and extent of the regression were higher when the immunoreactive fraction of serum was used as the measure of labeled insulin than when the TCA- precipitable isotope was used. The data obtained by the former method were therefore further analyzed.
932 CAMPBELL AND RASTOGI
Andysis of Data
Following the injection of labeled insulin, an initial phase of rapid fall in L-WI can be attributed to distribution in, and also to concurrent irreversible loss from, the fluid compartments involved (Fig. 2). The subsequent slower phase, or slow component. may be due chiefly to irreversible loss, if intermixing is substantially complete by this time. A method of analysis, for a system of two compartments, was applied.s.” For the slow component, the straight line of best fit, its slope (I?), and intercept on the zero time axis [L-IRI],, were computed from the relation
A dilution curve or “fast component” was derived by subtracting from the measured values in the initial phase of the curve, the respective values of the extrapolated slow component. A computer program was used in these calculations (IBM computer No. 7094). The apparent volume of distribution (PO, ml./Kg.) of labeled immunoreactive insulin was obtained from the relation
I’, = Q/[L-IRI],, (4)
The rate of irreversible loss of natural insulin in the body was calculated from the relation
U = k . [L-IRI] l V,, (5)
where U is the mass rate of irreversible loss of insulin, microunits/Kg. min.; k is the fractional disappearance rate constant of L-IRI in the slow component, fractionmin., IRI is the steady-state concentration of endogenous insulin in serum, micro-unit/ml.; and V, is the apparent volume of distribution of labeled insulin. ml./Kg. of body weight. This estimate involves the assumptions that in the body, both the isotopic and the natural, unlabeled insulins occupy the same space, that they are handled in the same way, and that the equilibrium level of unlabeled immunoreactive insulin remained steady in the test period. Under these circumstances, the rate of irreversible loss should equal the rate of production of insulin.ie
The data were also analyzed by an integral approach. The L-IRI concentrations were plotted on Cartesian coordinates, and the area under the curve was measured by planimeter, between the limits of 30 and 90 min. (Fig. 3). This area was designated (Y, microunit, min. If none of the insulin was removed in this time, the area (A) would be equal to [L-IRII,, l (f2 - tI), where [L-IRI],, is the concentration at time,,. The fraction removed was therefore (A - cu)/A, and the estimated mean fractional rate (k) in this interval was
k, fraction/min. = (A - ,a)/,4 (tZ - tr) (6)
The mass rate of irreversible loss of insulin (U, micro-units/Kg. min.) from the pool, with the assumptions given above, was obtained by
A--a u = A(t,?-_tl) . [IRI] . I’,,
As pointed out by several authors, !‘.I0 the integral method uses the data with equal weight, and is not dependent on constant fractional rate of removal in the test period.
Insulin Tolerance Test
About 18 hours after a meal, two samples of venous blood were taken. A standard dose of insulin (U.S.P., 0.1 unit/Kg. of body weight, in 0.04 M phosphate buffer, pH 7.4, containing 1 mg./ml. of plasma albumin) was injected intravenously. Blood
ACTIONS OF GROWTH HORMONE 933
IRI
Fig. 1 .-Effects of growth hormone, 2 mg./Kg. of body weight/day, on postabsorptive values in serum of dogs. Standard error of each mean is shown by vertical line, and probability Cp) of differ- ence from control value is given.
samples were obtained at intervals. Glucose,llJ” free fatty acids (FFA),‘” esterified fatty acids]:’ of serum, and the total lipids, and dry, fat free residue14 of tissues were determined. Immunoreactive insulin (IRI) in serum was assayed by a modification1 of the two-antibody method C of Hales and Randle.1”
&XJLTS
In four normal dogs under the standard regimen, postabsorptive levels of IRI, glucose, FFA and esterified fatty acids were determined. The rate of dis- appearance of immunoreactive isotopic insulin (L-IRI) from serum, and insu- lin tolerance were also tested. Growth hormone, bovine N.I.H., Lot B12, was then injected subcutaneously twice a day, in the dosage of 2 mg./Kg./day, for 6 consecutive days. The previous determinations were repeated during this treat- ment. Each dog therefore acted as its own control.
Postabsorptive Values
Growth hormone increased the postabsorptive level of IRI in serum by about 15-fold. The mean values, in micro-unit/ml., were: 20 initially, 304 (p = 0.005) after 4 days, and 350 (p = 0.001) after 6 days of treatment (Fig. 1). The effect occurred in each dog; range 6- to 29-fold. Serum glucose also in-
934 CAMPBELL AND RASTOGI
o.IoL 1 > 1 50 --i&F
TIME FROM INJECTlON OF INSULIN - f3’, mn.
Fig. Z.-Concentrations of labeled immunoreactive insulin (dots) and of TCA- precipitable 1311 (x’s) as fraction of dose per kilogram of body weight per liter of serum, following intravenous injection of lslI-insulin in dogs. Test was performed under control conditions, and after 4 days of injection of growth hormone, 2 mg./ Kg./day.
creased significantly during growth hormone treatment, from the mean (mg./ 100 ml.) of 88 under normal conditions, to 163 (p = 0.05) after 4 days, and to 186 (p = 0.01) after 6 days (Fig. 1). The insulinogenic index (IRU glucose, micro-unit/mg.) mean was 23 under normal conditions. Growth hor- mone treatment magnified the value lo-fold. The rise in IRI was therefore proportionately greater than the accompanying rise in glucose (Fig. 1).
A moderate, significant increase in serum FFA (60%) p = O.Ol), and a simi- lar significant increase in esterified fatty acids (65%, p = 0.01) were produced by growth hormone (Fig. 1). Serum ketone body concentration was not af- fected.
Body weight increased during the administration of growth hormone, with- out change in food intake (Table 1). In the liver, the total protein, as indi- cated by the dry, fat-free residue, and total lipid were increased significantly, in proportion to final body weight (Table 3).
Insulin Disappearance
The fast component curves show that the time required to reduce the L-IRI concentration differences to very low levels were 28-32 min., under the control conditions (Fig. 2). This may be considered an approximation to the inter-
* Values (mean 2 SE.) in 4 dogs under control conditions, and treated with growth hormone, 2 mg./Kg./day for 4 days.
T Equation 6. $ Equation 7.
Table 3,Effects of Growth Hormone on Organ Weights and Lipids*
Conditions Liver Kidney Weight DFFR t Total Weight DFFR + Total
Lipid Lipid -_. ___.~_~~ Gm./Kg. body weight
.- Normal 23.7 5.35 1.35 4.36 6-71 0.22 + 1.2 k 0.28 z!z 0.11 ?I 0.37 2 0.07 t 0.06
Growth hormone 45.0 9.74 2.83 4.38 0.66 0.21 t 5.0 -c 0.66 k 0.18 +- 0.59 IL 0.09 k 0.03
P 0.005 0.001 0.001 NS NS NS
* Dogs were under control conditions, or injected with growth hormone, 2 mg./Kg./day. for 6 days. Mean k SE for 4 dogs in each group.
t Dry, fat-free residue.
mixing time. -I,H During growth hormone treatment, the estimated intermixing
times were comparable (20-33 min.). From this information, and the near-
rectilinear shape of the curve after 30 minutes, the time span of the slow
component, relating to the irreversible loss of insulin, was taken as 30 to 90
minutes.
In dogs under normal conditions, the slow component data, analyzed by the
compartmenta method, gave the mean half-life value of 38 minutes and the
fractional rate of disappearance (k, % /min.) of 1.86 for labeled insulin (Fig. 1,
Table 1). The data for each dog were also analyzed by the integral method. The mean values on Cartesian coordinates are shown in Fig. 3. The fractional rate
of disappearance of insulin (mean k%/min.) during the 60 minute interval was
1.08 (Table 2). This value and its variance are less than were obtained by the compartmental method of analysis.
As pointed out by several authors,8J’J7 when the intermixing time is shorter
than the time that the tracer molecules spend in the pool, the estimate of the concentration of tracer substance at time zero can be low. In the case of in-
sulin, the intermixing time and the half-time are similar; therefore L-IRT,, may be under-estimated, and the apparent volume of distribution (Vo = 55.9%,
Table 1) may be somewhat higher than the actual insulin space. In comparison, values of the volume of distribution of insulin were 20 per centIS and 37 per
ACTIONS OF GROWTH HORMONE 937
Fig. 3.-Labeled immunoreactive insulin in serum, following intravenous injection of l;lll-insulin in 4 dogs. Mean values (Cartesian coordinates) with standard errors indicated by vertical lines are given. Test was performed under control conditions and after 4 clays of injection of growth hormone, 7_ mg.!Kg.!day.
cent*” in normal subjects, and 25.7 per cent in bilaterally-nephrcctomized
patients.‘!’ The value of the mass rate of irreversible loss of insulin ((i) from
the pool is therefore given as the numerical value of the product k l [IRl]
times the symbol V,,, and also including the value of V,]. Under the control
conditions, the value of U 0.357 l V,), or 202 micro-unit/Kg. min. by the compartmental method (Eq. 5), and 0.22 l V,, or 125 micro-unit/Kg. min.,
by the integral method (Eq. 7).
During the administration of growth hormone, the mean half-time, and the
fractional rate of disappearance of L-IRI were not appreciably altered (Tables
1, 2). The mass rate of irreversible loss of insulin was much greater, however, after 4 days of treatment with growth hormone than under control conditions:
6.10 0 V,,, or 3800 micro-unit/Kg. min. by the compartmental method of analysis and 3.34 l V,, or 2170 micro-unit/Kg. min. by the integral method
(Tables I. 2).
938 CAMPBELL AND RASTOGl
INSULIN o. untts,‘kq. I.“.
4 NORMAL -
GH. TREATED -
Fig. 4.-Insulin tolerance test. Mean values in serum with standard errors in 4 dogs, fol- lowing intravenous injection of insulin, 0.1 unit/Kg. of body weight. Test was performed under control conditions and after 6 days of growth hormone treatment, 2 mg./Kg./day.
0 20 40 60 80 100 120 TIME FROM INJECTION OF INSULIN, mtn.
Effects of growth hormone were also compared by the ratios of values in
growth hormone treatment to those in the control period. The ratios for T1,?,
k per cent and V, were not significantly different from unity (Tables 1 and 2).
However, the ratio for IRI was 19, and for U was 19-27. If I/, is considered
to be constant, and thereby eliminated, the ratio for U is about 19. The im-
portant factor in this massive increase in insulin utilization caused by growth
hormone was the great increase in the equilibrium level of endogenous insulin
in serum.
Insulin Tolerance
In the control period, the test dose of insulin produced the characteristic sharp fall in glucose concentration, to a minimum value in 20 minutes with
return to normal in about 90 minutes (Fig. 4). During growth hormone treat-
ment, the test dose of insulin was not followed by a prompt fall of glucose concentration; instead, a tendency to gradual decline from the initial high level
was found. Under control conditions, plasma FFA decreased very sharply in 10 minutes
after insulin injection, then rose in the next 10 minutes to supernormal level,
ACTIONS OF GROWTH HORMONE 939
and remained elevated for the remainder of the 2-hour period. Growth hor- mone treatment markedly altered the response. The FFA decreased rapidly, fol- lowing the test dose of insulin, and did not return to the initial high level within 2 hours (Fig. 4).
Insulin had no effect on the esterified fatty acid level, either under the control or the growth hormone treatment conditions (Fig. 4).
DISCUSSION
In this study of insulin metabolism, a comparison of methods indicated that antiinsulin bound isotope gave better representation of labeled insulin in serum than did TCA-precipitable isotope. Similar conclusions have been reached by other investigators.J~lp~“O If labeled insulin is to depict authentically the utiliza- tion of cndogenous insulin, it must be processed in the body in the same way. It is possible that molecular changes, which may be so subtle as to escape de- tection by ordinary methods, may influence recognition by the tissues. The insulin used contained less than one atom of Is11 per molecule, a degree of labeling that appears not to impair biological activity or antigenicity.?‘,“” How- ever, loss of biological activity in l”“I-insulin has been reported.‘” The design of the experiments permitted the use of the same preparation of labeled insulin in the same animals, under both control and growth hormone treatment con- ditions.
The fractional rate constant for insulin disappearance in the normal dog (k, %/min., 1.1-1.9) may be compared to the value of 2.0 found in man by Berson et a1.16 and by Samols and Ryder. l8 However, in patients with bilateral nephrectomy, a value of 9.6 (calculated from T,/-) was obtained,‘” and in normal subjects a value derived from the initial phase was 21.7.‘* The mass rate of irreversible loss of insulin in the fasting normal dog was 12.5-200 micro- unit/Kg. min., while in man the approximate range of 1 S-1 33 micro-unit/ Kg. min. may be estimated from the data of Stern et al.‘”
Growth hormone treatment did not alter appreciably the fractional rate of disappearance of labeled insulin in dogs. In a previous study, the rate of disappearance of unlabeled insulin was not changed in depancreatized and Houssay dogs-l by similar treatment with growth hormone. This rate therefore appears to be independent of growth hormone effects, which include marked increases in blood insulin, glucose, FFA and esterified fatty acids.
The mass rate of irreversible loss of insulin was greatly increased by the growth hormone treatment, however. The increases, approximately proportional to the increase in IRI, were of the order 19- to 25fold above the normal values. It may be emphasized that this signified an elevation to a higher steady- state of insulin utilization, maintained from day-to-day during the treatment. not a transient rise.
Factors that may or are known to be involved in the fate of insulin are represented in Fig. 5. Within plasma, combination with antibody, binding to a normal plasma protein, and enzymatic degradation of insulin are possible. Since the experiments were of short duration and the dogs were previously untreated, antibody activity of appreciable degree would not be expected in this study.
940 CAMPBELL AND RASTOGI
PANCREATIC PLASMA CELLS, FIXED AND CIRCULATING
INSULIN-ACTIVE SITE INTERACTION WITH COMPONENTS (ENZYMES?) OF:,
PLASMA MEMBRANE RIBOSOMES
plasma membrane
ENZYMATIC DEGRADATION
PROCESSES: t. AdsorptIon to capillary wall 1. Transport to interstitial fluid 3. Adsorption to cell membrane
KIDNEY FILTRATION REABSORPTION
4. Transport to cell fluid EXCRETION
Fig. S-Schematic representation of possible processes involved in utilization and destruction of insulin in the body.
Insulin appears to be free in plasma ,26 although evidence of normally-occurring bound forms has been presented. 2o Insulin was found to be stable in serum, under the conditions employed. Plasmatic factors therefore appear to be of minor im- port in the present study.
Insulin can pass through capillary wallslQJi and substantial amounts are filtered by the renal glomeruli .1D,27,2E( With regard to cellular processes, insulin may be destroyed enzymatically at cell surfaces as well as within cells, since it is rapidly degraded by isolated tissues in vitro, and by insulinase activity of tissue ex- tracts.2Q*32 Whether insulinase action is associated with the physiological effects of insulin is not yet understood.2Q The modes of utilization of insulin absorbed to cell surfaces, and within cells33 have not yet been fully explored. Although a number of possibilities must be considered, the evidence available appears to indicate that the loss of insulin from the circulation has relation to its phys- iological utilization in the body, and that the mass rate of irreversible loss may provisionally be called utilization rate (U) .
Insulin secretion rate (S) can be equated to insulin utilization rate (U) (if the blood insulin level remains steady during the test) plus the insulin loss rate in passage through the liver (U,,). In man, Stern et al.‘” found that plasma IRI was not appreciably altered following a trace dose of 1311-insulin. By a method for sampling of portal vein blood close to the liver, Campbell and Rastogi3 estimated that insulin secretion rate in unanesthetized dogs was of the order 430-570 pU/Kg. min. Growth hormone in the same dosage as in the present ex- periments, for 7-8 days, raised the estimate of insulin secretion to 5500- 8000 micro-unit/Kg. min. These investigations, therefore, show that growth hormone magnified both the secretion and the utilization of insulin, roughly to the
ACTIONS OF GROWTH HORMONE 941
same extents. Combining the results of the two investigations, the rate of hepatic utilization of insulin ( Uh = S - U) appears to be about 60 per cent of the total utilization, comparable to that in man. I8 In the normal dog, the rate appears to be of the order 200 micro-unit/Kg. min., and to be raised to 3000-4000 micro-unit/Kg. min. by growth hormone treatment for several days. These csti- mates are only rough approximations, but are of such magnitude as to demand attention.
The increase in the IRI/glucose ratio, despite the rise in serum glucose (Fig. 1): supports the suggestion that growth hormone increases the sensitivity to glucose of the insulin-secreting cells. l Growth hormone has been found, in this and other investigations, to decrease glucose tolerance and glucose utiliza- tion, and to diminish the effect of injected insulin on blood glucose.‘s”J’ :(;I A shift in the total metabolic pattern towards increased mobilization and utiliza- tion of lipid was indicated by the rises in FFA, esterified fatty acids and liver lipid. Thus, despite the plethora of insulin and glucose in blood, and the greatly increased transfers of insulin into and out of the blood, growth hormone sup- pressed the utilization of carbohydrate, and enhanced that of lipid.
In the normal dog, the initial fall in FFA produced by the test dose of insulin (Fig. 4) may be due to suppression of lipolysis in adipose tissue. The following sharp rise in FFA may have been induced chiefly by the fall in blood glucose, which has been reported to produce complex direct and indirect effects. in- cluding ( 1) increased lipolysis in adipose tissue, (2) enhanced production of catecholamines at sympathetic nerve endings in adipose tissue, (3) increased secretion of catecholamines by the adrenal medulla, and (4) increased secretion of ACTH and growth hormone. 3ti The alterations produced by growth hormone in the “FFA response” to insulin may be causally related to the persisting hi@
levels of insulin and glucose, which may limit the regulative processes I--L mentioned above. The results also suggest that growth hormone produced in adipose tissue an insulin insufficiency of a competitive type.
The finding in this and other investigations that the fractional rate constant (X-) of insulin disappearance was not affected by variations in blood insulin. glucose, FFA and esterified fatty acid levels.‘;.“,7 has important implications in
glucose and insulin homeostasis. According to this observation, for ins~lirr, the mass rate of utilization will depend chiefly on the concentration in blood. since k has little variation, and the volume of distribution has a relatively limited range. In the steady state, the blood level will correlate primarily with insulin secretion rate, and secondarily with insulin utilization rate. Alteration from the equilibrium level will be due directly to change in the rate of secretion, and secondarily to level-dependent utilization.
For glucose, concentration is a factor in utilization. More important, how- ever, are the influences of hormonal and other factors on the fractional rate constants for both utilization and production of glucose.‘Ji Further investigation of insulin uptake in various physiological states is required.
Several investigators have proposed that the inhibition of insulin action (carbohydrate oxidation and hypoglycemic effects) produced by growth hor- mone may be due to blockade of insulin, by direct or indirect means.‘).4Z.a:S These
942 CAMPBELL AND RASTOGI
suggestions appear to imply that the great fluxes of insulin, now known to be produced by growth hormone, would be compensatory, and functionally ne- gated.
In explanation of the action of growth hormone, we propose that the great amount of insulin made available is metabolically active and that it is used preferentially in protein synthesis. Insulin requirement for protein synthesis has been indicated in many investigations. 1~Z~34~38-40 Increased synthesis of liver protein, serum albumin and fibrinogen is associated with indications of increased hepatic utilization of insulin in the dog. +l Stimulation of protein synthesis can be expected to produce competition for insulin between enzyme systems of cells involved in the metabolism of carbohydrate, protein and lipid. The effect of growth hormone in suppressing the action of insulin on glucose uptake, sug- gests that the insulin-affinity of the enzyme systems for protein synthesis is enhanced, at the expense of enzymes for carbohydrate metabolism. The protein anabolic effect of growth hormone is accompanied by increased mobilization and utilization of lipid. These relations suggest that insulin’s action on protein syn- thesis is not necessarily linked to its action on carbohydrate metabolism. The several propositions included in this hypothesis are being tested in further in- vestigations.
ACKNOWLEDGMENTS
We gratefully acknowledge the work of Mrs. V. Lazdins and Mr. G. R. Green through- out this investigation. We thank Dr. R. Ninomyia for the computer program used in analysis of data, and Dr. G. Hetenyi, Jr., and Dr. C. C. Lucas for reading the manuscript critically.
REFERENCES
1. Campbell, J., and Rastogi, K. S.: and Bleicher, S. J.: Coated-charcoal im- Growth hormone-induced diabetes and high munoassay of insulin. J. Clin. Endocr. 25: levels of serum insulin in dogs. Diabetes 1375, 1965. 15:30, 1966.
2. DeBodo, R. C., and Altzuler, N.: The 8. Wrenshall, G. A.: Working basis for
the tracer measurement of transfer rates of metabolic effects of growth hormone and a metabolic factor in biological systems con- their physiological significance. Vit. Hor. 15: taining compartments whose contents do not 205, 1957.
3. Campbell, J., and Rastogi, K. S.: Aug- intermix rapidly. Canad. J. Biochem. Physiol. 33:909, 1955.
mented insulin secretion due to growth hormone. Diabetes 15:749, 1966.
9. Riggs, D. S.: The Mathematical Ap-
4. -, and -.: Effects of growth hor- preach to Physiological Problems. Baltimore,
mone on the rate of disappearance of insulin Williams and Wilkins, 1963.
from blood in depancreatized and Houssay 10. Stern, M. P., Farquhar, J. W., Silvers,
dogs. Metabolism 16: 562, 1967. A., and Reaven, G. M.: Insulin delivery
5. Campbell, J., Rastogi, K. S., Lazdins, rate into plasma in normal and diabetic
V., and Green, G. R.: Augmentation of subjects. J. Clin. Invest. 47:1947, 1968.
insulin utilization by growth hormone, as- 11. Nelson, N.: A photometric adaptation
sociated with hyperglycemia and suppression of Somogyi method for determination of
of glucose uptake. Fed. Proc. 27:433, 1968. glucose. J. Biol. Chem. 153:375, 1944.
6. Walfish, P. G.: Insulin I-131 disap- 12. Campbell, J., and Green, G. R.: Free
pearance rates following diazoxide, epineph- fatty acid metabolism in Chinese hamsters.
rine and glucose administration. Ann. N.Y. Canad. J. Physiol. Pharmacol. 44:47, 1966.
Acad. Sci. 150:292, 1968. 13. Stern, I., and Shapiro, B.: A rapid 7. Herbert, V., Lau, K.-S., Gottlieb, C. W., and simple method for the determination of
ACTIONS OF GROWTH HORMONE 943
esterified fatty acids and for total fatty acids in blood. J. Clin. Path. 6:158, 1953.
14. Best, C. H., Lucas, C. C., Patterson. J. M., and Ridout, .I. H.: The influence of biotin upon the relative lipotropic effects of choline and inositol. Biochem. J. 40:368, 1946.
15. Hales. C. N.. and Randle, P. J.: Im- munoassay of insulin with insulin-antibody precipitate. Biochem. _I. 88: 137, 1963.
16. Berson, S. A., Yalow, R. S., Bauman, .A.. Rothschild, M. A., and Newerley, K.: Insulin-It~il metabolism in human subjects. I. Clin. Invest. 35: 170. 1956.
17. Arnould. Y.. Cantraine, F., Ooms, H. .A.. Delcroix. C.. and Franckson, J. R. M.: Kinetics of plasma disappearance of labelled iodoinsulins following intravenous injection. Arch. Int. Pharmacodyn. 166:225, 1967.
IX. Samols. E.. and Ryder, J. A.: Studies on tissue uptake of insulin in man using ii differential immunoassay for endogenous and exogenous insulin. J. Clin. Invest. 40: 2092, 1961.
19. Rasio. E. A., Hampers, C. L.. Soeld- ner. J. S.. and Cahill, G. F., Jr.: Diffusion of glucose, insulin, inulin, and Evans blue pro- tein into thoracic duct lymph of man. J. Clin. Invest. 46:903. 1967.
20. Posner. B. I., Stotman, S. L., and Antoniades, H. N.: Insulin disposition in the isolated perfused heart. Effect of “bound” insulin. Diabetes 17:427, 1968.
21. Izzo. J. L.. Roncone, A., Izzo, J. J., and Bale. \V. F.: Relationship between de- gree of iodination of insulin and its biolog- ical, electrophoretic and immunochemical properties. I. Biol. Chem. 239:3749. 1964.
‘2. Lee, N. D.: Studies on insulin labeled with 11:“. Ann. N.Y. Acad. Sci. 70:94, 1957.
73. Arquilla. E. R., Ooms, H., and Finn. J.: Review of current research: genetic dif- ferences of combining sites of insulin anti- hodies and importance of C-terminal portion of the A chain to biological and immuno- logical activity of insulin. Diabetologia 3: I. 1966.
23. Stimmler. L.: Disappearance of im- munoreactive insulin in normal and adult onset diabetic subjects. Diabetes 16:652, 1967.
25. Yalow, R. S., Glick, S. M., Roth, J., and Berson. S. A.: Plasma insulin and growth hormone levels in obesity and dia- hetes. Ann. N.Y. Acad. Sci. 131:357, 196.5
26. Berson. S. A., and Yalow, R. S.:
Insulin in blood and insulin antibodies. Amer. J. Med. 40:676, 1966.
27. Rasio. E. A., Mack, E.. Egdahl, K. H., and Herrera. M. G.: Passage of insulin and inulin across vascular membranes in the dog. Diabetes 17:668, 1968.
28. Chamberlain, M. J.. and Stimmler. I .: The renal handling of insulin. J. Clin. ln\c\t. 46:991, 1967.
29. Mirsky, 1. A.: The metabolism of insulin. Diabetes 13:235, 1964.
30. Narahara, H. T.. and William\. I<. H.: Effect of protein added in vitro upt,n
insulin degradation and glucose uptake hb muscle. J. Biol. Chem. 333:1034. 19S8.
31. Schucher, R.: Studies on the met;rb- olism of insulin by pancreas in vitro, 1. Degradation of exogenous insulin by calf pancreas slices. Canad. J. Biochem. 43: 1 143.
196.5. 32. Tasaha, Y.. and Campbell, J.: Pan-
creatic insulinase and its inhibition by the A and B chains and partial hydrolysate oi insulin. Canad. J. Biochem. 46:483, 1WiX.
33. Edelman. P. M. and Schwartz. I. 1 .: Subcellular distribution of Ir”l-insulin in striated muscle. Amer. J. Med. 40:695. IY)hh.
34. Manchester. K. L., and Young:, I G.: Insulin and protein metabolism. C’ir Hot-. lY:Y5. 1961.
35. Kipnis, D. M.: Growth hormone and insulin antagonism. 111 Leibel, B. S., and Wrenshall, G. A., (Eds.) : Nature and Treat- ment of Diabetes. Amsterdam, Exerpta
Medica Foundation. 196.5, p. 2%. 36. Renold, A. E., and Cahill, G. F.. Jr.
(Eds.): Adipose Tissue. Section 5. Hand-
book of Physiology. Washington, D.(‘.. American Physiological Society, 1965.
37. Hetenyi. G., Jr.. and Wrenshall. G. A.: Rates of production, utilization, accun- ulation and apparent distribution space of glucose. Diabetes 10:304. 1961.
38. Milman. A. E.. Demoor, P., and Lukens. F. D. W.: Relation of purified pituitary growth hormone and insulin in regulation of nitrogen balance. Amer. J. Physiol. 166:354, lY51.
39. Salter. J. M.. Davidson, I. W. F.. and Best, C. H.: The effects of insulin and somatotrophin on the growth of hypophy- sectomized rats. Canad. J. Biochem. Physiol. 35:91?, 19.57.
40. Stirling. R. A. C.. and Campbell. J.: Metabolism in hypophysectomized-depan- creatized dogs. Metabolism 9:738. 1960.
944 CAMPBELL AND RASTOGI
41. Campbell, J.: Diabetogenic actions of 42. Stadie, W. C.: Current concepts of the
growth hormone. In Smith, R. W., Jr., Gaeb- action of insulin. Physiol. Rev. 34:52, 1954.
ler, 0. H., and Long, C. N. H. (Eds.) : The 43. Randle, P. J., Garland, P. B., Hales.
Hypophyseal Growth Hormone, Nature and C. N., and Newsholme, E. A.: The glucose
Actions. New York, McGraw-Hill, 1955, p. fatty-acid cycle. Its role in insulin sensitivity and the metabolic disturbance of diabetes
