the mechanism of action of insulin

The Mechanism of Action of Insulin. B y

H. WEIL-MALHERBE 1.

W i t h 9 F i g u r e s .

T a b l e o f C o n t e n t s . page

I. T h e E f f e c t of I n s u l i n o n G l u c o s e U t i l i z a t i o n . . . . . . . . . . . . . . . . 55

1. I n v i v o . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 2. I n v i t r o . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58

3- T h e P o i n t o f A c t i o n o f I n s u l i n . . . . . . . . . . . . . . . . . . . . 59

I I . O t h e r M e t a b o l i c E f f e c t s of I n s u l i n . . . . . . . . . . . . . . . . . . . 60

t . T h e E f f e c t of I n s u l i n o n G l y c o g e n F o r m a t i o n . . . . . . . . . . . . . . 60 a) L i v e r . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61

b) M u s c l e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62

2. T h e E f f e c t of I n s u l i n o n G l u c o s e O x i d a t i o n . . . . . . . . . . . . . . . 63 3. T h e E f f e c t o f I n s u l i n o n P h o s p h a t e M e t a b o l i s m . . . . . . . . . . . . . 65

a) E f f e c t s o n I n o r g a n i c P h o s p h a t e . . . . . . . . . . . . . . . . . . 65 b) E f f e c t s o n H e x o s e p h o s p h a t e s . . . . . . . . . . . . . . . . . . . 65

c) E f f e c t s o n H i g h - E n e r g y P h o s p h a t e s . . . . . . . . . . . . . . . . 65 d) E f f e c t s o f I n s a l i n o n R e a c t i o n s D e p e n d i n g o n H i g h - E n e r g y P h o s p h a t e s 66

e) C o m m e n t . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67

4. T h e E f f e c t of I n s u l i n o n t h e O x i d a t i o n of C o m p o u n d s O t h e r t h a n G l u c o s e �9 68

5. T h e E f f e c t o f I n s u l i n o n L i p o g e n e s i s . . . . . . . . . . . . . . . . . 70 6. T h e E f f e c t o f I n s u l i n o n N i t r o g e n M e t a b o l i s m . . . . . . . . . . . . . . 73

7. C o n c l u s i o n s . . . . . . . . . . . . . . . . . . . . . . . . . . . 75

I I I . T h e M e c h a n i s m of t h e I n s u l i n E f f e c t . . . . . . . . . . . . . . . . . . . 76

~. I n s u l i n a n d A T P C o n c e n t r a t i o n . . . . . . . . . . . . . . . . . . . 76 2. T h e E f f e c t o f I n s u l i n o n H e x o k i n a s e . . . . . . . . . . . . . . . . . 77 3. I n s u l i n a n d G l u c o s e C o n c e n t r a t i o n . . . . . . . . . . . . . . . . . . 79

a) T h e M a s s A c t i o n E f f e c t of G l u c o s e . . . . . . . . . . . . . . . . . 79 b) T h e P e r m e a b i l i t y T h e o r y o f I n s u l i n A c t i o n . . . . . . . . . . . . . . 82 c) S p e c i f i c i t y of t h e P e r m e a b i l i t y E f f e c t of I n s u l i n . . . . . . . . . . . 83

d) T h e I n s u l i n E f f e c t a n d t h e S u p p l y of E n e r g y . . . . . . . . . . . . 85

4. T h e STADIE E f f e c t . . . . . . . . . . . . . . . . . . . . . . . . . 86 5. T h e D i s t r i b u t i o n o f I n s u l i n in t h e T i s s u e s a n d in t h e Cell . . . . . . . . . 88

C o n c l u s i o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88

I V . T h e M o d i f i c a t i o n o f I n s u l i n A c t i o n b y O t h e r H o r m o n e s . . . . . . . . . . 88

1. I n s u l i n a n d t h e C o r t i c o s t e r o i d s . . . . . . . . . . . . . . . . . . . . ~

2. I n s u l i n a n d A d r e n a l i n e . . . . . . . . . . . . . . . . . . . . . . . 90

3. I n s u l i n a n d G r o w t h H o r m o n e . . . . . . . . . . . . . . . . . . . . 94

S u m m a r y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95

R e f e r e n c e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96

1 R u n w e l l H o s p i t a l , W i c k f o r d , E s s e x , E n g l a n d .

The Effect of Insulin on Glucose Utilization. 55

At the present time when the chemical exploration of the insulin molecule has yielded spectacular results (257--259) the physiology of its action is still a matter of speculation. Yet in this field, too, much new knowledge has been gained, mainly by the application of new techniques, such as the Use of isotopes and in vitro studies with surviving tissues. I t is the purpose of this article to examine the conclusions which may be drawn from the facts as they are known 1. Tile central point of the discussion will be the question of whether a single mode or multiple points of action are required to account for the various effects of insulin; having arrived at an answer in favour, essentially, of a single mechanism of action we shall analyse its possible nature. I t is not intended to review the literature completely -- a well-nigh impossible task in any case -- bu t to consider it only insofar as it seems relevant to the argument.

I. T h e Effect o f Insulin on Glucose Utilization.

1. In vivo.

The term "glucose utilization" is used to denote the uptake of glucose by the cells and its subsequent metabolic transformations. These comprise its breakdown by fermentation and oxidation as well as its incorporation, Wholly Or in part, i n to glycogen, proteins and lipids. Utilization is usually

Ineasured by the disappearance of glucose from the extracellular fluids or the suspension medium. Its rate is a function of glucose concentration, a Phenomenon which will be considered more fully in a subsequent section. For Comparative measurements, therefore, it is essential to keep the extracellular glucose concentration at a constant level. If, in experiments on the living animal, the blood sugar concentration is maintained at about its physiological level, neurohormonai influences affecting glucose homoeostasis are minimized. This is the basis of the compensation method of continuous glucose infusion, in which glucose utilization is estimated from the rate of glucose addition required to maintain a blood sugar level of about t00 rag-%. Moderate fluctuations of the blood sugar concentration may be allowed for by a mathe- matical correction (97). The method has been used extensively by BOtlCKAERT and his coilai~orators (33); With its aid they demonstrated that insulin greatly increases the rate of glucose utilization.

BOUCKAERT and I~E DUVE (33) were particularly interested to find out how the liver, on the one hand, and the extrahepatic tissues, on the other,

x This paper was written in • spring of t953 at a time when no recent comprehen- sive review of the subject was available. Owing to a delay in the publication of the CUrrent issue of the "Ergebnisse" the article was revised and brought up-to-date in .NOVember t954. Meanwhile several similar reviews had appeared (15, 137, 278), but it is hoped that the presentation of the facts, and the conclusions reached, are sufficiently different in this article to justify its publication.

56 H. WEIL-MALHERBE: The Mecbanism of Action of Insulin.

contributed to the total effect of insulin, at physiological and at saturation levels, on glucose utilization. The effect of insulin at physiological levels was estimated by the difference in glucose utilizations of the non-diabetic and the pancreatectomized dog; the effect of insulin at saturation level waS deduced from the glucose utilization of the insulinized dog. T o evaluate separately the effects of insulin on the liver and on the extrahepatic tissues the glucose utilization of the intact dog was compared with that of the hepatectomized dog. The hepatic glucose output of the fasting intact dog was assumed to be equal to the glucose utilization of the extrahepatic tissues, which could be measured in the hepatectomized dog. This figure was added to the hepatic glucose uptake of the insulinized dog, and the effect of a saturation dose of insulin on the glucose utilization of the liver was obtained by the following calculation: [glucose uptake of tl~e intact insulinized dog] [glucose uptake of the hepatectomized insulinized dog] + [glucose uptake of the hepatectomized dog without exogenous insulin~. The hepatic glucose output of the diabetic dog at a hypothetical physiological blood sugar level was not estimated directly and further assumptions were made to obtain an approximate figure (96).

Tab le t . Glucose utilization o[ liver and extrah~p:~tic tissues o[ the dog c,t a conMant blood sugar level o/about 80 rag- %. [ F r o m BOOCKAERT a n d DE DOVE (33).]

Insulin present

S a t u r a t i o n dose E n d o g e n o u s insu l in on ly . . . . . . . . . . .

Difference ( = effect of excess dose in n o r m a l dog)

E n d o g e n o u s insu l in N o n e ( p a n c r e a t e c t o m i z e d dog) . . . . . . . . .

Difference ( = effect of phys io logica l a m o u n t s of insul in) . . . . . . . . . . . . . . . . .

S a t u r a t i o n dose N o n e . . . . . . . . . . . . . . . . . . Dif ference ( = t o t a l effect of insulin) . . . . .

Glucose uptake (g/kg/hr)

Liver Extrahepatic tissues

+ 1 . 2 7 + 0 . 3 0 - -0 .24 + 0 . 2 4

+t .51 +0.06

- -0 .24 + 0 . 2 4 - -0 .64 + 0 . 0 7

+ 0 . 4 0 + 0 . t 7

+ 1 . 2 7 + 0 . 3 0 - -0 .64 + 0 . 0 7

+ t . 9 1 +0,.23

r

Ratio hepatic / extrahepatic insulin

effect.

25

2

Table t summarizes the results obtained by BOUCKAERT and DE DUVE (33). They indicate that the insulin effect on glucose utilization is apparently much greater in the liver than in the extrahepatic tissues. This disparity is especially marked at saturation levels of insulin.

The argument of BOUCKAERT and DE DOVE rests on the assumption that the glucose utilization of the extrahepatic tissues is not impaired by the

The Effect of Insulin on Glucose Utilization. 57

removal of the liver. The authors support their thesis by pointing out the good agreement between the glucose utilization of the hepatectomized dog (without exogenous insulin) and the hepatic glucose output of the intact dog, which has been determined by direct methods (82, 274). LANG, GOLD- STEIN and LEVINE (t82), however, compared the glucose utilization of the hind limbs "in intact and eviscerated dogs. Their results, at physiological blood sugar' levels, are shown in Table 2. While they do not indicate any significant effect of the liver on the glucose utilization of the limbs in the absence of exogenous insulin and at physiological levels of blood sugar, they do show a much stronger effect of a saturation dose of insulin on the peripheral tissues of the intact dog than on those of the liverless Tab le 2. Glucose utilization o/dog legs.

dog. If therefore the insulin e l - Means of resu l t s o b t a i n e d a t b l o o d glucose

feet is larger in the intact than levels below t63 rag-% [LANG, GOLDSTEIN and LEVINE (t82)] . in the hepatectomized dog, as

~OUCKAERT and DE D U V E had Glueose utilization

found, the reason is not so much (g/kg/hr) an excess of hepatic over peri- Insulin

- - +

pheral glucose utilization, but a

higher level for the maximum Intact dog . . . . 0.2t 1.45 glucose utilization of peripheral E v i s c e r a t e d d o g . . 0.20 0.54

tissues in the presence of the liver than in its absence. The probable hormonal nature of the hepatic faceor has been demonstrated by a cross-circulation experiment in which an intact and an eviscerated dog were joined together; under these conditions the hind legs of the eviscerated dog showed the same response to insulin as those of the intact dog, Further evidence for the existence of this factor comes from the brain perfusion experiments of GEIGER et al. (t18), who found that the glucose uptake of cat brain ceased when it was perfused with "simplified" blood consisting of Washed ox erythrocytes, electrolytes, serum albumin and glucose; glucose Uptake could be restored either by running the perfusion fluid through the cat's own liver or by adding fresh liver extract to it, The liver extract could aot be replaced by insulin.

It is tempting to speculate in this connection on the nature of the so- called "hunger diabetes", a syndrome which is undoubtedly of hepatic origin. Its symptoms include a. depression of peripheral glucose utilization (200, 20t, 340), an inhibition of glucose oxidation in liver slices (346) and a reduced efficacy of insulin (93). They might conceivably be linked with a diminished discharge of this hypothetical hepatic factor.

The experiment of BOIJCKAERT and DE DUVE has been repeated by I'DNDSGAARD (202), although he used cats instead of dogs. Insulin was found to treble the utilization rate of glucose, but the effect was the same whether

58 I-I. W'EIL-MALI~ERB~: The Mechanism of Action of Insulin.

intact, hepatectomized or eviscerated cats were used. The rate of glucose utilization shown in these experiments in the insulinized cat, whether eviscerated or not, was similar to the glucose utilization of the insulinized eviscerated dog. LUNDSGAARD'S results, therefore, while confirming the similarity of the insulin effect in hepatic and extrahepatic tissues, provide no evidence for the existence of the hepatic factor supposed to be required for max imum glucose utilization in the dog.

The technique of hepatic vein catheterization has made i t possible to measure directly the hepatic glucose output in human subjects. BEARN,

BILLING and SHERLOCK (9) found no" significant ~ l {nsu/,'n' ' t difference in the hepatic glucose output of diabetic

so0 - i ~ " ~t/ 'm ~ patients and heal thy controls. I t must be borne in

- - - - mind, however, that the blood sugar level was different ~sa~ in the two groups; a diabetic with a normal hepatic

10o ., ~gJ ~ glucose output at an elevated blood sugar level would ~lz~ : -~ - presumably have an increased hepatic glucose output

s0 ~ [I -~ at a physiological blood sugar level. An intravenous l ~ , ,'1 injection of insulin (0.~ unit/kg) led to an immediate

~0- / i , ~%, ~ lowering of the hepatic glucose ou tpu t which also ~ ' ' - / ~ was of a similar extent in diabetics and non-diabetics

zs6 -'-i\_h,,~,t,~ ~,,~ ~ (Fig. t). Diabetics could be divided into two groups ._6 X~pat~v~,~ J according to the response of the hepatic glucose

~ I ca~,v~:~>~-. J output to insulin: those who belonged to the insulin- [- ] --~-~ ",',,~. sensitive type [and whose diabetes, according to

1 8 0 1 i l ~ 0 10 20ro~n 30 BORNSTEIN a n d LAWRENCE (28), is d u e to a t r u e

Fig. 1. Reproduced from insulin deficiency~ showed a much more dramatic BEANN~ BILLING and

S~E~LOCK (9)- drop of the hepatic glucose output than those of the insulindnsensitive type. When insulin-sensitive dia-

betics became severely ketotic their reaction to insulin became more like that of the insulin-insensitive type. Presumably the lack of liver glycogen in these patients mobilized the hormonal insulin antagonists by a mechanism similar to that operating in "hunger diabetes".

BEARN, BILLING and SHERLOCK (10) have calculated that the mean glucose utilization of n o r m a l h u m a n subjects, in the first 30min after the intravenous injection of 0.t unit of insulin/kg, amounts to 10.3 g. Of this quant i ty 4A g were accounted for by the throt t l ing of the hepatic glucose output and 6.2 g by peripheral utilization.

2. In vitro.

The fact t h a t glucose utilization is decreased in diabetes and increased by insulin has been demonstra ted by experiments with isolated tissues or tissue slices. The effect of insulin has been studied either by injecting the

The Point of Action of Insu l in . 59

animal before death or by addition to the suspension medium. The excised diaphragm of the rat has become the classical object for in vitro studies of Various hormonal effects. Table 3 shows some of the results concerning the effect of insulin on glucose utilization obtained with this method.

Table 3. Glucose utilization o/the excised rat diaphragm.

State of animal

N~rmal . . . . . .

Diabetic . . . . . .

Normal . . . . . .


Normal . . . . . .


Glucose concentr. (mg- % )

100 100

200

200

Irxsulin eolteentr. (units/ml)

1.0 t.0

0.5

0.5

193 80

153 78

(in %

18.3

16.4

Glucose uptake (rag/to0 g tissue/hr)

200

200

1.0

! . 0

Insulin +

247

t 3 8

287

2 t 7

of initial radioactivity)

25.2

25.5

Reference

t 7 4

314

t l

In slices of liver and, to a smaller degree, of kidney the assessment of glucose uptake is complicated by the simultaneous glucose output, due to the presence of ghicose-6-phosphatase, but it has been shown with the aid of isotopically labelled glucose that the glucose uptake is about 50 % :lower in liver slices from diabetic rats than in those from normal rats (242, 243). Similar results were obtained with kidney slices (306); the impairment of glucose Utilization in tile kidney slices of diabetic rats was corrected by treating the animals with insulin before death.

KItAm. (t 70) showed that the utilization of glucose in slices of rat adipose tissue is stimulated by insulin in vitro, especially in tissue from diabetic rats.

3- The Point of Act ion of Insul in.

It was believed until recently that glucose was the only sugar whose Utilization was accelerated by insulin, but it is now known that insulin also acts on other monosaccharides. These effects of insulin will be discussed in a later section.

It is, however, well established that the utilization of fructose is unimpaired in the intact, diabetic organism (75, 84, 85, 213, 233, 335) and in the liver of diabetic animals (58, 59, 242). Similarly, when glucose-t-phosphate and glucose-6-phosphate were iniectea intravenously they rapidly disappeared from the blood of both nofinal and diabetic rabbits at a rate which could not be accounted for by the phosphatase activity of the blood (t 2). Glucose- l'phosphate was also shown to be utilized by the isolated rat diaphragm

60 H. WEIL-MAI.ItERBE: The Mechanism of Action of Insulin.

and to produce an increased formation of glycogen, unaffected by the addition of insulin (~2). The function of the enzyme systems responsible for the formation of lactic acid from glycogen is intact in liver and muscle of diabetic rats; insulin is without influence on it in either normal or diabetic animals (43)' The formation of glucose from glycogen, as well as the synthesis of glucose from pyruvate, are proceeding at an accelerated rate in liver and kidney of the diabetic rat (243, 306). These facts are relevant to the localization of the insulin effect, for if the diagram of enzymic reactions shown in Fig. 2 is considered it will be apparent that the only reaction which is impaired in the

Glycogen

Tt Glucose-I -Phosphate

Step controlled I I Glucose by insulin , Glucose-6-Phosphale �9 6-Phosphogluconate

Fructose-6- Phosphate Fructose

Fructose- t ,6-diphosphate ~ F~uctose- 1 -phosphate

3-Glyceraldehyde-, " Dihydroxyacetone- Glyceraldehyde phosphate phosphate

Phosphatase reaction

Fig. 2. Initial steps in the metabolism of glucase, glycogen and fructose.

diabetic organism and which is accelerated by insulin is the transformation of glucose to glucose-6-phosphate. Since this reaction is catalysed by hexokinase it will henceforth be referred to as the hexokinase reaction.

The question now arises whether the effects of insulin are entirely accounted for by its action on the hexokinase reaction or whether there are additional blocks in the metabolism of the diabetic. The more important metabolic effects of insulin will therefore be discussed from this angle.

II. Other Metabolic Effects os Insulin. 1. The Ef fec t of Insu l in on G l y c o g e n F o r m a t i o n .

With the exception of the reaction catalysed by the glucose dehydrogenase of liver, the metabolic significance of which is still obscure, the hexokinase reaction is the first step common to all subsequent metabolic transformations of glucose. Whether the synthesis of glycogen or the catabolic reactions of oxidation and glycolysis will be more prevalent will depend on various factors, such as the size of the hexose-6-phosphate pool, the concentration of inorganic phosphate, the ratio of adenosinetriphosphate (ATP) to adenosinediphosphate (ADP), hormonal influences and so on.

Other Metabolic Effects of Insulin. 6t

Stimulation of glycogen formation by insulin has been demonstrated ill muscle and liver. Since none of the reactions leading from glucose-6-phos- Phate to glycogen~is:impaired in the absence of insulin, it may be concluded that the increased synthesis of glycogen is a consequence of the increased SUpply of glucose-6-phosphate and that the r a t e of the hexokinase reaction is limiting for glycogen synthesis. Experiments on D,O-fed rats have shown (291) that :after the injection o f insulin a large proportion of glycogen is formed from glucose without a preliminary scission of' the 6-carbon chain; in the diabetic or the fasting rat, however, the synthesis of glycogen from glucose is largely replaced by a mechanism in which smaller units, such as lactic acid, are utilized for glycogen synthesis (35, 296). Similar results were obtained with liver slices of diabetic rats (243).

a) Liver.

The question whether insulin in, vivo increases the glycogen concentration of the .liver has remained controversial. In some of the earlier experiments hyPoglycaemia was allowed tO develop, but even when this complication: was prevented by the infusion of glucose the expected increase of liver glycogen Was not observed (2t, 40). SWENSSON (305), too, failed to find a significant effect of insulin on the liver glycogen of mice, provided hypoglycaemia did not OCcur. It isl doubtful whether these results can be explained entirely by the Possibility that the sample of insulin used was contaminated by glucagon (33). Paradoxically, the livers of diabetic animals often contain considerable amounts of glycogen and the livers of fasted diabetic rats, in particular, are USually richer in glycogen than those of controls which had been fasting for the same period i243, 260, 3 t0). This is presumably the result of an increased actiVit'y of pituitary and adrenocortical hormones leading to a more intense gluc~176 (2,43).

Experiments 'with liver slices in vitro are less ambiguous. Liver slices differ lrom other tissues, such as diaphragm or brain, in their inability to raaintain the intracellular level of potassium when suspended in an "extracellular" medium/such as Ringer solution (t09, t 77). This may be the reason Why~ an,~"intracel'lular" medium, {.e. a medium modelled on the ionic COmposition of tile intracellular fluid and therefore having a high concentration of potassium, calcium and magnesium, is required to demonstrate glycogen synthesis i n liver slices (t33, 135). While a hightpotassium, low-sodium medium promotes glYCogen synthesis and glucose uptake, 'a high-sodium, low-potassium m~dlur}i favours glycogenolysis and inhibits glucose uptake (135), The first medium, therefore, has effects similar to those of insulin, the Sec~ t0those Of insulin deficiency. But the insulin-like effects of a medium of the intracellular type are of no avail as far as liver slices of diabetic rats are Concerned. Compared with. normal, liver slices the glucose uptake of the

62 H. WEIL-MALHI~RBE: The Mechanism of Action of Insulin.

diabetic liver slices is reduced by about 98% and accordingly there is a decrease of their glycogen content instead of a rise (242, 243). While insulin is without effect o n the glycogen formation of normal liver slices in a high" potassium medium, it does restore the decreased glucose utilization and glycogen synthesis of the diabetic liver slices to normal values (134) 1 . In a low-potassium medium, on the other hand, the addition of insulin increases the glycogen synthesis of normal liver slices by 50 %, the glucose utilization by 40%, and it decreases the glucose output by t0% (t34).

b) Muscle.

The increased formation of muscle glycogen after the administration of insulin in vivo has been noted by many investigators (e. g. 297, 305). I t has also been demonstrated clearly in vitro with the aid of the excised rat diaphragm or of other sheets of striped muscle that are thin enough to allow a sufficient rate of oxygen diffusion. Some of the results obtained have been assembled in Table 4. Diaphragms of diabetic rats were found to form less glycogen than

Table 4. Glycoge~

State of animal

Norma l . . . . . . D iabe t i c . . . . . .

No rma l . . . . . . D iabe t i c . . . . . .

N o rma l . . . . . . D iabe t i c . . . . . .

N o rma l . . . . . . Mildly d i a b e t i c . . Severely d i a b e t i c .

Glucose concentration

(rag- %)

200 200

1 0 0

1 0 0

200 200

200 200 200

* D i a p h r a g m p re - incuba t ed

formation of r excised rat diaphragm.

Glycogen formation Insulin (mg/lO0 g tissue/hr)

concentration (units/ml) Insulin

- - +

0.5 0.5

0,8 0.8

0.5 0.5

0.1" 0.1" 0 . t *

for I rain in

! 7 62 14 61

18 50 26 43

t 0 200 ~0 70

69 35

- - t .2

insu l in-conta in ing solut ion, fol lowed by wash ing and incuba t ion in insulin-free solut ion,

Reference

3t4

3O9

32

284

those of normal rats; addition of insulin to the medium relieved the deficiency, even though incompletely.

The in vitro formation of glycogen by the rat diaphragm depends on numerous factors, especially the composition of the medium (t3, 290, 309), the previous dietary regime (t2t, t29, t65), the size and thickness of the tissue (194) and the rate of shaking during incubation (44). It has been

1 Elsewhere however , HASTINGS (132) s t a t e d t h a t insulin has no effect on d i ab e t i c l iver slices in t h e " in t r ace l l u l a r " m e d i u m .

The Effect of Insulin on Glucose Oxidation. 63

recommended to soak the diaphragm in bicarbonate-glucose-Ringer solution for a period before incubation (44). In view of so many variables it is not SUrprising to find that the results of different authors are not strictly comparable, although they do agree in showing an effect of added insulin.

In contrast to liver slices, the excised rat diaphragm has a smaller glycogen synthesis and a reduced insulin effect in a medium with a high potassium content (290, 309).

The enhancement of glycogen synthesis by insulin is accompanied by a shift of potassium from extracellular to intracellular spaces, in vivo (5 I, 345) and in vitro ( t59, 189). CALKINS, TAYLOR and HASTINGS (52) estimate that the deposition of t mg glycogen in the isolated rat diaphragm is associated with the uptake of 4.5/,moles potassium. The well-known decrease of the potassium concentration in plasma after the administration of insulin (130, 164) is probably connected with this effect.

2. The Effect of Insu l in on Glucose Oxidat ion.

Stimulation of glucose oxidation by insulin has been postulated by many Workers in the past, mainly because of a rise of the R. Q. after the injection of insulin, but the significance of this effect did not remain undisputed (cf. 276). It is generally recognized that insulin does not, as a rule, increase the net OXygen uptake of the living organism or of surviving tissues, though there are Some dissenting voices (5, t25, 136, t65). Unequivocal evidence has now been obtained with the aid of isotope experiments in which glucose uniformly labelled with 14C was administered; the specific activity of the respiratory CO2 served as an indicator of glucose oxidation. WIcI~ et al. (339) studied the effects of insulin on rabbits deprived of intestinal tract, liver and pancreas. The blood sugar of the preparation was maintained at the physiological level by a constant infusion of isotopic glucose of the same specific activity as that of the circulating plasma glucose. A priming dose of isotopic glucose of high Specific activity was injected at the beginning of the experiment, with the object of producing a rapid equilibration between the activities of plasma glucose and infusion glucose. In addition, some animals received 10 units of insulin intravenously at hourly intervals. Immediately after the administration of insulin the utilization of glucose rose, but the appearance of 14CO~ was slow at the start and reached its highest rate only after 6--8 hours. At that time the rate of glucose oxidation was 3-- t 5 times higher in the insulinized animals than in the controls (Table 5). The average increase ot glucose oxidation produced by insulin for the total period of all experiments was fourfold.

Similar in vivo experiments were done on normal and diabetic rats (105, 299, 300, 30t) and dogs (10"4), In the experiments of FELt.ER et al. (104, t05) a single dose of 1*c-glucose was injected; whereas the rate of glucose oxidation Was not much below normal in the diabetic rats, it was reduced by 60 % in the

64 H. WEIL-MALHERBE: The Mechanism of Act ion of Insulin.

T a b l e 5. E//ect o/insulin on the oxidation o/glucose.

t . P e r f u s i o n of e v i s c e r a t e d r a b b i t s (339).

W i t h o u t i n su l i n . . . . . . . . . . W i t h in su l in . . . . . . . . . . . .

2. R a t l ive r s l ices in vitro (57).

Glucose utilization I Glucose oxidation

(mg/l O0 g/hr)

12

50 3

2O

N o r m a l r a t . . . . . . . . . . . . D i a b e t i c r a t . . . . . . . . . . . .

% respiratory CO 2 derived from x4C-glueose (per g liver/3 br)

No insulin Insulin injection I hr before death

5 13 t.5 8

3. R a t d i a p h r a g m (314).

Normal rat . . . . . . . . . . . .

Diabetic rat

Glucose metabolized to CO~ (rag/100 g/hr)

No insulin insulin added i x vilrol

t 1 22 8 It

diabetic dogs, in spite of a high blood sugar, and was restored to normal by the injection of insulin. The "glucose pool" was much larger in these experb ments than in those with eviscerated rabbits, presumably owing to the presence of the liver. In the experiments of STETTEN et al. (299--301) rats were given a constant intravenous infusion of 14C-glucose at s u c h a rate that continuous glycosuria resulted. In diabetic animals the oxidation of glucose was reduced by about 60 %.

In vitro experiments yielded similar results. CHERNICK e~ al. (58, 60) showed that the oxidation of 14C-glucose ', but not that of ~4C-fructose, was lowered in liver slices from diabetic rats (cL Table/5). This was confirmed by RENOI.D et al. (242, 243), who foUnd a decrease of two thirds in the glucose oxidation of diabetic liver slices. VILLEE and HASTINGS (3!4), working with diaphragms of normal and diabetic rats, f0{ind that the oxidation of ~4C-glucose was decreased in diabetic diaphragms; addit ion of insulin to the medium increased glucose oxidation in both normal and diabetic diaphragms (Table 5). SACKS and SINEX (255) confirmed that the oxidation of glucose by the diaphragm of non=diabetic rats was increased when insulin was .added to the suspension medium, but BELOFF-CHAIN el all (tl) failed to find this effect.

The Effect of Insul in on Phospha te Metabolism. 55

The effect of insulin on the oxidation of glucose can be understood if it is assumed that the conversion of glucose to glucose-6-phosphate is the rate: limiting step.

3. The Effect of Insu l in on Phosphate Metabolism.

a) Effects on Inorganic Phosphate.

One of the oldest observations concerning the effects of insulin is the lowering of the concentration of inorganic phosphat e in plasma (cf. 70). The effect is especially marked when glucose and insulin are administered together (77, t79). I t has been shown (190) that an intravenous injection of glucose accelerated the rate of disappearance of inorganic s2p from human plasma by a factor of 3, an injection of insulin (0.t u./kg, i. v.) by a factor of 4. Most of the phosphate which disappears seems to go into the tissues rather than into the red blood cells (t65, 33t), though some increase of the acid-soluble phos- Phate fraction of red blood cells has also been reported (i 63).

b) Effects on Hexosephosphates.

DI~ DUVE et al. (98, 99) showed that the injection of insulin into rabbits Which received sufficient glucose to maintain normoglycaemia led to the deposition of hexosemonophosphate in skeletal muscle, the increase amounting to 22--44% in 80 minutes. This was confirmed by HAUGAARD, MARSH and STADIE (138) with the excised rat diaphragm ./n vitro; after an incubation of t hour an increase of 34 % was found in the presence of insulin. SACKS and SIN~x (255) also found that the concentration and the turnover of the hexosemonophosphate fraction of the isolated rat diaphragm were increased When insulin was added to the suspension fluid. In rabbits injected with 3~p, iUsulia raised the Specific activity of the hexosemonophosphate fraction of rauscle (t63). Muscle does not normally contain any hexosediphosphate and none is found after the injection of insulin (75, 87).

Similar results were obtained with liver tissue. Insulin treatment increases the specific activity of the barium-soluble phosphate fraction of liver in rabbits ilaiected with ~2p (t63). Fasting, or a high-fat diet, causes a reduction of the Phosphate ester fraction of rat liver, an effect that can be reversed by the administration of glucose or insulin (t60, 217, 239). In the liver of diabetic rats injected with a2p the specific activity of glucose-l-phosphate is smaller than in that of normalcontrols (254).

c) Effects on High-Energy Phosphates.

Insulin increases the turnover of phosphocreatine and ATP in resting skeletal muscle of the cat (253) and the rat (124), and in the isolated rat diaphragm (255).

~rgebnisse der Physlologie, Bd. 48, 5

66 H. WEIL-MALHERBE: The Mechanism of Action of Insulin.

Both glucose and insulin increase the turnover of acid-labile phosphate in human blood (t 79).

Insulin injection induces a rise of the ATP concentration in the liver of fasting rats (~62). At the same time it stimulates the incorporation of 3~F into the labile phosphate groups of ATP, an effect that can also be produced by the injection of glucose and that is especially large when glucose and insulin are combined (160). In livers of diabetic rats, on the other hand, the turnover of acid-labile phosphate groups is reduced (254). Fasting, or a high-fat diet, has results opposite to those of insulih or glucose. I t produces in rat liver an increased breakdown of acid-labile phosphate groups (16t,

2~7, 239). GORANSON and ERULKAR (123) studied the phosphorylation of creatine

in vitro, using minced heart and homogenized brain~of the rat, with succinate or malate as substrate. They found that the reaction was depressed in the tissues of diabetic rats and that it could be restored by a previous t reatment of the animals with insulin. I n vitro addition of insulin to brain homogenates of diabetic rats increased the synthesis of phosphocreatine with both substrates. This result is remarkable not only because it is one of the few examples of an insulin effect on a broken-cell tissue preparation, b u t also because it was apparently obtained in the absence of glucose.

d) Effects of Insulin on Reactions Depending on High-Energy Phosphates.

The acetylation of p-aminobenzoic acid is deficient in diabetic rats. The defect is due to a relative deficiency of ATP, since it can be repaired by the injection of ATP. Administration of insulin has the same effect (54). The synthesis of p-aminohippuric acid by liver homogenates of diabetic rats is 15 % lower than in those of normal controls (48).

A depression of thiamin phosphorylation in diabetic animals has been described by FoX, SMITH and WEINSTEIN (t 13), they found that the intravenous injection of thiamin led to a rapid rise of the diphosphothiamin concentration in the blood of the normal dog. The ability to phosphorylate thiamin was enhanced by the simultaneous administration of insulin, diminished after the removal of the pancreas and again restored by insulin. These results agree with those of SILIPRA~CDI and SILII'RA~DI (163), who showed that administration of thiamin led to a marked increase of the diphospothiamin concentration in normal, but not in diabetic, rat liver. The defect can be restored by t reatment of the diabetic rat with insulin (264). In a second publication Fo~ et al. (tt3) also found a lower ratio of phosphorylated to free thiamin in the liver of diabetic rats. Their claim to have shown an effect of insulin on the rephosphory- lation of thiamin by liver homogenates in vitro is, however, not convincing. Insulin appeared to inhibit the dephosphorylat!on of thiamin phosphates, but it is open to question whether this inhibition is to be at tr ibuted to insulin itself

The Effect of Insulin on Phosphate Metabolism. 67

or to some impurity such as traces of heavy metal. Even if it was caused by insulin itself, it is hardly sufficient evidence for an effect of insulin on thiamin phosphorylation in the homogenate. When thiamin was added to liver homogenates from diabetic rats, the mean thiamin phosphate content per 3 ml rose from an initial value of 2.2/~g to 4.4 #g in the absence of insulin and to 4.8 pg in the presence of insulin. No figures were given which allow the significance of this small increase to be assessed.

Stimulation by insulin of oxidative phosphorylation in a liver homogenate Was also reported by Ru~Fo and CE~AMO (250), but a closer analysis of the effect by RuFFo, D'ABRAMO and GULLINO (251, 252) revealed that it was due not to insulin itself, but to an impurity, probably glycerol, which acted as phosphate acceptor.

e) Comment.

It appears from the foregoing review that insulin decreases the concentration of inorganic phosphate in plasma and extracellular fluid and increases the concentration and the turnover of hexosephosphates and of high-energy phosphate groups in the tissues. The same effects may be obtained, under suitable conditions, with glucose alone, but only in the normal, not in the diabetic, organism.

The fall Of the inorganic phosphate concentration in plasma which is observed in the normal animal after glucose administration is absent in the diabetic (277). On the other hand, the administration of fructose produces the same reduction of the phosphate concentration of plasma in diabetics and non-diabetics (t92). Since the utilization of fructose is unimpaired in diabetes, while that of glucose is inhibited, the change in plasma phosphate COncentration is clearly secondary to the phosphorylation of the hexose and is therefore not a direct effect of insulin.

The increased rate of glucose phosphorylation which follows upon insulin administration accounts for the increased concentration of hexosephosphates in the tissues. The accelerated turnover of high-energy phosphates is also Consistent with a higher rate of glucose catabolism, since there is a net gain of 2 high-energy phosphate bonds, even when glucose is only converted to lactic acid. SACKS and SINEX (255) studied phosphate transfer and glucose Uptake by incubating the excised rat diaphragm in a solution containing 32p and glucose uniformly labelled with 14C. They found that for each molecule of glucose about two molecules of phosphate were taken up by the tissue. Insulin speeded up both rates to about the same degree. On the other hand, the reduced carbohydrate metabolism in starvation, diabetes or prolonged hyPoglycaemia seems to result in a depletion or a reduced turnover of high- energy phosphate groups, at least in liver (t6t, 217, 239, 254) and brain (2t8). This is understandable as far as brain is concerned, since its energy metabolism

5*


is dependent on glucose. I t is less obvious in liver and is, in fact, in disagreement with the results of ENNOR and STOCKEN (t09) who found an increase of high* energy phosphate groups in the fa t ty livers of non-diabetic guinea pigs poisoned with carbon tetrachloride.

4. The Ef fec t of Insu l in on the Oxidation of C o m p o u n d s Other t h a n Glucose.

It is not difficult to recognize that the insuhn effects decribed so far are connected with a higher rate of glucose utihzation. This connection is less evident when certain other effects of insulin are considered.

KREBS and EGGLESTON (t76) found that insulin (ca. t unit/ml.) delayed the fall of respiration of minced pigeon breast muscle when it was incubated in a medium containing boiled muscle extract and a component acid of the citric acid cycle. The effect was most pronounced in the third and fourth hour of the experiment when respiration had almost s topped in the control experiment; at the beginning of the experiment the effect was hardly noticeable. These results have been confirmed (262, 29t, 293), although other investigators found smaller effects than KREBS and EGGLESTON. According to S~IORR and BARKER (262) slices of pigeon breast muscle which contain many intact fibres and have a steady and vigorous respiration do not respond to insulin. STADIE, ZAPP and LUKENS (29t) found no effect of insulin in minced muscle from depancreatized pigeons two days after the operation, but STARE and BAUMANN (293) found it to be enhanced in the second week after pancreatectomy; after four weeks the pigeons had apparently recovered from the operation and at this time the insuhn effect could no longer be detected.

�9 RICE and EVANS (245) showed that the utilization of pyruvate was increased in minced or sliced pigeon breast muscle after pre-incubation with insulin (1 unit/ml.). This effect, too, has been confirmed (285).

These effects of insulin are restricted to pigeon muscle. They could not be obtained with muscle from other species, whether mammalian or avian (262, 285, 29t), nor did they appear after pancreatectomy in dogs (262) or cats (29t). As pointed out by STARE and BAUMANN (293) the pigeon is exceptional in having normally a high blood sugar and in being unusually resistant not only to the removal of the pancreas, but also to enormous doses of insulin. It is therefore doubtful whether these observations are of general significance, especially in view of the fact that the utilization of pyruvate is normal in diabetes (t t2, 2t2) and unaffected by insulin injection (47). It has further been shown that the combustion of isotopic lactate, pyruvate and acetate to CO 2 proceeds at the normal rate in liver slices of diabetic rats (106, t07, 2t9) and that the oxidation of acetate by the extrahepatic tissues of the rabbit is unaffected by insulin (9~). A voluminous literature exists on

The Effect of Insulin on the Oxidation of Compounds Other than Glucose. 69

the utilization of acetoacetic acid; most authors agree that it is not impeded in the diabetic and not influenced by insulin (17, 49, 53, t0t, 2t6, 292), Thi~ is not to say that an inhibition of ketolysis can be excluded as a contributory cause of severe diabetic ketosis.

MASoRo and ABI~AMOVlTCH (207) recently reviewed the literature on the effect of insulin on the metabolism of ethanol. Their own experiments in Which slices of rat kidney and liver were incubated with 14C-ethanol disclosed no difference in the rate of ethanol metabolism in slices prepared from insulin- treated rats or from untreated controls.

There are however some observations which seem, at first sight, to imply an action of insulin on the utilization and oxidation of pyruvate and acetate (229, 315, 3 t 6). In heart slices from diabetic rats the utilization and oxidation of pyruvate was found to be reduced by about 46 % ; insulin in vitro had no effect on the inhibition. The utilization and oxidation of acetate, on the other hand, was not reduced. In the excised diaphragm of the diabetic rat the Oxidation of both pyruvate and acetate was found to be depressed. The Oxidation of pyruvate, but not that of acetate, was restored to normal by the addition of insulin in vitro. Insulin had no effect on the utilization or oxidation of pyruvate by the diaphragm of the non-diabetic rat.

Those effects that were not corrected by the in vitro addition of insulin, via. the oxidation of pyruvate in diabetic heart and that of acetate in diabetic diaphragm, are presumably secondary results of the diabetic condition. This is known to entail a depletion of high-energy phosphate groups and other coenzymes and a shortage of the supply of C4-dicarboxylic acids without Which the citric acid cycle cannot function. Incidentally, it is these factors Which are responsible for the reduced ketolysis of the diabetic.

The question, however, arises as to whether there is a primary effect of insulin on the oxidation of pyruvate in the diaphragm of the diabetic rat. The following facts suggest that this assumption need not be made: it is known that the oxidation of pyruvate and other substrates is coupled with the formation of energy-rich phosphate bonds and that, in the intact cell, the rate of oxidation is limited by the discharge of the energy stores. One of the most efficient acceptor systems for the disposal of high-energy phosphate is the combination of glucose and hexokinase; its addition to suitable tissue preparations greatly increases the rate of oxidation of pyruvate and other Substrates (t 83, 238)..The effect o4 insulin on pyruvate oxidation might thus result from its activation of glucose phosphorylation and the higher rate of discharge of high-energy phosphate groups. It is true that the effect of insuhn OCCurred without the addition of glucose, but the tissues of these diabetic rats must have contained a more than negligible amount of glucose in blood and extracellular fluid. It might have been largely washed out of the more finely divided heart slices during preliminary storage, but it would probably

70 H. WXzLLMALHERBTt: The Mechanism of Action of Insulin.

have been retained to some extent in the intact diaphragm. This would account for the difference observed in these two tissues.

I t is emphasized that this interpretation is not applicable to the insulin effect in minced pigeon breast muscle, since this preparation contains hardly any glucose (289).

5. The Effect of Insu l in on Lipogenesis .

One of the most important reactions initiated by the uptake of glucose in the tissues is the conversion of glucose to fat. Since insulin increases the utilization of glucose in general, it is only to be expected that it also activates the synthesis of fat from glucose and that, conversely, this process is inhibited in diabetes and again improved under insulin treatment (296--298).

The capacity of rat liver slices for lipogenesis from isotopic glucose was found to be severely curtailed after the induction of diabetes as well as after prolonged starvation or deprivation of carbohydrate (60, t42, 208); it could be restored by a preliminary treatment of the diabetic rats with insulin (57). An injection of insulin, given to normal rats one hour before death, led to a tenfold increase of fat synthesis from isotopic glucose by liver slices (57). While slices of adipose tissue of normal rats actively convert glucose into fatty acids, this capacity was lost in slices of adipose tissue from diabetic animals (t42).

There is, however, in diabetes a reduction of fat synthesis not only from glucose, but also from lactate, pyruvate and acetate. I t was this observation in particular which seemed incompatible with the single-point theory of insulin action (38, 58, 106, t07, 219). In vitro addition of insulin increases lipogenesis from acetate in liver slices of the normal, well-fed rat (18, 37, 39, 210), but observations on its effect in liver slices of the diabetic rat are contradictory (38, t40, 219). Treatment of diabetic rats with insulin in vivo restores the ability of liver slices for fatty acid synthesis from lactate, pyruvate, acetate or acetoacetate (56, t06, t07, 2t9).

A closer analysis of the phenomenon revealed the following facts. The formation of fatty acids from acetate by liver slices of non-diabetic rats was greatly reduced after fasting. The deficiency was not relieved by a fat or protein meal, or by insulin in vitro; it was, however, corrected by the feeding of glucose or by its addition in vitro and even more so, when the medium contained both glucose and insulin (204, 209). In similar experiments MEDES, THOMAS and WEINHOUSE (210) found that addition of insulin in vitro did not affect the conversion of acetate into fatty acids in liver slices of fasting rats, but that a response could be obtained in the liver slices of fed rats. The action of insulin is thus clearly shown to be secondary to its activation of glucose utilization; the effect of feeding suggests that the insulin effect also depends on the presence of glycogen in the liver. HAUGAARD and

The Effect of Insulin on Lipogenesis. 71

STADIE (~t39) compared fatty acid synthesis from acetate in liver slices from rats on low- and high-carbohydrate diet~ and found that lipogenesis was a linear function of the initial glycogen content of the slices. Although liver slices of starved or diabetic rats have an undiminished capacity for the oxidation of fructose, the formation of fat from fructose is as severely restricted as that from glucose (58, 346). But when fructose is fed to diabetic rats, the capacity of liver slices for lipogenesis is restored whether fructose or glucose is used as substrate (204). I t can be assumed that fructose is utilized by the diabetic rat for the formation of liver glycogen and that the resumption of lipogenesis is due to this effect.

A prior injection of cortisone into normal rats inhibits lipogenesis from acetate by liver slices (39); this is in harmony with the general antagonism of insulin and cortisone. However, a synergistic action of cortisone and insulin has been observed in experiments in which lipogenesis from acetate by the perfused rat liver was studied. Neither cortisone nor insulin added singly had any marked effect, but a large increase was observed when both hormones Were added together (2, 333). A possible explanation would be that the increased rate of glycogen formation from non-carbohydrate precursors in the liver which cortisone is known to produce might be a prerequisite for the action of insulin.

Since the pathway of liposynthesis or diabetic rat, lactate, pyruvate and (106, t07, 2t9). Insulin reverses this

is blocked in the liver of the fasting acetate are oxidized at a faster rate situation by enabling more .of these

substrates to be fed into the channels of lipogenesis. The variations in the rate at which the 2- and 3-C-compounds are oxidized in the liver are accompanied by similar variations in the degree of ketogenesis.

Two questions now arise. If the point of action of insulin is at the level of the hexokinase reaction, then the utilization of glycogen must be independent of insulin. How can this be reconciled with the fact that the action of insulin on lipogenesis depends on an adequate level of glycogen in the liver ? It is suggested that this apparent contradiction is due to the presence of glucose- 6-phosphatase in liver. The concentration of glucose-6-phosphate in liver is the result of a dynamic equilibrium between the activities of hexokinase arld glucose-6-phosphatase and if the rate of the hexokinase reaction is diminished owing to insulin deficiency, the activity of glucose-6-phosphatase raay predominate; .thus the utilization of glycogen may become inhibited. This interpretation is supported by the results of HAUGAARD and STADIE (140} who found that lipogenesis from acetate was decreased by hyperglycaemic factor or adrenaline, both of which have a stimulating effect on glycogenolysis and therel~y increase the leakage of glucose (304).

The second question is: by what mechanism does the utilization of glycogen or glucose promote the synthesis of fat from acetate and related compounds ?

7~ H. WEIL-1V~ALHERBE: The Mechanism of Action of Insulin.

Important information relevant to this problem has come from the study of liposynthesis in lactating mammary tissue (t 15). Slices of lactating mammary gland of rodents (mouse, rat, rabbit) were found to utilize acetate for the synthesis of fatty acids, but only in the presence of glucose, not in its absence. This process was stimulated by the in vitro addition of insulin, again only if glucose was present. A similar activation of lipogenesis from acetate could be obtained by the addition of glycerol, though this effect was not accompanied by an increased utilization of glucose, such as was observed when insulin was added. The effects of insulin and glycerol were not additive.

On the basis of these observations ]3ALMAIN and FOLLEY (5) and BALMAIN, FOLLEY and GLASCOCK (6) suggested that liposynthesis from acetate may be limited in the lactating mammary gland by the availability of glycerol and that the effect of insulin may consist largely in accelerating the formation of glycerol from glucose.

A somewhat different situation is met with in the lactating mammary gland of ruminants (sheep, goat, cow). Here fatty acid synthesis from acetate proceeds in the absence of glucose, though it is accelerated if glucose is present. Addition of insulin to the medium has no effect, whether glucose be present or not. Addition of glycerol, on the other hand, increased fatty acid synthesis from acetate alone, but had no effect in a medium containing both glucose and acetate (6). These observations are in agreement with known peculiarities 6f ruminant metabolism; the ruminant udder is capable of synthesizing hexoses (8t) and glycerol (237) from acetate, while the breakdown of glucose to 2-carbon compounds is negligible (7). Each of these facts tends to reduce the influence of added insulin or glycerol. Moreover, ruminants have normally low blood sugar values (35--65 mg-%) and are relatively resistant to the induction of experimental diabetes, whether by pancreatectomy or by alloxan (69, 205). Insulin thus seems to be a factor of minor importance in their metabolism.

The hypothesis that liposynthesis from acetate is limited by the supply of glycerol also explains some of the observations on liver slices. The fact that the insulin effect depends on an adequate level of glycogen or, in the absence of glycogen reserves, on the presence of glucose in the medium is understandable if the effect is due to an increased supply of glycerol. Such a mechanism is compatible with the known action of insulin on the hexokinase reaction and thus eliminates the need for the assumption of a second point of action. It finally explains another observation made by several investigators: whereas the synthesis of fatty acids from acetate is inhibited in the liver and other tissues of the diabetic rat, the synthesis of cholesterol from acetate is unimpaired or even accelerated, i~, vivo (46) and in vitro (38, 45, 154). This fact shows that it is not lack of available energy which is responsible for the reduction of lipogenesis in diabetic liver, since in that case presumably

The Effects of Insul in on Nitrogen Metabolism, 73

all synthetic processes would be similarly affected. The insulin effect must therefore be connected with a more specific mechanism which is not applicable to cholesterol synthesis. The limitation of fatty acid synthesis by glyceride formation is such a mechanism.

In their la tes t publication BALMAIN, FOLLEY and GLASCOCK (7) have Somewhat modified their views. They carried out ingenious experiments in which they incubated rat mammary gland slices in a solution containing glucose uniformly labelled with 14C and acetate labelled in the carboxyl group with 13C and in the methyl group with tritium; from their results they concluded that both insulin and glycerol increased the rate of incorporation of acetate carbon and of glucose carbon into fatty acid~, but, whereas insulin Stimulated both processes more or less to the same extent, glycerol increased the utilization of acetate more strongly than that of glucose carbon. They found it difficult to reconcile this result wi th the hypothesis that the two mechanisms are identical. I t may be argued, however, that the action of insulin is followed by two distinct events: (1) by an increased formation of glycerol and (2) by an increased formation of 2-carbon c o m p o u n d s . The Second effect will compete in lipogenesis with the acetate present and will thus reduce the incorporation of acetate carbon into lipids. While the addition of glycerol itself results in increased lipogenesis, its oxidation to 2-carbon COmpounds may be slower than that of glucose in the presence of insulin; Since glycerol, unlike insulin, presumably does not increase the supply of 2-carbon compounds from glucose, any increase in the utilization of 2~carbon COmpounds for lipogenesis may have to be met mainly by the available acetate.

6. The Effects of Insu l in on Ni t rogen Metabol i sm.

The ramifications of insulin action extend into the metabohsm of nitrogenous compounds. Again the problem is whether these effects can be explained by the effect o f insulin on the hexokinase reaction or whether different mechanisms have to be postulated.

The concentration of amino nitrogen in plasma, like that of potassium and inorganic phosphate, is decreased after an injection of insulin, a fact that was recognized early in the history of insulin (167, 168, 199 and others). This decrease may be due to a lower rate of formation or a higher rate of Withdrawal, i. e. to art inhibition of proteolysis or to the promotion of proteosynthesis. The indications are that both of these mechanisms are operating, With the accent on increased proteosynthesis. I t is, of course, a well-established clinical observation that the proteins of the body may serve as fuel and are drawn upon in emergencies, auch as fever, hunger or diabetes. In these situations insulin, or insulin plus carbohydrate, will, by promoting the utilization of glucose, exert a sparing action on nitrogen metabolism. According


to HOBERMAN (149) the stimulation of proteolysis in diabetes is attributable to adrenocortical hormones and the inhibition of proteosynthesis to lack of insulin.

The plasma level of amino nitrogen is usually higher in venous than in arterial blood, showing that amino nitrogen is liberated from the tissues. This difference is accentuated in diabetes (t 69). The drop in plasma amino nitrogen which occurs after insulin administration can be understood as an inhibition of proteolysis due to the promotion of glucose utilization and this interpretation is supported by the observation that glucose administration alone produces changes in the same direction (t3t, 266, 308). BONE and REID (26), on the other hand, report that the fall of plasma amino nitrogen in hypoglycaemic cats can be prevented, not only by glucose, but-also by other oxidizable substrates, such as olive oil. In View of the multiplicity of factors influencing the plasma amino-N level, i .e. the rates of proteolysis and proteosynthesis, of amination and deamination, occasional discordant results of this kind are not surprising.

That the fall of plasma amino-N level after insulin administration is not only due to an inhibition of proteolysis, but also to an increased proteosynthesis, is suggested by the findings of LOTSPEICH (t96). He studied the decrease of the t0 essential amino acids in the blood of dogs after the injection of insulin a n d found that they were removed in proportions closely corresponding to the composition of muscle protein. He later found (197) that identical changes could be initiated by growth hormone, but only in the normal, not in the diabetic dog, and he suggested that growth hormone required the synergistic action of insulin to stimulate protein synthesis, a conclusion previously reached by MI~SKY (2t 5).

The increase of the amino acid level in plasma which takes place after hepatectomy (25) is suppressed by insulin (1t7, 156, 214). This effect is duplicated by glucose at hyperglycaemic levels ( t l 0 ) o r by insulin plus glucose ( i lQ, which again points to the acceleration of glucose metabolism as the mechanism responsible.

KRAHL (t 71, t 72) studied, on normal and diabetic rats, the incorporation of isotopic glycine into glutathione by liver slices and the incorporation Of isotopic glycine and phenylalanine into protein by liver slices and by the excised diaphragm, with or without the addition of insulin to the medium (0.t unit/ml). He found a reduced synthesis in the tissues of fasting rats, which could be restored by the addition of glucose, but not of insulin, to the medium. Insulin only acted on the tissues of well-fed animals. Synthesis was still more reduced in the tissues of diabetic rats: in liver slices insulin, without glucose, had no effect; with glucose present 14C-incorporation was increased, and even more so with insulin plus glucose. In the diabetic diaphragm glucose alone was sufficient to relieve the inhibition and insulin plus glucose produced

The Effects of Insulin on I~itrogen Metabolism. 75

only a slight additional effect. These results demonstrate tha t there is not only an increased proteinbreakdown, but especially a reduced proteosynthesis in diabetes, and further, that the effect of insulin is exerted via its effect on glucose utilization. The incorporation of isotopic methionine (1 t 6) and alanine (265) into muscle protein has also been shown to be stimulated by insulin. Whether a higher rate of glucose utilization activates protein synthesis by an increased Supply of energy or by a more specific mechanism remains to be decided.

It has been suggested that insulin inhibits the deamination of amino acids. This view is based on the experiments of BACH and HOLMES (4) who found a decreased formation of urea b y liver slices, with or without added DL-alanine, when insulin was present in the medium. Since the inhibition of urea formation was not accompanied by the appearance of ammonia, the authors attributed it to an inhibition of deamination. STADIE, LUKENS and ZAPP (288) showed that insulin did not inhibit urea formation in the absence of added alanine and this has since been confirmed (67). Neither was there any inhibition of urea formation from natural L-amino acids by insulin. Only the urea formation from the non-natural D-isomers was inhibited. In liver slices of diabetic cats, but not in those of hypophysectomized diabetic cats, urea formation was increased by 46 %. The amino acid dehydrogenase activity of tissue extracts was not affected by the addition of insulin.

KREBS and EGGLESTON (t76) suggested that the effect of Baca and HOLMEs was caused by the sparing action of insulin on non-carbohydrates, but such an action would not be expected, to affect specifically the non- natural amino acids. Whatever the explanation, it seems improbable that an effect of insulin o n the metabolism of the non-natural amino acids can contribute much to the understanding of its principal physiological functions.

7. Conclusions.

The discussion of the metabolic effects of insulin has shown that, with two possible exceptions, they are consistent with the theory that insuhn has a single point of action; its primary effect is an acceleration of the hexokinase reaction which brings about a higher rate of glucose utihzation, Most of its other effects are secondary consequences of the primary effect. The two exceptions which cannot, as yet, be satisfactorily connected with the hexokinase reaction are: the activation of pigeon breast muscle respiration with pyruvate or other members of the citric acid cycle as substrate, and the inhibition of the deamination of non-natural amino acids. In the first case, the effect is restricted to a single Species with an unusual behaviour towards insulin; in the second case, the effect concerns a reaction whose metabolic function is unklm'wn. The physiological significance of these effects is problematical; it is unlikely that they are involved in the general metabolic tasks of insulin and they can, for the present, be regarded as side effects.


III. The Mechanism of the Insulin Effect.

Having fixed the locus of insulin action at the level of the hexokinase reaction we may now consider its mechanism in greater detail. The hexokinase reaction is formulated b y the following equation:

Hexokinase Glucose + A T P - - *Glucose-6-phosphate + ADP.

There are three participants of the reaction: glucose, ATP and catalyst. The reaction rate may be limited by the concentration of any one of them, and consequently three possibilities present themselves: t. an effect of insulin on the supply of ATP, 2. an effect of insulin on the hexokinase molecule and 3. an effect of insulin on the supply of glucose.

x. Insul in and A T P Concentrat ion.

The ATP concentration of animal tissues varies between t -- 8 • 10 -3 mol/kg. Since ATP is not present in the extracellular fluids and since muscle tissue, for instance, contains about 64 %, and heart tissue about 54 %, of intracellular

w a t e r (198), the actual intracellular concentration of ATP is of the order of 0 .2--1.2Xt0-2mol/1. The MICHAELIS constant of animal hexokinase for ATP was estimated to be 8 • -4 M by WEIL-MALHERBE and BONE (3t9) and t.3 • t 0-* M b y SoLs and CRANE (268). Even if the larger of these values is assumed as correct, it is clear that the intraceUular concentration of ATP is sufficient to saturate the enzyme, unless, of course, free access is restricted by intracellular barriers. WALAAS and WALAAS (3 t8) found no indications that ATP concentration was a factor which limited the rate of glucose uptake in diaphragms of normal o r diabetic rats under aerobic conditions. Even anaerobically, when the ATP concentration was reduced to about 10% of normal, the rate of glucose uptake was not reduced.

POLLS et al. (235) studied oxidative phosphorylation in a mitochondrial preparation of rat liver, with ketoglutarate as substrate, and they found that the addition of insulin resulted in a significant increase of the P/O-ratio, while the addition of a preparation of soluble adenosinetriphosphatase inhibited both oxygen uptake and phosphorylation. The authors further claimed that insulin relieved the inhibitory effect of ATPase and suggested that insulin acted b y inhibiting cellular ATPase, thereby increasing ATP concentration. The reversal of inhibition by insulin was in fact variable and was complete in only one case out of three. In the other two experiments the effect was slight. Some of the P/O-ratios reported in this paper are improb- ably high. The alleged inhibitory effect of insulin on ATPase act ivi ty could

not be confirmed by BROH-KAHN et al. (41).

The Effect of Insul in on Hexokinase. 77

2. The Effect of Insulin on Hexokinase . If insulin is acting directly on the hexokinase molecule, an effect demon-

strable in tissue extracts and solutions of purified enzyme would be expected. The experiments of COLOWICK, Co~I and SLEIN (68) provided some evidence Qf such a mechanism. These authors decribed an inhibition of the hexokinase activity of brain and muscle extracts by an unstable protein fraction of the anterior pi tui tary gland. The inhibition was enhanced by adrenocortical extract which by itself had no effect on the muscle hexokinase of normal rats, although it did inhibit the muscle hexokinase of diabetic rats. These inhibitions were reversed by insulin in vitro, whereas insulin alone did not affect the activity of the uninhibited hexokinase. The observations of COLO- Wlci~ et al. account for some hormonal actions on intact tissues, but not for others: since insulin increases the glucose uptake of the diaphragm of hyPophysectomized or hypophysectomized-adrenalectomized rats which were presumably deprived of hormonal inhibitors (29, 175, 230, 3t4), an activation of uninhibited hexokinase by insulin might have been expected.

When the experiments of COLOWICK et al. were repeated by other investigators these effects were found to be inconstant and exceptional (42, 240, 267). STADIE and HAUGAARD (279), working with muscle extracts of diabetic rats, and STADIE, HAUGAARD and HILLS (280), using muscle extracts of depancreatized cats, failed to find a decrease of hexokinase activity in these preparations or any effect on it of adrenocortical extract or insulin, singly or together. CHR~STENSEN et al. (63) studied the hexokinase activity of cytolysed rat blood corpuscles and found no significant difference with blood from normal, diabetic and hypophysectomized animals, either in the presence or in the absence of insulin or adrenocortical extract. Coal himself (71) referred to the fact that the experiments showing hormonal effects on hexokinase in solution did not have the desired degree of reproducibility.

A factor occurring in red blood ceils, muscle and possibly other animal tissues which activates the hexokinase reaction in brain extracts has been described by WEIL-MALHERBE and BONE (320, 321). It was found to be a protein and to be Specific inasmuch as it acted only on hexokinase, but not, for example, on the related enzyme phosphohexokinase, and was not replace- able by other proteins. No evidence was found for the assumption that the activation was due to the removal of the inhibitory product of the reaction, glucose-6-phosphate, or to the protection of functional groups [cf. STERN, (295)].

The same authors (322) found activators and inhibitors of hexokinase in human plasma . They collected consecutive blood samples from the same subject at regular intervals' for a period of several hours and measured the effect of the plasma on the hexokinase activity of rat brain and muscle extracts. No significant effects were observed with plasma from subjects under basal

?8 H. WEIL-I~ALHERBE" The Mechanism of Action of Insulin.

conditions, b u t activators or inhibito!s of hexokinase were found to appear in response to metabolic or stressful stimuli, such as a glucose meal, a convulsion or insulin hypoglycaemia. A correlation was established in glucose tolerance tests between the appearance of a hexokinase inhibitor and a high blood sugar rise, on the one hand, and between the appearance of a hexokinase activator and a slight blood sugar rise, on the other. The addition of insulin in vitro modified the plasma effects considerably. Activation effects were moderately, inhibition effects more strongly, depressed. In about half of a small number of diabetic patients deprived of exogenous insulin an inhibitory factor appeared in plasma in response to a mixed meal. This inhibitory response tended to decline or disappear after the patient had received an injection of insulin. The presence~of an inhibitory factor in the plasma of some diabetics has been confirmed by GERRARD (t 20) who concluded from his results that the effect showed some correlation with ketosis.

BORNSTEIN and PARK (30) and BORNSTEIN (27) reported that the fl-lipoprotein fraction of the serum of diabetic rats inhibited the glucose uptake of the isolated diaphragm of normal fasted rats. The inhibition was reversed by insulin in vitro. Adrenalectomy or hypophysectomy of diabetic rats caused the inhibitor to disappear from plasma; neither was any inhibitor found in the blood of normal rats. Injection of growth hormone and cortisone together into hypophysectomized diabetic rats restored the inhibitory effect of serum, while either substance alone was ineffective. The in vitro addition of the hormones, combined or singly, or the injection of one hormone and the in vitro addition of the other were equally ineffective.

While these results, which were obtained with an intact tissue, have no immediate bearing on the possible interaction between the hexokinase molecule and insulin, KRAHL and BORNSTEIN (173) later investigated the effect of lipoprotein fractions from diabetic plasma, anterior pi tui tary and other tissues on the hexokinase activity of muscle extracts. They obtained an inhibition, although the amounts of plasma lipoprotein required varied from sample to sample. The addition of insulin, o. t unit]ml, gave very irregular results, reversal of the inhibition being observed in 12 tests out of 42. These observations, therefore, are in agreement with those of WEIL-MALHERBE and

BONE (322). ZAcco (348) and ZACCO and SEVAG (349) found an inhibitor of hexokinase

in the freshly collected plasma of tuberculous patients and also in about 14 % of normal plasma specimens. The inhibitor was quickly inact ivated on standing, but could be reactivated by dialysis against distilled water. Even plasma specimens which were inactive before dialysis showed an inhibitory effect after dialysis. The effect of the plasma inhibitor was not counteracted by insulin. These observations have been confirmed and extended by STERN

in the writer's laboratory (295).

Insulin and Glucose Concentration. 79

ABOOD and GERARD (1) described an inhibitor of hexokinase in nerve homogenates. A repetition of their work by STERN (295) did not confirm the existence of an inhibitory action separable from ATPase activity. The effect of the plasma inhibitor, on the other hand, could not be accounted for by ATPase activity.

REIss and REEs (24t) found that t0--12 days after hypophysectomy the hexokinase activity of rat brain homogenates was increased by 37--t40 %. Rats that were adrenalectomized 2--5 days previously showed a similar increase. Anaerobic glycolysis was also increased in brain slices of hypophysectomized rats, but respiration was unchanged.

From the evidence here reviewed we may conclude that the hexokinase activity of extracts is subject to the influence of specific inhibitors and acti- Vators occurring in blood and tissues. The effect of these factors is sometimes modified by insulin in vivo or in vitro, but the activity of insulin is variable and its reproducibility is unsatisfactory. Moreover, the effect of insulin is riot always in the expected direction. These phenomena therefore do not provide a sufficient basis for the mechanism of action of insulin.

3. i n s u l i n and Glucose Concentrat ion .

a) The Mass Action Effect of Glucose.

Perfusion experiments (i4, 182, 202, 203, 275, 343) and experiments With isolated tissues or tissue slices (58, 60, t19, t45, 228, 282, 290, 314, 318) have established the fact that glucose utilization increases with increasing glucose concentration in perfusion fluid or suspension medium. Fig. 3--6 illustrate this effect. Fig. 3 is based on the data obtained by SOSKIN and LEVINE (276) in infusion experiments on eviscerated dogs; Fig. 4 shows the glucose utilization of the hind legs in intact and eviscerated dogs, according to LA~G et al. (t 82); Fig. 5 shows the oxidation of isotopic glucose in normal and diabetic rat liver slices, as measured by the radioactivity of the respiratory CO2 (6), and Fig. 6 contains the results of several authors on glycogen formation by the excised diaphragm of normal rats. In all these experiments the effect measured increased with rising glucose concentration, not only in the absence of insulin, but also in its presence.

Table 6. Glucose u t i l i za t ion o

Blood sugar level

Lt~w Normal . . . . . . . . . . . .

High (ca. 1ooo mg-%) . . . . .

eviscerated rabbits [WmK and DRURY (338)].

Glucose utilization (mg/kg/hr)

% respiratory CO~ coming from injected glucose

Insulin

-- + -- +

183 2t3

220 448 848

20 24

34 44 63

80 H. WEIL-MALHERBE: The Mechanism of Action of Insul in.

The results of LANG et al. (t82) among others (276, 343) give a clear indication that a saturation value was reached; a maximal utilization of

r . . . . . . . glucose was attained at levels x

N/~g/hr of about 100 rag-% in the pre-

about 600 mg- % without in- "~ sulin. They thus support the

~oc contention of SOSKII~ and " / " / ~ ' / LEVlNE (276) that insulin

z~ / / promotes maximal glucose la~ utilization at low glucose con-

centrations and that the same r J a 1oo 2oo J~o ~ 5oa Joo z~o goo level "of glucose utilization can

alood sugar /eve/ r~~ be reached in the absence of Fig. 3. Glucose utilization of the eviscerated dog at varying exogenous insulin and even in blood sugar levels. Data of SosKt~ and LEVINE ["Carbo- hydrate Metabolism" {276), Table 25, p. 203). Curve 1: t h e diabetic animal, provided • non-diabetic dog, insulinized. Curve 2: e - - e non-

diabetic dog. Curve 3: O- -O pancreatectomized dog. the blood sugar level is suffi, ciently pushed up. This view

is not shared by WICK and DRtlRY (336, 358) who, after investigating the utilization of isotopic glucose in eviscerated-nephrectomized rabbits, with

In/at/dog Ev/scera/e# doff ~ $ 0 0 ~ I T ' fO I I I I r J' I I I I

~ ~~176176 l ~ 8ooL ] ~ o

~ G O 0 o o o o

ot/ / ~ . "

U ZOO �9 �9

~ " J I i i , V I P i I 1 j i 0 100 200 300 r 500 800 0 /Od 200 300 ~00" ~r00 SO0 700

Blood-glucose rrt~% Fig. 4. Glucose utilization of dog hind legs. Data of Lm~a, GOLDSTEI~I aEld LEWNE {182}.

• • insulinized dog, e - - o without exogenous insulin.

and without insulin, concluded that no conceivable blood sugar level could raise the rate of glucose utilization to the values observed in the/nsulinized preparation. Table 6 gives a summary of their results which differ from those obtained in dogs mainly by the fact that insulin does n o t raise

Insul in and Glucose Concentrat ion. 8t

the utilization of glucose to its maximum level. Species differences may account for this.

ZILVERSI~IT et al. (350) found that nephrec- 12o0o tomized diabetic rats were able to oxidize c.p.~. glucose at about the normal rate when a ~oooo blood sugar level of 900 to t 000 rag-% had been reached. The in vitro experiments with .,~ 8non rat liver slices and with the excised rat diaphragm which are illustrated in Fig. 5 ~'soao and 6 show no definite evidence of a saturation level up to glucose concentrations of ~ anna 800~t000 mg-%, nor do they support the claim that the utilization of glucose tends to ~aoa reach the same ceiling in the absence and in the presence of insulin.

0 Further evidence for the mass action effect

of glucose is provided by observations on the arteriovenous difference of glucose, which, at a constant rate of blood flow, is a measure

at a physiological blood sugar

I I I I t I I

/ d/dle, C ' e ~

1 I I 1 I I I l 200 ~00 600~0% 800 tee- Glucose eoncen/mhbn

Fig. 5. Glucose oxidation by rat liver slices at varying glucose concentration. Data of CHERNICK, CHAIHOFF, ]~ASORO

and ISAEFF (60).

of glucose uptake. According to SOMOGYI (270, 27t) the arteriovenous difference increases with increasing blood sugar level and vice versa.

The important point of the discussion is the fact a~o! that even in the presence of ~g~ insulin maximum glucose .~ r utilization is only reached .~

at glucose concentrations ~aoo of at least 100 mg-%, .2

%-

whereas in its absence the .~ 2o~ Same rate is not at tained ~

at much higher concen- 100 trations. Since the rate of the hexokinase reaction is presumably the limiting 0 factor for many or all of the subsequent reactions of glucose, full saturation of the hexokinase system With respect to glucose

i i i I I i t i ~ 1

~ , - L - - ' ' ' ~ ' ~ " " 3 . . . "

I I I I t ] I �9

200 r 600 1000 rn,0% 3000 Glucose concenfcuttbn

Fig. 6. Effect of glucose concentration on glycogen synthesis in the diaphragm of the normal rat. • 2 1 5 insulin added in vitro; e - - o in the absence of insulin. Curve 1 : Data of GEMMILL and HAMMArr (119). Curves 2: Data of STADIE and ZAPP (290). Curves 3: Data of STADIE, HAUGAARO and MARSH (282). Curve 4:

Data of PARSES and WERTHEmER (225).

only occurs at glucose concentrations that bring about the maximum rate of glucose utilization. Now the affinity of brain hexokinase -- and probably of other animal hexokinases -- for glucose is unusually high. A reliable estimate

]Zrgebnisse der Physiologie~ Bd. 48. 6

82 H. WEIL-MALHERBE; The Mechanism of Action of Insulin,

of the MICHAELIS constant has only recently been obtained by an indirect method: it is 8 • -s M according to SoLs and CRANE (269). Hence it can be calculated that the enzyme is fully saturated at glucose concentrations of about 0.3 rag-%. For maximum glucose utilization, therefore, a concentration gradient of at least 10o/0.3 is required in the presence of insulin and a much higher one in its absence. The existence of these concentration gradients and the effect of insulin thereon clearly imply an action of insulin on diffusion processes.

b) The Permeability Theory of Insulin Action.

The theory that insulin acts by increasing the permeability of the cell membrane for glucose is old. As early as 1914, HOEBER (/50) suggested that the primary defect in diabetes was an inhibition of the entry of glucose into the cell. LOEVr and his collaborators (88, 143, 144, t95) a t tempted to demonstrate that insulin promotes the diffusion of glucose into red blood cells i~z vitro. However, it is now generally recognized that there is no evidence of such an effect (t48, 294).

LEVINE et al. (t9t) at tacked the problem from anew approach. They based their experiments on the fact that galactose is not significantly metabolized by muscle tissue. Using eviscerated, nephrectomized dogs they showed that when galactose is infused it distributes itself in a space corresponding to about 45 % of the body weight and remains constant at this level for several hours. When insulin is administered the galactose space increases to 70 % of the body weight, corresponding approximately to the entire intra- and extracellular fluid. Similar experiments on rats showed that practically all the galactose infused could be recovered from the carcass at the end of the experiment. WICK and DRURu (337) perfused eviscerated rabbits with galactose-t-14C. They found, in agreement with LEVINE et al., that, in the absence of insulin, galactose was quickly distributed in the extracellular space, viz. about 25 % of the body weight, and entered the cells very slowly. The administration of insulin greatly speeded up the rate of transfer from extra- to intracellular compartments. Some oxidation of galactose by the extrahepatic tissues definitely took place, and this was about doubled by insulin.

The volume of distribution of glucose itself, the "glucose space", corresponds to the extracellular space, whether insulin is present or not (90, 261), but this is due to the fact that, normally, glucose is metabolized immediately on entering the cell. However, an intracellular accumulation of glucose can be demonstrated under suitable conditions. PARK (224) found that the effect appeared in the excised rat diaphragm at a glucose concentration in the suspension medium of 600 mg- %, when insulin was present, but without insulin a concentration of about 2000 rag-% was required. At a temperature

Insuhn and Glucose Concentration. 83

of 10 ~ insulin may cause an intracellular accumulation of glucose at physiological levels of glucose concentration. When eviscerated rats were given a continuous intravenous infusion of glucose for t hr, the glucose content of excised muscle Was consistent with a purely extracellular distribution. When, however, insulin was administered as well, the glucose content of the muscle was, a t comparable blood sugar levels, 2-- 5 times higher, showing that a large fraction must have been intracellular. Muscle of diabetic rats contained no intracellular glucose, even at very high blood sugar levels, unless insulin was administered. Muscle of normal fasted rats conta ined no intracellular glucose either, but muscle of fed non-diabetic rats did contain some, probably because of the action of endogenous insulin (225). An increase of intracellular glucose has also been demonstrated in the perfused rat heart when insulin was added to the perfusion fluid (t08).

Ross (248) succeeded in demonstrating an effect of insulin on the rate of transfer of glucose from blood into the anterior chamber of the eye across the blood-aqueous barrier. The "permeabili ty constant", which is proportional to the rate of transfer per minute, was t .9 • 10 -~ for the normal rabbit, t .0 • t 0 -~ for the diabetic rabbit and 4 . 7 • -~ for the insulinized rabbit. He later showed (249) that insulin accelerated the uptake of glucose by the decapsulated lens in vigro. W i t h o u t insulin the mean rate of glucose uptake was 0.202 mg]g/hr, while in the presence of insulin the figure was 0.71 mg/g/hr -- an increase of 350%. Galactose did not enter the le.nticular tissue in the absence of insulin, but with insulin present it was taken up at the rate of 0.t l 'mg/g/hr.

c) Specificity of the Permeability Effect of Insulin.

Some substances, such as urea, quickly reach equilibrium between extra- and intracellular fluid even in the absence of insulin. Others do not enter the cells whether insulin is present or not. GOLDSTEIN et al. (t22) studied the Specificity of the insulin effect on the transfer mechanism of monosaccharides in eviscerated dogs. L-Arabinose and D-xylose were found to behave like I)-galactose, b u t the distribution or utilization of D-arabinose, L-rhamnose. D-sorbitol, D-mannose, D-fructose, and L-sorbose was not affected by insulin. The authors concluded from their results that the insulin effect is limited to those monosaccharides that possess the chemical configuration of glucose on their first 3 carbon atoms (Fig. 7). Subsequent work has made it clear that this is an oversiml~lification. DmJ~u and WICK (92) found, in contrast to GOLDSTEIN et al. (t22), that insulin increases the space of distribution of D-mannose in the eviscerated rabbit from about 23 % to about 58% of the body weight. In agreement with GOLDSTEIN et al. they failed to observe any utilization of fructose, even in the presence of insulin. Other workers, however, found that fructose is taken up by the excised rat diaphragm and that insulin promotes the process (86, 128, 206, 232). [-[&FT, MIRSI~Y and PERISUTTI (t28)

6*

84 H. WEIL-MALHERBE: The Mechanism of Action of Insulin,

t. Responsive to insulin

CH0 I H--C--0H t H0--C--H

H0--~--H H--~--0H

~H2OH

[D-galactose [

CHO H--~--OH

HO--~--H H - - ~ - 0 H

H - ~ - - 0 H

~H2OH

[ D-glucose]

2. Not responsive to insulin

CHO CH2OH CH2OH I ~ = o I HO--C--H C=O

~oA_~ ~oA_~ ~o_~_~ l ~ - ~ - o H H - ~ o ~ H--C--OH

~.o~ ~.o~ ~,.o~ [D-mannose ]]"DifructoseJl L-sorbose I

CHO CH0 H--~--0H H--~--0H

~OA-H HoA-~

IH~0H [ CH,OH

I D xylose I I L-ar~binose I

CHO CHO CH20H

~_~-oH ~o~_~1 ~_L-o~ ~.,o~ ~o_~_~ ~_~_o~

I "-~rabin~ II ~-rhamn~ I .-sorbi,o, ] Fig. 7. Relation of chemical structure of sugars to the action of insulin. Reproduced from GOLDSTEIN,

HENRY, HUDDLESTUN and LEVlNE (122}.

further observed that D-galactose, D-xylose and L-arabinose approached maximal values of diffusion into the intracellular compartment of the rat diaphragm, but, while the transfer of D-galactose and L-arabinose was accelerated by insulin, that of D-xylose was not.

I t is interesting to note that the monosaccharides that appear to be responsive to insulin are, on the whole, also those that are able to penetrate, unaided by insulin, across cellular membranes. Ross (247) has shown that -- besides glucose -- galactose, xylose and 3-methylglucose were transferred across the blood-aqueous barrier at a preferential rate, not accounted for by their lipid solubility. The following sugars -- besides glucose -- are taken up by human erythrocytes: D-fructose, D-galactose, L-sorbose, D-mannose, D-xylose and L-arabinose (t 86), whereas L-xylose and D-arabinose are unable to penetrate (344).

Although it may therefore be necessary to modify the original conception of GOLDSTEIN et al., it remains nevertheless true that the transfer mechanism which is the target of insulin action is endowed with a configurational specificity of the kind familiar from enzyme studies. In addition, the kinetics of the transfer, such as it exists in human red cells, have been shown to be similar to those of enzyme reactions and have led to the hypothesis of a mechanism

Insulin and Glucose Concentration. 85

mediated by a "ferry" or "carrier" and involving carrier-substrate complexes (t86, t87, 34t, 342). That these carrier-substrate complexes are of importance for the insulin effect is suggested by observations of competitive inhibition of the transfer of one sugar by another, in the absence and in the presence, but sometimes only in the presence, of insulin. Thus it has been shown that the addition of glucose inhibits the intracellular transfer of galactose (337, t08), of mannose (92), of fructose (206, 342) and of sorbose (342). The fact that insulin increased the utilization of fructose by the rat 's diaphragm When the medium contained fructose as the only substrate, but not when it contained glucose and fructose, was interpreted by MACKLER and GUEST (206) as showing that hexokinase was stimulated by insulin, while fructokinase was not. A competition between glucose and fructose for a common transfer carrier would be a simpler assumption and would be in line with other examples of competition referred to above.

It is not impossible, of course, that hexokinase is a constituent of the cell membrane and somehow implicated in the transfer mechanism. The specificity of brain hexokinase has been studied by SoLs and CRANE (269) and shown to be less restricted than had been assumed, yet comparison of hexokinase specificity with the specificity of the transfer system reveals significant differences: while glucose and mannose have high affinities for both systems, galactose has a high affinity for the transfer system, but a very low affinity for hexokinase. Fructose reacts readily with hexokinase, whereas L-sorbose does not, yet both have similar, though low,, affinities for the carrier of the erythrocyte membrane (342). D-xylose inhibits brain hexokinase, but it is readily transferred across cellular membranes; D-arabinose, on the other hand, is phosphorylated .by brain hexokinase, but it is unable to penetrate the cell membrane. There is thus no ground for the assumption that insulin affects the transfer mechanism via membrane-bound hexokinase.

d) The Insulin Effect and the Supply of Energy.

If a substance is absorbed by the cell against a concentration gradient the process is called "active transfer"; it is, of course, dependent on the supply of energy. If transfer occurs in the direction of the concentration gradient, but at a faster rate than transfer by diffusion, the process is called "facilitated transfer". Inasmuch as the maintenance of this mechanism is bound up with the metabolism of the living cell it, too, will suffer if the energy production of the cell is interfei-ed with. There are indications that the insulin effect, in particular, is dependent on the integrity of the energy-producing reactions. ~-~AFT and MIRSKY (t27) and DEMIS and ROTHSTEIN (86) found that, although the glucose uptake of tile excised rat diaphragm was the same aerobically and anaerobically, insulin increased the rate of uptake only aerobically, not anaerobically. The insulin effect can be restored by returning the tissue to

86 H. WEIL-MALHERBE: The Mechanism of Act ion of Insulin.

aerobic conditions and allowing it to recover for a period of 15--30 min. The effect of anoxia manifests itself only after exposure to anaerobic conditions for about ~o--t5 min. The divergent results of WALAAS and WALAAS (3'18) w e r e probably due to the fact that their observations were made too soon after the exclusion of oxygen.

Some poisons have a similar effect: although iodoacetate does not diminish the rate of glucose uptake by the rat diaphragm, it abolishes the insulin effect (t26). 2: 4-Dinitrophenol, an inhibitor of oxidative phosphorylation, reduces the utilization of glucose and inhibits the effect of insulin in rat diaphragm (3t3) while a closely related compound, 4:6-dinitro-o-cresol, increases the utilization of glucose by mouse ascites tumor cells, both aerobically and anaerobically (64).

While these findings, therefore, suggest that the insulin effect depends on a high level of energy-rich compounds, a different conclusion has been reached by ISSEKUTZ, HETI~NYI and WINTER (157). These authors studied the effect of an intravenous injection of 2: 4-dinitrophenol in the living dog; they found that it increased the glucose uptake of the thigh muscles in the intact, but not in the depancreatized dog. In addition, it led to the loss of high- energy phosphate groups in the tissues and increased the output of inorganic phosphate, in both control and diabetic animals. The injection of insulin increased the uptake of glucose and of inorganic phosphate by the muscles poisoned with dinitrophenol in the control as well as in the diabetic animals. The authors therefore concluded that the insulin effect is not prevented by dinitrophenol and that it lies outside the main lines of energy production.

Whatever the final verdict will be in this controversy, it seems to be fairly well established that the effect of insulin on the rate of the hexokinase reaction must itself be secondary to its effect on the diffusion mechanism. By governing the rate of entry of glucose into the cell insulin controls the degree of saturation of hexokinase. Such a mechanism is obviously dependent on the integrity of the membrane barrier and it is in the cellular membranes that the point of action of insulin is to be sought.

4. The STADIE Effect.

In this connection the observations of STADIE and his collaborators are of particular significance. In a series of papers (281, 282, 284) they showed that a short exposure of the excised rat diaphragm to a solution containing insulin, followed by thorough washings, was sufficient to produce the essential effects of insulin, viz. an increase of glucose uptake and glycogen formation, on subsequent incubation in an insulin-free medium, and they concluded that a rapid, difficultly dissociable combination of insulin with some structural unit of the diaphragm had taken place. For a maximum effect the diaphragm required to be left in contact with the insulin-containing medium for only

The STADIE Effect. 87

t - -2 rain, but even shorter exposures sufficed for an effect to be demonstrated. STADIE, HAUGAARD and VAUGI-IAN (286) prepared isotopically labelled insulin by treatment with H285S04 or 1311 without changing its hormonal activities. With its aid they showed that, at an insulin concentration of t unit/ml, the amount which entered into combination with the diaphragm was about t/zg/g. With higher concentrations a saturation value was approached; a one-minute exposure to a concentration of 3 units/ml, followed by 30 rain washing, resulted in the fixation of about t .7/lg/g. The combination of insulin with diaphragm in the ratio of t #g/g brought about an extra formation of glycogen of 3.6 :k 0.76 mg/g/hr in normal rats of the WIsTAR strain (287).

CHAYEN and SMITH (55) prepared conjugates of insulin with fluorescent dyes which they injected into rats. \Vhen the rats were killed 2 hr later, the diaphragms were removed, teased with needles and studied under the fluorescence microscope. The fluorescent insulin conjugate was seen to be associated with the reticulin network of the muscle. The reticulin of spleen and adipose tissue also showed a marked affinity for insulin conjugates. Collagen fibres, on the other hand, did not fix the insulin conjugates, nor was there any combination of reticulin with the fluorescent conjugates of serum albumin, lactoglobulin or other proteins. The authors therefore suggest that the site of the STADIE effect is the reticulin of the connective tissue.

Following upon this suggestion OTTAWAY (222) incubated the rat diaphragm with well-washed reticulin fibres prepared from spleens of diabetic and non-diabetic rats. Reticulin of normal rats increased the glucose uptake of the diaphragm, but that of diabetic rats did not. OTTAWAY concluded that endogenous insulin, like the fluorescent insulin conjugates, forms a slowly dissociating combination product with reticulin.

The STADIE effect is not confined to nmscular tissue, but has also been demonstrated with slices of adipose tissue and lactating mammary gland. Short exposure of adipose tissue to insulin leads to an increased oxygen uptake (t36) whereas lactating mammary gland slices respond with a rise of RQ (t47). BLEEHEN and FISHER (~6), on the other hand, studied the effect of insulin on perfused rat heart and noticed that the insulin effect d id not persist after washing out the preparation. W'hen the heart was perfused with inulin, which is mainly confined to the extracellular space, it was washed out at about the same rate as insulin. The authors, therefore, think that the STADIE effect does not exist in rat heart; moreover, they are of the opinion that the STADIE effect is as consistent with slowness of diffusion as with fixation by the tissue.

Although the results of STADIE and Iris colleagues have no immediate bearing on the mode of action of insulin, they emphasize the importance of the structural element and thus fit in well with the other deductions re~ched.

88 H. WEIL-1ViALHERBE: The Mechanism of Action of Insulin.

S- The Distribution of Insulin in the Tissues and in the Cell.

Isotopically labelled insulin has been used to study its distribution in the body after subcutaneous or intravenous injection. The greatest concentration was found in kidney cortex where 15 rain after intravenous injection it was 36 times higher than the total body concentration (t02). The next highest concentration was found in the liver. There was also some accumulation in heart and muscle, but the concentration in brain was very low. No insulin was found in red blood cells. The plasma level after intravenous injection rapidly dropped to a steady level which was maintained for about 2 hr (t41); after a subcutaneous injection the plasma level reached a maximum after about 3 hr, but this peak value did not exceed one half of the steady level found in the plasma after intravenous injection. All the radioactivity appearing in the urine was associated with dialysable breakdown products (t 41 ).

LEE and WILLIAMS (t 85) studied the intracellular localisation after perfusion of rat liver with labelled insulin. The radioactivity was firmly bound to the mitochondrial and microsome fractio~s and some was also found in the supematant solution. The nuclear fraction contained but little radioactive material. It has however not been established that the radioactive material associated with intraceUular fractions was identical with the integral insulin molecule.

Conclusion.

The acceleration of the hexokinase reaction brought about by insulin cannot be explained either by an increase of the intracellular concentration of ATP or by a direct effect on the hexokinase system, but appears to be due to an increased saturation of hexokinase with glucose. The point of action of insulin is the transfer system localized in the cell membrane, which has the characteristics of an enzyme system.

IV. The Modification of Insulin Action by Other Hormones.

A complete discussion of the complicated interactions of insulin and other hormones is outside the scope of this review. However, in so far as they tend, by their homoeostatic effects, to obscure or modify the action of insulin, their consideration cannot be entirely omitted. Several examples of this kind have already been mentioned in the preceding parts.

1. Insulin and the Corticosteroids.

It is well known that adrenalectomy, like hypophysectomy, attenuates the hyperglycaemia and glycosuria of an existing diabetes, while injection of adrenocortical extracts or of certain pure corticosteroids has the opposite

Insulin and the Corticosteroids. 89

effect and may even induce hyperglycaemia and glycosuria in non-diabetics, animal or human. I t is believed that many of the effects on carbohydrate metabolism produced by the 1 l-oxy-cortieosteroids follow from an increased rate of gluconeogenesis from non-carbohydrate precursors, especially proteins. This process raises the glycogen concentration of the liver, if insulin is available; in the diabetic it results in increased hyperglycaemia and glycosuria. Whether, in addition, corticosteroids exert an inhibitory influence on glucose utilization is still under discussion. Experiments in which isotopically labelled glucose was infused into intact rats pretreated with cortisone for several days did not show any significant decrease of the rate at which glucose was oxidized to CO S (332). In eviscerated rabbits neither adrenalectomy nor the administration of adrenocortical extract was found to have a significant effect on the oxidation of glucose (340). On the other hand, the blood sugar level was found to be lower after adrenalectomy when glucose was given by COnstant infusion to eviscerated rats (t55), and BOOTWELL and CHIANG (34) reported an inhibition of glucose utilization and oxidation in normal rats 4--6 hr after an injection of cortisone. COHN et al. (66) found that the utilization of glucose by the eviscerated dog was increased after adrenalectomy; in spite of this the utilization of total carbohydrate was less. They explained this effect by an inhibition of muscle glycogenolysis.

I n vitro experiments have shown that after adrenalectomy the glucose utilization of the excised rat diaphragm was unchanged (174) or moderately increased (314) while glycogen formation was normal (31 t) or inhibited (2t t, 314). The action of insulin was unchanged (174, 3t4). In the diaphragm of the diabetic rat glucose utilization and glycogen synthesis were restored to their normal level after adrenalectomy, or even beyond (32, t74, 314), and both effects were enhanced by insulin. The effects observed in the diaphragm of the non-diabetic rat are consistent with an inhibition of glycogen mobilisation after adrenalectomy, but this alone is hardly sufficient to account for the improvement of the diabetic symptoms; it suggests rather a more direct antagonistic function of corticosteroids towards insulin.

The improvement of diabetic symptoms which is observed after adrenalectomy is enhanced by a combination of adrenalectomy with hypophysectomy (32, 314). A synergistic action of cortisone and growth hormone has been postulated by several authors (30, 227, 231, 283, 307); it remains to be determined whether the combined effects of adrenalectomy-hypophysectomy are such that they cannot be accounted for by a simple summation of effects.

Adrenocortical extract inhibits anaerobic glycolysis in the excised rat diaphragm (65) and glycogenolysis in rat liver slices (62). Glycogen synthesis m rat liver slices was increased by the in vitro addition of deoxycorticosterone (I)OC), corticosterone and cortisone (6t). On the other hand, VERZ~,R and WENN~.R (3t t, 3t2), working with rat diaphragm, found that DOC and other


steroids completely inhibited the synthesis of glycogen from glucose and cancelled the insulin effect; they suggested that the steroids acted by accelerating glycogenolysis. BARTLETT, WICK and MACKAY (8) confirmed the effect of DOC and corticosterone on the rat diaphragm, but favoured an inhibition of glycogen synthesis. Similar results were also reported by LI et al. (193) and PLETSCHER and PLANTA (234).

2. Insul in and Adrenaline.

The glycogenolytic effect of adrenaline on liver and nmscle (cf. 70) has been studied in vitro. RIESSER (246), TUERKISCHER and WERTHEIMER (309) and WALAAS and V~rALAAS (31 7), using rat diaphragm, and BozovI~, LEtlPIN and V~RZ~,R (36), using mouse abdominal muscle, showed that addition of adrenaline to the medium not only prevented glycogen synthesis, but increased the breakdown of glycogen. Adrenaline also inhibited the stimulat~ion of glycogen synthesis by insulin. SUTHERLAND and CORI (304) demonstrated an analogous effect on rabbit liver slices. Half maximal stimulation of glycogenolysis occurred at a concentration of about I part of adrenaline in ! 5 million, a concentration which is about 30 times higher than the adrenaline level in venous plasma (325). L-Noradrenaline had only about one sixth of the activity of L-adrenaline. SUTHERLAND and CORI showed conclusively that the action of adrenaline consisted in accelerating the phosphorylase reaction; moreover, they were able to show that this effect was due to an increase in the concentration of active phosphorylase. In muscle, phosphorylase exists in two forms, phosphorylase a and phosphorylase b (78). Phosphorylase a is the active form of the enzyme. It is reversibly transformed to phosphorylase b when muscle is stimulated to fatigue or, in vitro, through the activity of the so-called PR-enzyme. Phosphorylase b can be reactivated by the addition of adenylic acid. Liver phosphorylase, too, exists in an active and inactive folan which, however, differ from the corresponding form s of muscle phosphorylase (302). The inactive form of liver phosphorylase is not reactivated by adenylic acid. As in muscle, the transformation of the active into the inactive form is catalysed by an enzyme. SUTHERLAND and CORI (304) found that the activity of this enzyme was not inhibited by adrenaline and they assumed therefore that the action of adrenaline consists in promoting the resynthesis of active phosphorylase. StlTHERLAND and CoI~I (cf. 303) further found that a similar reaction occurs in muscle: adrenaline increases the amount of phosphorylase a in rat diaphragm and stimulates the reconversion of phosphorylase b into phosphorylase a.

There has been some discussion as to whether adrenaline inhibits glucose utilization in extrahepatic tissues. Such an effect was first described by CoRI and CORI (72) and it received strong support from the observation that the glucose arteriovenous difference was decreased after adrenaline administration,

Insulin and Adrenaline. 91

in spite of hyperglycaemia (73, 74, 79, 272, 273). DE DUVE, MARIN and ]~OUCKAERT (t00) showed directly that the insulinized rabbit utilized t.22 g glucose/kg/80 min; when adrenaline was infused, this figure was reduced to O. 77 g/kg/80 min.

The inhibition of glucose utilization by adrenaline may be interpreted as a consequence of its action on glycogenolysis. The increased rate of glycogen breakdown leads to an increased concentration of hexose-6-phosphates in muscle (76, t46). Since glucose-6-phosphate is a powerful inhibitor of brain hexokinase (3t9) and of animal hexokinases generally (83), its accumulation will result in an inhibition of the hexokinase reaction and, possibly, in the accumulation of intracellular glucose.

COHEN (65) demonstrated all inhibition of glucose utilization in the excised diaphragm of rats that had been injected with adrenaline before death. Since the effect was unchanged after adrenalectomy, but abolished after hypophysectomy, he concluded that growth hormone was implicated.

Many authors have reported that the glycogenolytic effect of adrenaline is inhibited after hypophysectomy (e. g. t9, 82, t8t). According to DJIN, VLASBLOM and COHEN (89) this inhibition is due to the depletion of liver glycogen and is prevented, in rats, by forced feeding. Unfortunately, the experiments of DJIN et al. were performed several days after the operation, whereas the effects of hypophysectomy require several weeks to develop. DE BODO, BLOCtt and GRoss (t9) expressly deny that lack of liver glycogen accounts for the reduction of the adrenaline effect in hypophysectomized dogs. DE BODO and SINKOFF (22) further state that the glycogenolytic effect of adrenaline is also inhibited after adrenalectomy and that treatment with cortisone restores the response to adrenaline in both hypophysectomized and adrenalectomized dogs, while growth hormone is ineffective. LEONARD and I{INGI.ER (188) agree that adrenaline mobilizes less muscle glycogen in rats after hypophysectomy, but they find no difference if the initial glycogen is taken into account and the result is expressed as percentage loss. They further find that both growth hormone and small doses of cortisone inhibit the mobil- ization of muscle glycogen by adrenaline in hypophysectomized rats, while thyroxine potentiates it.

The action of adrenaline as an insulin antagonist is accounted for by the acceleration of the phosphorylase reaction which, in the postabsorptive state, will bring about increased glycogenolysis, hyperglycaemia and inhibition of glucose utihzation. Adrenaline thereby reinforces a tendency resulting from the action of insulin itself, since insulin, by pushing increased amounts of glucose into tile cell, activates glycogen synthesis while the blood sugar is high, particularly in muscle; during the later phases of insulin action, when the blood sugar is low, glycogenolysis is increased, particularly in the liver. Both phases of insulin action therefore lead to an increase of phosphorylase activity.

92 H. WEII.-MALBERBE: T he M e c h a n i s m of Ac t ion of Insu l in .

There is evidence that the utilization of adrenaline in the tissues is increased after the administration of insulin. H6KFELT (152) found an increased fixation of adrenaline in the liver and heart of rats injected with insulin. Most animals went into shock after about 3 hr; they then received I or 2 injections of glucose. After 3 hr the adrenaline concentration in liver had risen from an initial value of t.8/~g/kg to 50/~g/kg, that of heart from 22 to 350yg/kg. Later, after the animals had received some glucose, the adrenaline concentration slowly returned to its normal level in both tissues.

~ 1 i p r i i i i i I

- ,~ !

l' ....--- \ ~ / ~ . s ~ I ~ ' S ~

-60 ~ /

-70

_~0 0 I I I I J I I I 10 20 30 r 50 60 70 80min.~

Fig. 8. Mean percentage concentration Change of drenaline (full line) and glucose (broken line) in plasma after an intravenous injection of insulin (0.1 unit/kg) in h u m a n subjects. The blacked-in circles indicate s tandard errors. Data of WEIL-

MALHERBE and BouE (326).

An increased utilization of adrenaline is also indicated b y the observations of WEIL-MALHERBE and BoNE (324, 326). They found that an intravenous injection of insulin in fasting human subjects (0.t unit/kg) elicited an immediate lowering of the plasma adrenaline concentration, which pre- ceded the fall of the. blood sugar. This was followed by a spontaneous return to the initial level within about an hour (Fig. 8). The determination of the arteriovenous difference of adrenaline revealed a transitory increase which was presumably due to the increase of utilization. When coma

doses of insulin were injected, the plasma level of adrenaline remained depressed until the coma was termi-

nated by the administration of glucose or other means; the termination of coma was accompanied by a rapid rise of the plasma adrenaline concentration (Fig. 9). The effect of glucose administration was not confined to insulin hypoglycaemia, but was also observed under the conditions of the simple glucose tolerance test (327).

In view of the part played by adrenaline in the resynthesis of the active form of phosphorylase, it is suggested that an acceleration of the phosphorylase reaction involves an increased utilization of adrenaline. This would explain the increased utilization of adrenaline resulting from insulin injection, since insulin can be expected to cause an acceleration of phosporylase activity. Conversely, the administration of glucose, especially during hypoglycaemia or fasting, will reduce phosphorylase activity since exogenous glucose will replace glucose mobilized from glycogen. This may account for a decrease of adrenaline utilization and for the increase of the plasma adrenaline level.

I t is a well-established fact that insulin hypoglycaemia entails an increased

discharge of adrenaline from the suprarenals. Evidence has been obtained

I n su l i n a n d Adrenal ine . 93

both by histological methods (15t, 158, 236, 244) and by the analysis of extracts (3, 50, ~52, 223, 256, 334) that the adrenaline stores of the adrenal medulla are gradually depleted during insulin hypoglycaemia. An increase of the adrenaline concentration in the blood of the suprarenal vein during insulin hypoglycaemia has been demonstrated by LA BARRE and HotlssA (t 80) and by other investigators (95, 347). The effect is usually regarded as a response to the lowering of the blood sugar level, and it need not be denied that the height of the blood sugar level is one the factors that regulate the discharge of adrenaline -- a fact that has been demonstrated by DUNt~R (94). But the observations of WEIL-MALHERBE and BONE suggest another possibility: that the increased discharge of adrenaline is a homoeostatic adjustment to the increased utilization of adrenaline in the tissues and to the fall of the level of circulating adrenaline.

The results of WEIL-MALHERBE and BONE (324, 326) were obtained with a fluorimetric method of estimation (323, 325), the validity of which has been carefully checked (328). ~-~OLZBAUER and VOGT (t 53), however, obtained entirely different results with a biologicalmethod of assay. They found not only a much lower basal value for the concentration of adrenaline in human and canine plasma, but also a large increase after the injection of insulin, reaching values of 3--6/~g/1 in the dog. The lower basal v a l u e s o{ H O L Z B A U E R and VOGT can be explained, at least in part, by a difference in the method of blood collection, since about 50-- 70 % of the adrenaline estimated by the fluorimetric method was shown to be associated with the

m. 9" ~ blood svggr 4":

I201 ~ { "/ Im i'

#

10o #

0 - i0 20 dO ~ S#min60 Fig. 9. Mean, percentage eoncer~- tration changes of p lasma adrenaline (full line) after a n intravenous injection of glucose (50 ml 33,3 % sol,) into human subjects during hypoglycaemic coma, The blacked-in circles indicate s tandard errors. The blood sugar curve (broken line) shows the mean concentration change in absolute units (mg-%). After arousal the patients received more glucOSe by mouth. Data of WEIL-M&LHI~RBE

and BO~E (326),

blood platelets under basal conditions (3 29) and this fraction was discarded by HOLZBAUER and VOGT. Preliminary experiments (33 0) indicate that the platetets lose their adrenalineduring insulin hypoglycaemia; the decrease of the concentration of the free adrenaline in plasma is therefore smaller than that of the sum of free and platelet-bound adrenaline. But this does not account for the increase found by HOLZBAUER and VOGT. While it is impossible to be certain that the fluorimetric met-Hod estimates nothing but adrenaline, it is considered highly improbable that it would have failed to detect an increase of adrenaline of the order reported by HOLZBAUER and \rOCT.


3. Insul in and Growth Hormone . The anterior pituitary produces two insulin antagonists or "diabetogenic"

hormones, the adrenocorticotrophic homlone (ACTH) and growth hormone (GH). Hypophysectomy therefore leads to an improvement of the diabetic condition, as shown, for instance, by the increased glucose uptake (t 75, 3t4) and the increased glycogen formation(32 , 314) of the excised rat diaphragm. Similar changes occur after adrenalectomy, and they are still further enhanced by a combination of adrenalectomy and hypophysectomy. Since ACTH is inactive in the absence of the adrenal cortex, the additional effect of hypophysectomy can only be attributed to GH. Other observations have shown that the effects of hypophysectomy aud adrenalectomy on carbohydrate metabolism are not identical: while the diaphragm of the adrenalectomized rat gives a normal insulin response (283), the diaphragm of the hypophysectomized rat shows a greatly increased insulin response (32, 28t, 283). The hypersensitivity towards insulin of the hypophysectomized dog is greater than that of the adrenalectomized dog (23); it can be reduced to normal limits and may even be changed into insulin resistance either by ACTH or by prolonged treatment with GH (20, 24, 184). The diabetogenic action of crystalline GH has been studied particularly by YOUNG and his associates (80).

STADIE et al. (284) have showax that injection of purified GH before death inhibits or prevents the insulin effect on glycogen formation in the excised rat diaphragm, while in vitro incubation of the diaphragm with GH prior to or simultaneously with insulin has no effect. In experiments with radioactive insulin STADIE, HAUGAARD and VAUGHAN (287) showed that the same amount of insulin is fixed by the diaphragms of intact and hypophysectomized rats. The higher activity of insulin when tested on diaphragms of hypophysectomized rats is therefore not due to the availability of a greater number of reaction sites, but presumably to the removal of an inhibitor.

Passing reference has already been made to examples of synergism between GH and other hormones, particularly cortisone. STADIE, HAUGAARD and MAI~SH (283) found that the effect of insulin on the excised diaphragms of adrenalectomized or hypophysectomized rats was inhibited if the animals were injected with both GH and cortisone before death, neither substance being active alone. The experiments of PARK and KRAHL (227) and of BORN- STEIN and PARK (30) may be interpreted as showing the same effect. PARK et al. (226) have further shown that the anti-insuhn effect of pure GH only becomes apparent several hours after its injection ; they suggest that the latent period is required for the GH to undergo some modification in the tissues. The indispensableness of the adrenal cortex for the anti-insulin activity of GH has not been confirmed by others (22, t84) . DE BODO and SINI~OFF (22) found that cortisone, far from potentiating the action of GH, inhibited its toxic effects, such as the development of diabetes and of insuhn resistance,

Insul in and Growth Hormone. 95

which were observed after prolonged treatment of hypophysectomized dogs with large doses.

In contrast to the slowly developing anti-insulin activity of GH, an immediate effect has been noted, in vivo and in v~tro, consisting of an enhancement of glucose uptake and a fall of blood sugar (t78, 226). OTTAWAY (220, 22i) found that the stimulation of glucose uptake which is observed in the normal diaphragm was absent when diaphragms of diabetic rats were used, but could be restored by insulin treatment of the diabetic animals. He therefore suggests that GH liberates insulin from an inactive complex present in diaphragm. The occurrence of the effect in eviscerated rats (226) and in freshly pancreatectomized dogs (178), while ruling out a stimulation of the islet tissue, is not inconsistent with this hypothesis.

Summary.

The principal metabolic effects of insulin have been reviewed. They are the following:

t. Insulin promotes glucose utilization in vivo and in vitro. The effect is basically similar in the liver and in the extrahepatic tissues.

2. The only reaction in the initial metabolism of glucose which is impaired in diabetes and stimulated by insulin, is its phosphorylation to glucose-6- phosphate.

3. Insulin promotes the conversion of glucose into glycogen, in. vivo and in vitro. Insulin similarly stimulates lipogenesis from glucose.

4. Insulin promotes the oxidation of glucose to CO2, in vivo and in vitro. 5. Insulin promotes the utilization of inorganic phosphate and the formation

of hexosephosphates. It increases the concentration and the turnover of high-energy phosphate groups. These effects are shown to be secondary to an increased rate of glucose utilization.

6. The utilization and oxidation of lactate, pyruvate and acetate in mammalian tissues are unimpaired in diabetes and unaffected by insulin, according to most observers. Some divergent observations may indicate secondary results of the diabetic condition, since they are not corrected by the in vitro addition of insulin; others are probably due to a lower rate of discharge of high-energy phosphate groups in the absence of insulin.

7. Insulin promotes lipogenesis from lactate, pyruvate and acetate by liver slices. The effect depends on the presence of an adequate level of liver glycogen. It is suggested that insulin stimulates the utilization of liver glycogen (but not of muscle glycogen) by inhibiting the leakage of glucose into the extraceUular fluid, and t-t{'at the increased utilization of liver glycogen enhances lipogenesis from acetate by making available a larger supply of glycerol for the synthesis of glycerides.

96 H, WEIL-MALHERBE: The Mechanism of Action of Insulin.

8. Insulin promotes the synthesis of protein, not only by a "sparing

action" on nitrogenous substrates, but also by a positive stimulus. The effects are secondary to an increased utilization of glucose.

I t is concluded from the discussion of the main metabolic actions of insulin that there is a single primary effect which results in the acceleration of the hexokinase reaction. Possible exceptions are: the activation of pigeon breast

muscle respiration and the inhibition of the deamination of non-natural amino acids. The physiological significance of these effects is obscure.

In analysing the mechanism of the insulin effect the following possibilities were considered:

t. That insulin increases the intracellular concentration of ATP. No theoretical or experimental foundation for such a mechanism could be demonstrated.

2. That insulin increases the activity of hexokinase by a direct effect on the enzyme. Although the hexokinase activity of tissue extracts is subject to the influence of inhibitors and activators of unknown nature, these phenomena do not provide a sufficient basis for the mechanism of insulin action.

3. That insulin increases the intracellular concentration of glucose. To obtain complete saturation of intracellular hexokinase a concentration gradient of at least 1o0/0.3 is required in the presence of insulin and a much higher one in its absence. The existence of these concentration gradients and the effect of insulin thereon imply an effect of insulin on transfer mechanisms across a membrane barrier. The specificity of the transfer mechanism is different

from the specificity of hexokinase. The importance of the structural element

for the insulin effect is emphasized by the rapid formation of a difficultly dissociable combination of insulin with the tissue. Insulin thus accelerates

the rate of the hexokinase reaction by the simplest mechanism possible: by increasing the degree of saturation of the enzyme with substrate.

In a final section the interactions of insulin with corticosteroids, adrenaline and growth hormone, and the modification of its effects thereby, are briefly reviewed.

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References: 97

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the mechanism of action of insulin

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