Role of synalbumin insulin antagonist in pathogenesis of diabetes mellitus

Role of Synalbumin Insulin Antagonist in Pathogenesis of Diabetes Mellitus By MAYERB. DAVIDSONAND Continued interest in the possible pathogenetic role of the SIA in diabetes mellitus has been perpetuated primarily by three observations: (1) that plasma and sera from many diabetic subjects and their close relatives contain higher levels of the SIA than most nondiabetics. (2) that the SIA appears to affect glucose metabolism of muscle but not adipose tissue, possibly accounting for the increased prevalence of obesity in adultonset diabetics; and (3) that the reduced B-chain of insulin when bound to albumin reproduces most of the in vitro and in vivo metabolic and physiochemical characteristics of the SIA. On the other hand, a critical appraisal of the present state of knowledge reveals serious obstacles to acceptance of a role for synalbumin in diabetes mellitus. These are: (1) the potential presence of a dialysis casing artifactual insulin antagonist; (2) the spectrum of antagonistic, neutral and stimulatory activity in albumin fractions

R

PHILIP

L. POFFENBARGER

prepared by methods not utilizing dialysis indicating that preparative technique can significantly affect the degree of measured insulin antagonism; (3) the failure of the SIA to demonstrate an unequivocal in-vivo effect; (4) a mechanism of action inconsistent with the metabolic alterations observed in diabetes mellitus; (5) the use of the relatively insensitive and imprecise rat hemidiaphragm bioassay to assess qualitative and quantitative differences in insulin antagonism; and (6) the lack of specificity since albumins of other species as well as egg albumin and gelatin are indistinguishable from the SIA in the assays currently in use. Although these difficulties do not absolutely deny a physiological role for the SIA, they do urge a very cautious interpretation of the available data until a much more selective means of assessment becomes possible. (Metabolism 19: No. 9, September, 66% 686, 1970)

ECENT TEXTBOOKS

continue to accord the albumin-bound insulin antagonist, synalbumin, a prominent role in the pathogenesis of diabetes mellitus.1~2 This theory was first promulgated in 1961 by John Valiance-Owen:’ and has been discussed numerous times by its author.4.‘I However, much experimental information concerned with the biochemistry and physiology of the synalbumin insulin antagonist (SIA) has appeared since the earlier reviews and ~._.____

-

.- -

_____~__

-.---

___~_.

From the UCLA School of Medicine, Los Angeles, Calif.. and the Department of Medicine, Harvard Medical School and The Peter Bent Brigham Hospital, Boston. Mass. Received for publication March 6, 1970. Supported in part by Grant 4-444934-44867 f ram the Diabetes Association of Soufhern California. MAYER B. DAVIDSON, M.D.: Assistant Professor of Medicine, UCLA School of Medicine: V.A. Clinical Investigator, Wadsworth Veterans Administration Hospital, Los Angeles. Calif. PHILIP L. POFFENBARGER.M.D.: Research Fellow, Department of Medicine, Harvard Medical School and The Peter Bent Brigham Hospital, Boston, Mass.: Special Fellow I-F03-AM-30777-01, Institute of Arthritis and Metabolic Diseases, USPHS. 668

METABOLISM, VOL. 19, No. 9 (SEPTEMBER), 1970

SYNALBUMIN INSULINANTAGONIST

669

has not been included in the more recent ones. As more investigators have been drawn into this challenging area, data both to support and refute the original hypothesis have accumulated. For these reasons, we think it appropriate to review the entire area of synalbumin at this time to provide a comprehensive basis for present assessment of its role in the pathogenesis of diabetes mellitus. We shall also offer criteria which any circulating insulin antagonist should meet to be of physiological significance. BACKGROUND Insulin will stimulate glucose uptake and glycogen deposition in rat diaphragm incubated in vitr0.l” When this tissue is incubated in plasma or serum, the increase in these parameters reflects the e&ctive concentration of serum insulinlike activity (ILA) , i.e., the biological sum of insulin and insulin-like substances on the one hand and antagonists on the other. Studies of insulin antagonism have been primarily concerned with the inhibitory effect of plasma or protein fractions on the action of insulin added in vitro. The observation that added insulin could not always be quantitatively recovered led to the conclusion that a circulating antagonist must be present. Since these early experiments are of fundamental importance, they will be presented in detail. The early studies employing the rat hemidiaphragm bioassay revealed that plasma from normal fasting subjects contained measurable ILA which markedly increased after administration of oral glucose. I3 This ILA, presumed to be insulin, was also found in plasma from obese, mild diabetics not receiving insulin.14 In contrast, plasma from hyperglycemic, nonketotic, insulin-requiring diabetics demonstrated no effective ILA, and yet many patients had received an injection of insulin one hour before sampling. l4 In addition, the activity of insulin added to their plasma could not be recovered. l-I When plasma from this same type of patient was examined after control had been established, effective insulin concentrations were found and the recovery of added insulin was comp1ete.l’ It was postulated that when insulin-requiring diabetics received sufficient insulin to overcome a plasma antagonist, the diabetes was brought under control. Although insulin antibodies were not well appreciated at the time these experiments were conducted, their presence in the insulin-treated diabetic15 might have been the insulin antagonist measured. For example, in plasma from obese uncontrolled diabetics not receiving insulin, no antagonism to insulin could be demonstrated, but antagonism was found in plasma from patients in better control on insulin therapy.r4 Fractionation of plasma revealed that the insulin antagonist resided in the albumin fraction.16 Albumin from normal subjects and insulin-requiring diabetics was antagonistic at concentrations of 3.5-5.5 Gm. per cent, but only diabetic albumin retained its antagonism at 1.25 Gm. per cent.16 Further studies demonstrated that albumin preparations from five obese, noninsulin requiring diabetics and six “prediabetics” were also antagonistic at 1.25 Gm. per cent.” Since the inhibitory effect of these albumin preparations could be removed from the albumin molecule by passage through a cellulose column,17 the term “synalbumin” was applied to this insulin antagonist. Based largely on the foregoing

670

DAVIDSON

ANDPOFFENBARGER

evidence, the SIA was postulated to be an albumin-bound substance responsible for “the primary abnormality in essential diabetes mellitus.“3 CLINICALSTUDIES Many investigators have attempted to confirm these potentially important findings with varying results. Valiance-Owen used an acid-ethanol extraction procedure’* for isolating albumin and although this led to interpretive difficulties, most workers have followed his lead. For example, in certain hands acid-ethanol extracted albumin preparations exhibited marked ILA which made evaluation of insulin-antagonistic properties difficult. lg~‘OFor purposes of collation, the following scheme was devised. If glucose uptake in the presence of insulin alone was not significantly different than the uptake with albumin alone, then that preparation was considered to have stimulated the diaphragm maximally and no examination of its antagonistic properties could be made. However, if the glucose uptake in the presence of insulin alone was significantly higher than with albumin alone (even if ILA were present), then that preparation could be evaluated for antagonism by comparing the glucose uptakes between flasks containing insulin alone and albumin plus insulin. Table 1 summarizes the available data on acid-ethanol extracted albumin preparations from normal and diabetic subjects. The results show that at albumin concentrations of less than 3.0 Cm. per cent, 107 of 161 diabetics manifested insulin antagonism as compared to only 24 of 121 normals. At albumin concentrations of 3.0 Gm. per cent or greater, 38 of 51 normals and six of 18 diabetics manifested insulin antagonism. Insulin-like activity of these albumin extracts was a prominent feature of many studies in which the effect of albumin alone was assessed. It is generally accepted that increased SIA is present in a subject if his albumin preparation manifests insulin antagonism when tested at a concentration of 2.0 Gm. per cent or less. Increased SIA has also been noted in families of patients with diabetes mellitus. In the most extensive study to date, Valiance-Owen has found increased SIA in 58 of 97 close relatives of diabetics. w Sixteen of these people had carbohydrate intolerance and three suffered from “spontaneous hypoglycemia.” Excluding the 16 patients with abnormal glucose tolerance tests, 42 of the remaining 81 had increased SIA as compared to 25 of 98 randomly selected hospital patients. In another report, 26 of 48 siblings and five of nine parents of diabetics manifested increased SIA.“g In less extensive studies, increased SIA has also been demonstrated in the following proportion of close relatives; three of sevenjja four of eight,)” two of four,‘):’ and one of one. :(:,Of the 20 sibships suitable for analysis in the families reported by Valiance-Owen,“’ 38 subjects had increased SIA while 26 did not, a ratio which did not differ significantly from 1: 1. It was proposed that increased SIA provided a genetic marker for diabetes mellitus and that the pattern of inheritance is one of autosomal dominance.“Y However, Sherman, who feels that idiopathic hypoglycemia of infancy may characterize the prediabetic state,“R could not find increased SIA in the plasma of 8 of these childrena Not only did repeat testing fail to reveal increased SIA in three of these patients even after they developed clinical diabetes, but increased SIA could be demonstrated in only three of fifteen parents.:‘” It is of interest that two

(1965)

(1965) (1965) (1966) (1966) (1966)

(1966) (1966) (1966) (1966) (1967) (1967) (1968) (1968)

23

24 25 26 27 28

29 30 31 32 33 34 35 36 83

$ Effect 5 When

* Denominator t Albumin

(1961) (1963) (1964) (1964)

(Year)

21 20 19 22

References

(3.54.0%)? (3.0%) (3.5%)1 (1.25%)1 (2.5%) (3.0-3.5%) (5.0%) (1.25%) (5.0%) (1.25%) (1.25%) (4.0% ) t (1.25%) (1.5%) (4.0%) (1.25%)? (2.0%) (1.5%) (3.0%)

5/5*

2/5 4/4 O/6 O/11 2/3 5/5 l/4 6/6 O/l O/33 6/6 2/10 7/19 2/4

4/4

o/13

10/20 6/13

SIA

1 .-Summary

NCXllldS

Table

(1.25%) (1.5%) (1.25%) (2.0%) (1.25%)i (2.0%) (2.0%) (1.25%) (1.5%) (3.0%)

5/11 9/9 O/2 l/7 O/6 7/8 6/9 O/4 o/17 6/17

5 per

(1.25%) (1.25%)

2/3 51/54

2.0%)

(1.25%)

(I

(1.25%)+

(3.5-4.0%

lO/lO

9/14

7/7

O/l

Diabetics

of SIA in Normal

)

and Diabetic

20/20 16/16

(1.25%)t

4/4

(1.5%) (3.0%)

4

(4YO%)

o/4

$

(5.0%) $ (5.0%) (1.25%) (1.25%)? (4.0% ) t

2/5 2/6 O/l O/8 o/4

(2.15-4.0%) (3.0%) (3.5%)? (1.25%)P (2.5-3.5%)

13/13 8/8 o/4 O/6 l/14

1

Normals

Population

5

ILA

$

6/6

;

4

(1.25%);

$ $ $

(I

o/14

2.0%)-F

(1.25%)i

(2.5-4.0%)

O/7

13/15

Diabetics

672

DAVIDSON

ANDPOFFENBARGER

groups”3,“”

who have been able to find increased SIA in a large percentage of diabetics have not been able to demonstrate increased SIA in close relatives of these patients and have challenged its role as a genetic marker. Increased SIA has also been found in conditions often associated with the “prediabetic” state. Thus, 19 of 28 patients who had suffered a myocardial infarction three months previously exhibited increased SIA as compared to only six of 28 control subjects. :WIncreased SIA was also noted in 10 of 13 patients with angina pectoris and electrocardiographic changes of “diffuse myocardial lesions” but no evidence of diabetes mellitus.:“’ In the same study. 26 of 33 patients who had suffered a myocardial infarction within the past year also manifested increased SIA, but it is not clear which of these subjects had overt diabetes. In one review,“) Valiance-Owen mentioned that 35 of 40 patients with ischemic heart disease had increased SIA, but no further details were given. Major congenital anomalies are more common in offspring of women with diabetes.“” Increased SIA was found in 24 of 36 mothers of children born with cleft palates and/or lips,“l in 13 of 14 mothers delivering babies with major spinal deforrnities,l” and in 15 of 18 mothers delivering babies with major extremity deformities.-” In the control group used for these two studies, only 14 of 50 women had increased SIA. 41 However, six of the children with extremity anomalies were “thalidomide babies” and since five of their mothers had increased SIA, one may question the causative role of the insulin antagonist in producing these deformities. Fifteen of 50 women with unexplained intrauterine deaths during the third trimester also had increased SIA,“:’ but again the postulated role of synalbumin in these cases is questionable since the prevalence here is the same as in the previous unselected control group of women. Increased SIA was also demonstrated in five of 10 normal women during the third trimester of pregnancy.“:’ However, these women did not deliver large babies nor did a “family history of diabetes seem to play a role in those pregnant women who showed greater than normal inhibitory activity.““:$ Increased SIA was present in four patients with necrobiosis lipoidica diabeticorum and normal glucose tolerance tests.11 It appears that increased SIA may be found in a high percentage of conditions associated with diabetes mellitus but its validity as a genetic marker remains unproven. PHYSIOLOGYOF SYNALBUMIN Circulating

Levels

Several hormonal and metabolic manipulations have been found to affect the circulating level of synalbumin. Valiance-Owen could demonstrate no insulin antagonism in albumin fractions (4.0 Gm.% ) from three hypophysectomized patients. l7 Albumin preparations from one of these patients had exhibited insulin antagonism before his operation. However, this finding could not be confirmed in another seven hypophysectomized patients”l or in hypophysectomized rats*4 with albumin concentrations of 2.5-4.0 Gm. per cent. Albumin fractions (4.0 Gm.% ) of two adrenalectomized patients, from whom cortisone had been withheld for 50 hours, did not antagonize insulin whereas insulin antagonism returned after cortisone replacement. 45 Levels of the SIA fall at thirty and sixty

SYNALBUMIN

INSULIN

ANTAGONIST

673

minutes after an oral glucose load and at fifteen and thirty minutes following an intravenous glucose challenge. 46 This apparent decrease in synalbumin was shown not to be a masking of insulin antagonism by endogenous insulin secretion. On the other hand, synalbumin levels rise following intravenous tolbutamide and insulin35 and it was concluded that the SIA may be a product of insulin degradation. This explanation is supported by the demonstration of increased insulin antagonism in albumin preparations of rat serum after perfusion through rat liver and a further augmentation of this antagonism after insulin was added to the perfusate. 44 However, marked insulin antagonism in an albumin preparation from a pancreatectomized animal not receiving insulin44 seems inconsistent with this hypothesis. This explanation is clearly at variance with the results following glucose administration where insulin levels are high but SIA is diminished. A cohesive explanation for the varying levels of synalbumin cannot be formulated on the available fragmentary and contradictory data. Mechanism of Action

The mechanism of action of synalbumin will be examined from two viewpoints: its possible mode of action and its site of action in the metabolic pathways of carbohydrate metabolism. The role of synalbumin as a physiological insulin antagonist may then be judged in the context of the available information. Since free fatty acids (FFA) have been shown to inhibit the insulin effect on glucose uptake in rat diaphragm and heart preparationq4’ several investigators have examined the possibility that this albumin-bound substrate is the SIA. However, no correlation has been found between the FFA content of various albumin preparations and their antagonistic activity.23J4,27+46FFA added to non-antagonistic albumin preparations do not induce insulin antagonism.24~27~48 Most workers agree that albumin prepared by several different methods will cause a significant lowering of pH in Krebs Ringer bicarbonate buffer,24,33,48,4g although one group noted no change. 5o Since acidosis will decrease insulinstimulated glucose uptake in vitro, 51 the possibility that synalbumin acts by altering the pH of the incubation media has been examined. However, significant antagonism remains after pH adjustment.24*33,4s,4gThe increase in hydrogen ion concentration of unaltered albumin solutions must play some part though since after pH adjustment, not only was the antagonistic effect attenuated,24,48g4s but basal glucose uptake was enhanced by the albumin preparations in three2”*“*r49 of four33 studies. The tissue interaction of synalbumin and insulin has also been studied in various ways. Increasing albumin in the presence of a constant insulin concentration resulted in more antagonism.“3~52 Conversely, increasing insulin a thousand-fold in the presence of a constant albumin concentration did not appreciably change the level of insulin antagonism in one study52 although another group noted decreasing antagonism in the presence of ten times the usual concentration of insulin.z3 Preincubation of hemidiaphragms with insulin did not protect the tissue from the effect of synalbumin33p5z nor did washing after exposure to the SIA before incubation with glucose and insulin.52 The SIA also had no effect on the binding of 1311-insulin to diaphragms.23 Antagonistic albumin preparations

674

DAVIDSON

AND POFFENBARGER

have no insulin-destroying properties as measured by the preservation of trichloroacetic acid precipitable insulin after incubation.“” Preincubation of tissue with synalbumin (as well as “bound” insulin, crystalline insulin or diabetic sera) actually resulted in a delayed disappearance of immunoreactive insulin during a subsequent period of incubation.53 It may be significant that after incubation in the presence of synalbumin, the hemidiaphragms are pale and rigid.33$0,54,55

Although albumin binds calcium and magnesium ions, inhibition of the insulin effect on glucose uptake could not be duplicated by reducing the concentrations of Ca++ and Mg++ in the incubation media.‘i In spite of the fact that fraction V albumin preparations contain citrate”H and increased intracellular concentrations of this substrate decrease muscle glycolysis by inhibiting phosphofructokinase,5B incubation of diaphragms in citrate-enriched media did not block insulin-stimulated glucose uptake.4s The mode of action of the SIA, then, does not appear explicable on the basis of FFA or citrate content, bivalent cation or pH effects, or insulin-degrading activity of the albumin preparations. Their antagonistic potential far exceeds the known physiologic limits of insulin concentrations but the precise manner by which this is accomplished remains unclear. Since in-vitro studies of glucose metabolism in adipose tissue have demonstrated either no insulin inhibition?l or ILA in the albumin preparation itse1f,Z9~32,53 the rat diaphragm has been utilized almost exclusively to study the site of action of the SIA. The initial transport of substrate across the cell membrane seems to be affected by synalbumin and some of the evidence for this also suggests that insulin inhibition per se may not be involved. Thus, certain albumin preparations not only antagonize basal glucose uptake (i.e., in the absence of insulin)27sU.48,” but also abolish”” the anoxic enhancement of glucose entry into muscle.“’ The SIA depresses basal as well as insulin-stimulated a-aminoisobutyric acid (AIB) uptake.52 (In another muscle system, the isolated levator ani preparation of the rat, both antagonistic and nonantagonistic albumin stimulated basal AIB transport.31) Since transport of this nonutilizable amino acid occurs in the absence of glucose,5H the action of synalbumin on the intracellular transfer of this substrate is probably independent of its effect on glucose transport. Insulin-stimulated d-xylose transport is also inhibited by synalbumin although in this study basal transport is slightly increased by the SIA.4R However, since many albumin preparations do not affect basal glucose uptake, but block only insulin-mediated uptake, it may be that it is only when the intracellular transfer of glucose is maximal that the antagonism of transport by these preparations become manifest. The effect of synalbumin on intracellular metabolic pathways has also been examined. Insulin-stimulated glycogen synthesis was inhibited by the SIA to the same extent48 or much lessZ3.“’than the inhibition of glucose uptake. More direct evidence that glycogen synthesis is not affected by the SIA was provided by studies on glycogen synthetase. This enzyme exists in two forms, one of which is regulated by insulin and substrate independent while the other is substrate dependent and apparently not influenced by insulin.“” Neither form of glycogen

SYNALBUMIN

INSULIN

ANTAGONIST

675

synthetase was inhibited when measured after exposure of rat diaphragm to the SIA although insulin-stimulated glucose uptake was markedly depressed.s0 However, other intracellular pathways are modified by synalbumin. Insulin increases protein61*62 and RNA synthesise3-65 in the absence of glucose and independent of the transport of their respective precursors. The SIA inhibits insulin-stimulated incorporation of radioactive phenylalanine into proteiP and adenine into RNA.6o It also decreases oxygen consumption,66 a process which is not influenced by either glucose or insulin in the media. When glycolytic intermediates were measured in the presence of albumin or an antagonistic subfraction, there was a decrease in pyruvate and an accumulation of phosphoenolpyruvate and earlier intermediates suggesting inhibition of pyruvate kinase.50 The fact that there were no changes in glycolytic intermediates of adipose tissue50 supports the causal relationship between this change in the glycolytic pathway and the diminution in glucose uptake induced by the SIA in muscle. Insulin-stimulated lactate production was also decreased by the SIA.48,4Q However, this may have been secondary to the inhibition of glucose uptake since there were no changes in lactate production or glucose transport under basal conditions48,4Q and nonantagonistic albumin had no effect on insulinmediated lactate production. 4Q Synalbumin also inhibited insulin-stimulated glucose incorporation into lipid, 4Qbut this again is probably secondary to decreased glucose transport. A recent study has extended both the ILA and insulin antagonistic properties of albumin preparations to lipolysis .67 Adipose tissue incubated in the presence of SIA exhibited a dimunition in corticotropin( ACTH) -induced glycerol release, an insulin-like effect. However, when the tissue was exposed to ACTH, synalbumin and insulin, inhibition of ACTH-induced lipolysis by insulin was prevented, an insulin-antagonistic effect. In-vitro experiments reveal that the SIA acts primarily on muscle, probably through an irreversible alteration of the tissue. It blocks transport of glucose, AIB and xylose, insulin-stimulated protein and RNA synthesis but has no effect on the glycogen pathway. The SIA also depresses oxygen consumption and appears to inhibit glycolysis at the pyruvate kinase step. Synalbumin has an insulinlike effect on both glucose metabolism and lipolysis of adipose tissue although its insulin antagonistic effect has also been demonstrated on lipolysis. In Vivo Effects

An in-vivo effect of the SIA must be demonstrated before it can be accepted as a physiological insulin antagonist. To that end, Jervell injected antagonistic albumin and insulin intraperitoneally into rats and demonstrated diminished glycogen content of their diaphragm when compared to the effect of insulin and nonantagonistic albumin. 26Incorporation of glucose-C?* into radioactive glycogen was also decreased by the antagonistic preparation under these circumstances.26728 When the testes were retracted into the abdominal cavity and the inguinal canal sutured 24 hours before injection, no effect was noted on radioactive glucose incorporation into epididymal fat pad glycogen.68 YoungGQ has also seemingly demonstrated inhibition of insulin-stimulated incorporation of glucose-Cl4 into rat

676

DAVIDSON

AND

POFFENBARGER

diaphragm glycogen after intraperitoneal injection of serum or acid-ethanol extracted albumin. However, appreciable amounts of ILA were present in the sera and since the parameter being compared was the differential effect of added insulin, the results must be interpreted cautiously. For example, although the addition of insulin to the control solution and test sera resulted in nearly identical activities, antagonism was claimed for the sera since the total effect minus the sera ILA was significantly less than the total activity of the test solution minus the minimal effect of the control in the absence of insulin (Figs. 3 and 5, Reference 69). Although no data on the effect of the albumin extracts alone were furnished in this study, albumin prepared in this manner can possess marked ILA when injected intraperitoneally. W*After intraperitoneal administration, the usual vascular pathways of introducing substances to target tissues are not followed so that the normal detoxifying mechanisms of the body are circumvented. The effect of synalbumin on glucose metabolism was studied in a number of experiments in which the intravenous route of administration was used.” A markedly insulin-antagonistic preparation (human fraction V) was infused over a three hour period into rats anesthetized with pentobarbital. Endogenous plasma insulin concentrations were unaffected as well as the establishment of steadystate blood glucose levels in fed normal rats and the disposal of exogenous glucose in fasted normal rats. The SIA also had no effect on steady-state blood sugar or the handling of exogenous glucose in animals with limited insulin reserve (partially pancreatectomized) and the hypoglycemic response to exogenous insulin was unaltered. In vitro experiments demonstrated that insulin insensitivity was nor induced in the diaphragms of infused rats nor did their plasma inhibit the action of insulin on normal diaphragms. The concentration of human fraction V albumin achieved in these experiments was 7.4 Gm. per cent at two hours and 5.5 Gm. per cent at three hours. It was concluded that the failure of synalbumin to affect glucose metabolism in vivo was due to neutralization of the SIA by the intact animal. A recent attempt to demonstrate an in-vivo effect of synalbumin after intravenous administration was more successful. Insulin-stimulated incorporation of radioactive glucose into diaphragm glycogen was inhibited by the SIA when measured thirty minutes after all three (insulin, glucose-Cl4 and albumin) were injected into the tail vein of unanesthetized rats .71 One reason for the discrepancy between this injection study and the infusion experiments may lie in the different rates of intravenous administration. If the postulated clearing mechanism were rapidly saturated, synalbumin might then exert an in-vivo effect for a short period of time. Since the response of the diaphragm seems to be irreversibly altered after exposure to the SIA (see above), the metabolic activity of this tissue over the entire 30minute period could be influenced by the initial contact. TO resolve this question, it would be necessary to know whether plasma harvested 30 minutes after injection inhibited glucose metabolism of normal diaphragms in vitro.

SYNALBUMIN

677

INSULIN ANTAGONIST THE BIOCHEMISTRYOF

SYNALBUMIN

Problems of Isolation

Although some information is available concerning the physiochemical properties of the SIA, interpretation of these data is hampered by methodological problems surrounding the isolation of this insulin antagonist. After initial characterization studies revealed that the plasma ultrafiltrate contained no insulin antagonism,T2 the SIA was subsequently localized in the albumin-containing supernatant of sodium sulfate treated plasma.ls A more pure preparation of plasma albumin was obtained by a modificatiorP of a trichloroacetic acid-ethanol extraction method16 which has been widely copied. However, these acid-ethanol extracted albumin preparations also contain small amounts of alpha-28,30,73and beta-73globulins, nonsuppressible insulin-like substances (NSILA) 1g~20~24~26~27~32~ Table 2 lists these 33,55proinsulin and variable amounts of insulin. 1%2WZ55,75,r6 substances for comparison. (The problem that ILA causes in evaluating these fractions for insulin antagonism has been discussed above.) Dialysis of these acid-ethanol extracts, which is an integral part of this method of isolating albumin, introduces another serious difficulty. Untreated dialysis casing contains a low molecular weight sulfur-containing compound55g77 which binds to albumin under acid55 but not neutral conditions.7s This substance can be removed by boiling the dialysis casing55p77and after recovery will antagonize insulin in vitro.55 This artifactual insulin antagonist is probably responsible for the conversion of non-antagonistic albumin to antagonistic albumin after reextraction and dialysis.48z55 The interaction between untreated dialysis casing and an acidic albumin solution does not entirely explain the SIA since synalbumin is not only recovered from extracts dialyzed through boiled casing 55,78but can also be demonstrated in human albumin preparations obtained by methods not requiring dialysis. These include fractionation by cold ethanol (Cohn method)1s,23%3,48,54,‘1,79 ether precipitation,16 acid methanol extraction24 and isolation of albumin by sodium acetate-ethanol treatment. 8o The SIA can be found in human albumin prepared by ammonium sulfate precipitationz4 zone electrophoresis,24 and treatment with hexadecyl pyridinium bromide, 6g all of which utilize dialysis of fairly neutral solutions. Specificity and Reproducibility

The specificity of the SIA for human diabetes mellitus may be questioned since insulin antagonism can be demonstrated in pig,6g 0x,69 bovine,271sg rat44;6g and egg27,33 albumin as well as the collagen protein gelatin.33 The spectrum of antagonistic, neutral, and stimulatory activity in different preparations of bovine fraction V2? suggest that certain minor unknown variations in preparative technique can significantly affect the degree of measured insulin antagonism. Glitzers1ps2 found some ILA and no insulin antagonism in approximately 200 separate albumin fractions prepared by either acid-ethanol or acid-methanol extraction. His experience indicates that problems in studying synalbumin may also lie outside of isolation procedures since both hes2 and Cameron32 were unable to demonstrate the SIA in albumin preparations that manifested insulin

Table

~____

____

10-4 M 69,000 anionic not adsorbed denatures 4.9 denatures

liver

Albumin

~-

2.-Comparison

* Properties listed may be found in references

Origin Concentration in blood Molecular weight Dowex 50 chromatography Cellulose chromatography Heat stability pH stability Isoelectric point Disulfide cleavage

Property

~~~ _~ NSILA

of Proteins

cited in text.

extrapancreatic unknown 45,000 cationic unknown stable alkali inactivates 8.0 inactivates

~~~~~~

of Properties

in Acid-Ethanol

pancreas lo-11 M 9800 anionic adsorbed stable unknown 5.3-5.4 inactivates

Proinsulin -___

Present

of Serum:’

pancreas lo-10 M 6000 anionic adsorbed stable unknown 5.3-5.5 inactivates

Insulin

Extracts

_.___

liver (?) unknown 4000-I 0,000 cationic adsorbed stable alkali inactivates unknown inactivates

SIA

SYNALBUMIN

INSULIN

ANTAGONIST

679

antagonism in other laboratories. Significant ILA was found in an albumin preparation that was neutral in another laboratory.s3 Thus, inexplicable variations in both preparative and assay techniques hinder progress in this challenging area. Physiochemical Properties

Some information exists concerning the physiochemical properties of synalbumin prepared by acid-ethanol extraction,45~73~78,84 Cohn fractionation37s73 and hexadecyl pyridinium bromide precipitation. 6s However, firm conclusions should not be drawn from these data since the nature of the substance under study may be influenced by its method of isolation. The SIA can be removed from the albumin molecule by heat denaturation, 45,6s by extraction with isooctaneacetic acid,Zrr45 chloroform”” and hot ethanolt5 by chromatography through partially acetylated cellulose17,84 or sulfonic acid polystyrene resin,78J4 by disulfide bond cleavage with thioglycollic acid&l and by dextran gel filtration in 0.02 M sodium chloride in one laboratory73 but not in another.55 The SIA can be recovered in the supernatant after heat denaturation but loses its activity after three days at 2O C.O” Gel filtration suggests that this substance is a polypeptide with a molecular weight between 4-10,000. 8~6s,73This polypeptide appears to be stable in acid but not in alkali,@ is adsorbed onto oxycellulose and eluted with acid,6s passes through DEAE cellulosecs and is dialyzable.45~69~73 Further identification and purification of the SIA has not been accomplished to date. B-Chain Hypothesis

A number of workers have indirectly approached the identification problem by testing the hypothesis originally suggested by Ensinck, Mahler, and VallanceOwen that the B-chain of insulin may indeed be the polypeptide antagonist.@ In studies of the transport of the A- and B-chains of insulin in serum, these investigators demonstrated that B-chain binds to albumin whereas A-chain apparently remains unassociated with a transport protein.85 Subsequently it was shown that both chemically and enzymatically derived reduced B-chain rendered non-antagonistic albumin antagonistic when measured by the in vitro bioassay.% This observation has been confirmed in vivo68,86-88and both confirmedss and denied@ in vitro. Valiance-Owen has prepared immune rabbit gamma globulin by injection of the heat denatured supernatant fraction from antagonistic albumin preparations and in a preliminary report has stated that this antisera inhibits the in vitro insulin antagonism of normal, diabetic and B-chain albumin.g Although B-chain has not been identified by other chemical means in antagonistic albumin preparations, similar ion-exchange chromatographic behavior,% molecular weight,8*69,‘3electrophoretic mobility,24~sj and its insulin antagonistic activity all lend indirect support to the B-chain hypothesis. On the other hand, A- and B-chain immunoassay data of human sera have failed to show elevation of B-chain in diabetic subjects.so~sl And one post mortem study of human hepatic insulin glutathione transhydrogenase revealed no difference in enzymatic activity between non-diabetic and diabetic subjects.s’

680

DAVIDSON AND POFFENBARGER CRITERIA

Although the past decade has seen much effort expended in examining the role of the SIA in diabetes mellitus, there are no generally accepted guide lines for physiological insulin antagonism against which synalbumin can be measured. We propose the following four criteria for any circulating insulin antagonist to be of physiological significance and hence of possible importance in the pathogenesis of diabetes mellitus. These criteria have been formulated to be applicable even if the antagonism of insulin can not be directly demonstrated in vitro, but requires prior administration to the test animal (as with growth hormone). The four conditions that must be fulfilled are the following: ( 1) The antagonist should display greater activity in plasma from diabetics and prediabetics and be absent or exhibit lesser activity in plasma from nondiabetics. Differential levels of the albumin-associated antagonist have been found

and consequently synalbumin does meet this first criterion. (2) The demonstrated mechanism of action of the antagonist should be con-

by

many

investigators","",Z~,~,j,~7-:~0.~~.~~,8~

sistent with the qualitative and quantitative metabolic alterations found in diabetes mellitus. Qualitatively, the in-vitro depression of oxidative metabolismfj6

and insulin-stimulated RNA synthesis by the SIA”” suggest impairment of two vital biological functions, neither of which seem altered in diabetes mellitus. Quantitatively the in-vitro antagonism of large amounts of insulin (up to 0.5 U/ml.) by synalbumin in an irreversible manner suggests that muscle tissue of diabetics should be completely refractory to circulating insulin. However, not only does the forearm muscle of diabetics respond to less than 0.005 U/ml. of exogenous insulinD3 but glucose uptake by forearm muscle in maturity-onset diabetics is comparable to controls after the intra-arterial injection of 0.1 U of insulin.94 Since the SIA can neutralize such large amounts of insulin in vitro, there should be no assayable insulin in the plasma of diabetics when measured on the rat diaphragm.“” Again, this is not the case as several investigators have demonstrated normal or increased levels of insulin in plasma from diabetics by this bioassay method. 14~96,07 For these reasons, synalbumin fails to meet this second criterion. (3) The in-vivo administration of the antagonist should be capable of decreasing sensitivity to endogenous insulin. The inability of infused synalbumin to alter glucose homeostasis in vivo under a variety of conditions as well as the failure of both the tissue and the plasma from infused rats to demonstrate insulin antagonism provide direct evidence that this antagonist is not active in the living animal.rO The reasons for withholding acceptance of a physiological invivo effect by albumin preparations injected intra-peritoneally or as a single intravenous dose have been discussed above. Thus, we feel that synalbumin also fails to meet this third criterion. (4) Glucose intolerance or insulin insensitivity should be reversed if the antagonist can either be neutralized in vivo or its source removed (assuming that B cell function is not irreversibly altered). This condition can only be studied

once the third criterion has been fulfilled and consequently vestigated with synalbumin.

has not been in-

SYNALBUMIN

INSULIN

ANTAGONIST

681

The general validity of these criteria can be examined by applying them to three other circulating insulin antagonists : growth hormone (GH) , FFA, and insulin-binding antibodies. The first two are accepted physiological antagonists which have also been considered as of possible pathogenetic significance in diabetes while the third is an acquired insulin antagonist with a well-defined mechanism of action. The crux of the argument about the role of GH and FFA in human diabetes centers around the first criterion. Fasting GH levels are similar in controls, prediabetics, noninsulin dependent and insulin dependent diabetics.34vQs-106The blunted GH response of maturity-onset diabetics to oral glucose,107J08 insulinlo and arginineQQ,100,105 may be related to their associated obesity. Studies in insulindependent diabetics have revealed norma1,101J05J06 diminishedloo or enhancedlo GH responses to arginine, normal GH release after insulinlo and a blunted rebound after oral glucose.1o2 Prediabetics, on the other hand, have shown an increased GH response to intravenous tolbutamidelM and a heightened rebound after either oraP4~Qsor intravenous lo4 glucose. Elevated fasting FFA concentrations as well as a delayed fall after glucose are noted only in the obese diabeticlo suggesting that adiposity may be more important than abnormal carbohydrate metabolism. However, it is possible that the relatively normal concentrations of these two insulin antagonists (GH and FFA) may actually be abnormal in the presence of the hyperglycemia and hyperinsulinism of early diabetes. Since insulin-binding antibodies are only found after insulin treatment,ls they obviously do not fulfill this condition. Thus, none of these substances unequivocably meets the first criterion. With respect to the second criterion, the changes induced by GH administrationllo and the metabolic changes in spontaneous acromegaly provide good evidence that GH does meet this condition. Furthermore, the usual increase in GH three to five hours after an oral glucose challenge will interfere with the disposition of a subsequent glucose meallll indicating that changes in GH secretion alter carbohydrate metabolism under normal physiological conditions. The present evidence from in-vitro biochemical studies concerning impairment of glucose metabolism and insulin effects by FFA, although incomplete, is also compatible with this criterion. 47 Since insulin-binding antibodies specifically neutralize insulin, the resulting metabolic abnormalities must parallel those seen in diabetes. Thus, the second criterion is met by each of these insulin antagonists. In contrast to synalbumin, GH,12 FFA113 and insulin antibodies114 are all capable of altering insulin sensitivity and glucose homeostasis in vivo and therefore meet the third criterion. All three of these circulating antagonists can be shown to fulfill the fourth condition. With respect to GH, the Houssay phenomenon is readily demonstrable in diabetes mellitus. Abnormal glucose tolerance tests in the presence of high levels of FFA induced by norepinephrine can be converted to normal when the rise in fatty acids is blocked by nicotinic acid.l15 If animals made hyperlgycemic by the infusion of insulin antibodies are given sufficient insulin, normal glucose metabolism can be restored. 114Therefore all three antagonists fulfill this fourth condition.

682

DAVIDSON

Table 3.-Characteristics Criteria

1. 2. 3. 4.

Differential levels Mechanism of action In vivo activity Reversal of glucose intolerance

of Selected Circulating ~__ Synalbumin G.H. Yes No No *

? Yes Yes Yes

AND POFFENBARGER

Insulin Antagonists FFA ? ‘, Yes Yes

Insulin Ab. No Yes Yes Yes

* Can only be tested if third criterion fulfilled.

Table 3 summarizes how well these circulating insulin antagonists meet these criteria. Each condition is met by at least one antagonist, yet no substance meets all four. Thus, although each condition is necessary, only fulfilling all of them is sufficient to implicate a circulating insulin antagonist in the pathogenesis of diabetes mellitus. Although the first three criteria by themselves are compelling, until the in-vivo effect of an antagonist could be reversed or its source identified, its physiological significance must remain in doubt. This is true because the first three criteria could be met by a substance artifactually produced during in-vitro preparation from plasma. Since synalbumin meets only the first criterion, it should not be assigned a causative role in diabetes mellitus on the basis of the available evidence. ACKNOWLEDGMENT The authors are deeply grateful to Dr. C. J. Goodner for his guidance in making the initial transition from the bedside to the laboratory bench and for suggesting the fourth criterion. REFERENCES 1. Scott, R. B.: (Ed.) Price’s Textbook of the Practice of Medicine (ed. 10). London, Oxford University Press, 1966, p. 375. 2. Williams, R. H.: The pancreas. In Williams, R. H. (Ed.): Textbook of Endocrinology (ed. 4). Philadelphia, W. B. Saunders Co., 1968. 3. Valiance-Owen, J., and Lilley, M. D.: Insulin antagonism in the plasma of obese diabetics and prediabetics. Lancet 1: 806, 1961. 4. Valiance-Owen, J. : Diabetes mellituscausation. Proc. Roy. Sot. Med. 55:207, 1962. 5. -: Synalbumin insulin antagonism. Diabetes 13:241, 1964. 6. -: Insulin antagonists and inhibitors. In Levine, R., and Luft. R. (Eds.): Advances in Metabolic Disorders, Vol. I. New York, Academic. 1964, p. 200. 7. -: Synalbumin antagonism in obesity and maturity onset diabetes mellitus. Ann. N.Y. Acad. Sci. 131:315, 1968. 8. -: Insulin antagonists. In Leibel, B. S., and Wrenshall, G. A. (Eds.): On the nature and treatment of diabetes. Amsterdam,

Excerpta Medica Foundation, 1965. p. 340. 9. -: Synalbumin insulin antagonism. Folio Endocr. Jap. 44:455, 1968. 10. -: Synalbumin insulin antagonism in diabetes. In Ostman, J., and Milner. D. G. (Eds.): Proceedings of the Sixth Congress of the International Diabetes Federation. Amsterdam, Excerpta Medica Foundation, 1969, p. 243. 11. -: Diabetes mellitus. Brit. J. Derm. 81:9, 1969. 12. Gemmill, C. L., and Hamman, L., Jr.: The effect of insulin on glycogen deposition and on glucose utilization by isolated muscles. Bull. Johns Hopkins 68:50. 1941. 13. Valiance-Owen, J., and Hurlock, B.: Estimation of plasma-insulin by the rat diaphragm method. Lancet 1:68, 1954. 14. -, Hurlock, B., and Please, N. W.: Plasma-insulin activity in diabetes mellitus measured by the rat diaphragm technique. Lancet 2:583, 1955. 15. Berson, S. A., Yalow, R. S., Bauman, A., Rothschild, M. A., and Newerly, K.: Insulin-I-131 metabolism in human subjects. Demonstration of insulin-binding

SYNALBUMIN

INSULIN

ANTAGONIST

globulin in the circulation of insulin-treated subjects. J. Clin. Invest. 35:170, 1956. 16. Valiance-Owen, J., Dennes, E., and Campbell, P. N.: Insulin antagonism in plasma of diabetic patients and normal subjects. Lancet 2:336, 1958. 17. -, -, and -: The nature of the insulin-antagonist associated with plasmaalbumin. Lancet 2:696, 1958. 18. Debro, J., Tarver, H., and Korner, A.: The determination of serum albumin by a new method. J. Lab. Clin. Med. 50:728, 1957. 19. Cameron, J. S., and Keen, H.: Insulin activity of normal plasma and plasma acidethanol extracts. Lancet 1:74, 1964. 20. Keen, H.: Properties of human “albumin.” A metabolic study of albumin extracts from normal and diabetic plasma. Diabetes 12:406, 1963. 21. Lowy, C., Blanshard, G., and Phear, D.: Antagonism of insulin by albumin. Lancet 1:802, 1961. 22. Alp, H., and Recant, L.: Effect of the insulin-inhibitory albumin fraction from normal and diabetic subjects on adipose tissue. Metabolism 13:609, 1964. 23. -, and -: Studies of the insulininhibitory effect of human albumin fractions. J. Clin. Invest. 44:870, 1965. 24. Jervell, J.: The antagonistic effect of human plasma albumin on the insulin stimulated glucose uptake of the isolated rat diaphragm. Acta Physiol. &and. 65:33, 1965. 25. Ashton, W. L.: The glucose uptake induced in rat hemidiaphragms by insulin in the presence of human albumin. J. Endocrin. 33: 103, 1965. 26. Jervell, J.: Insulin antagonistic effect of human plasma albumin on protein synthesis in vitro and on glycogen synthesis in vivo in the rat diaphragm muscle. Acta Physiol. Stand. 66: 300, 1966. 27. Davidson, M. B., and Goodner, C. J.: Assay of insulin antagonism by serial incubation of paired rat hemidiaphragms. Diabetes 15:380, 1966. 28. Sherman, L.: The Valiance-Owen (“synalbumin”) insulin antagonist. Reproducibility and reliability of results in nondiabetic and diabetic humans. Diabetes 15: 149, 1966. 29. Ehrlich, R. M., and Martin, J. M.: Presence of synalbumin antagonist in sib-

683 lings of diabetic children. Diabetes 15:400, 1966. 30. Kammerer, L., Bretan, M., Nemesanszky, L., Jakali, L., and Virag, S.: A plasmasynalbumin antagonist. Lancet 2: 1366, 1966. 31. Arvill, A., Westberg, G., Jonsson, K. A., Hood, B., and Ahren, K.: Insulinlike activity and insulin-inhibitory effect of human albumin fractions studied with two biological test methods. Diabetologia 2: 253, 1966. 32. Cameron, J. S., Boyns, D. R., Jarett, R. J., and Keen, H.: The properties of trichloroacetic acid-ethanol extracts of human plasma. 1. Bioassay and immunoassay of plasma and extracts. Diabetologia 2:86, 1966. 33. Mahler, R. J., and Szabo, 0.: Effects of normal human albumin upon glucose uptake by the isolated rat diaphragm in the presence and absence of insulin. Metabolism 16:853, 1967. 34. Levin, M. E., and Recant, L.: Hormonal and biochemical studies in a diabetic family. Ann. Int. Med. 66:69, 1967. 35. Devrin, S., Kilo, C., Voyles, N., and Recant, L.: Effects of tolbutamide and insulin on insulin inhibitory activity associated with plasma albumin. Diabetes 17:746, 1968. 36. Sherman, L., and Rosenbloom, A.: Synalbumin insulin antagonism in idiopathic hypoglycemia of infancy. Diabetes 17:557, 1968. 37. Valiance-Owen, J.: The inheritance of essential diabetes mellitus from studies of the synalbumin insulin antagonist. Diabetologia 2:248, 1966. 38. Rosenbloom, A. L., and Sherman, L.: The natural history of idiopathic hypoglycemia of infancy and its relation to diabetes mellitus. New Eng. J. Med. 274:815, 1966. 39. Valiance-Owen, J., and Ashton, W. L.: Cardiac infarction and insulin antagonism. Lancet 1:1226, 1963. 40. Pederson, L. M., Tygstrup, I., and Pederson, J.: Congenital malformations in newborn infants of diabetic women. Lancet 1:1124, 1964. 41. Valiance-Owen, J., Braithwaite, F., Wilson, J. S. P., Edwards, J. R. G., and Murice, D. G.: Cleft lip and palate deformities and insulin antagonism. Lancet 2: 912, 1967. 42. Wilson, J. S. P., and Valiance-Owen,

684 J.: Congenital deformities and insulin antagonism. Lancet 2:940, 1966. 43. Connon, J. J.: Unexplained foetal deaths and synalbumin insulin antagonism. Diabetologia 5: 188, 1969. 44. Voyles, N., Penhos, J. C., and Recant, L.: Hepatic production of an albuminassociated insulin inhibitor. Proc. Sot. Exp. Biol. Med. 30:635, 1969. 45. Valiance-Owen, J., and Lilley, M. D.: An insulin antagonist associated with plasma albumin. Lancet 1:804, 1961. 46. Jervell, J., and Valiance-Owen, J.: Variations in synalbumin insulin antagonism during glucose-tolerance tests. Lancet 1: 1253, 1967. 47. Randle, P. J., Garland, P. B., Hales, C. N., and Newsholme. E. A.: The glucosefatty acid cycle. Its role in insulin sensitivity and the metabolic disturbances of diabetes mellitus. Lancet 1:785, 1963. 48. Buse. M., McMaster. J., and Buse, J.: Effect of human serum albumin on the glucose metabolism of isolated rat diaphragm and its response to insulin. Diabetes 15:388, 1966. 49. Davidson, M. B., and Goodner, C. J.: Unpublished observations. 50. Karl, I., Voyles, N., and Recant, L.: Effects of plasma albumin on glycolytic intermediates in rat diaphragm muscle. Diabetes 17:374. 1968. 51. Rogers, T. A.: Inhibition of glucose uptake by acidosis in vitro. Proc. Sot. Exp. Biol. Med. 97:646, 1958. 52. Davidson, M. B.. and Goodner, C. J.: Studies on the mechanism of insulin antagonism by albumin in rat diaphragm. Diabetes 15:373, 1966. 53. Antoniades, H. N., and Gershoff, S. N.: Inhibitory effects of “bound” insulin on insulin uptake by isolated tissues. Diabetes 15:655, 1966. 54. Davidson, J. K., and Haist, R. E.: Inhibitors of insulin in plasma fractions and mouse hemidiaphragm glycogen metabolism. Diabetes 15:532, 1966. 55. Ensinck, J. W., Poffenbarger, P. L., Hogan, R. A., and Williams, R. H.: Studies of insulin antagonism. 1. An artifactual antagonist to insulin and plasma nonsuppressible insulin-like activity occurring in preparation of “albumin.” Diabetes 16:289, 1967. 56. Garland, P. B.. Randle, P. J.. and Newsholme, E. A.: Citrate as an inter-

DAVIDSON AND POFFENBARGER mediary in the inhibition of phosphofructokinase in rat heart muscle by fatty acids, ketone bodies, pyruvate, diabetes and starvation. Nature 200:169, 1963. 57. Randle. P. J.. and Smith, G. H.: Regulation of glucose uptake by muscle. 1. Effect of insulin, anaerobiosis and cell poisons on the uptake of glucose and release of K+ by isolated rat diaphragm. Biochem. J. 70:490. 1958. 58. Kipnis. D. M.. and Noall, M. W.: Stimulation of amino acid transport by insulin in the isolated rat diaphragm. Biochem. Biophys. Acta 28:226, 1958. 59. Villar-Palasi. C.. and Larner, I.: Insulin treatment UDPGand increased glycogen transglucosylase activity in muscle. Arch. Biochem. Biophys. 94:436, 1961. 60. Davidson, M. B.. and Goodner, C. J.: Effect of antagonistic albumin on insulinstimulated intracellular metabolic pathways. Diabetes 15:835. 1966. 61. Wool, 1. G., and Krahl. M. E.: Incorporation of C-l4-amino acids into protein of isolated diaphragm: an effect of insulin independent of glucose entry. Amer. J. Physiol. 196:961, 1959. 62. Manchester. K. L., and Young, F. G.: The effect of insulin on incorporation of amino acids into protein of normal rat diaphragm in vitro. Biochem. I. 70:353. 1958. 63. Wool. I. G.: Insulin and incorporation of radioactivity into nucleic acid fraction of isolated diaphragm. Amer. J. Physiol. 199:719, 1960. 64. -: Effect of insulin on nucleic acid synthesis in isolated rat diaphragm. Biochem. Biophys. Acta 68:28, 1963. 65. -, and Munro, A. J.: An influence of insulin on the synthesis of a rapidly labeled RNA by isolated rat diaphragm. Proc. Nat. Acad. Sci. 50:918, 1963. 66. Davidson, M. B., and Goodner, C. J.: Effects of insulin antagonistic albumin on transport, glycogen synthetase. RNA synthesis and QO, in rat diaphragm. Clin. Res. 14: 176, 1966. 67. Biener, J.. and Valiance-Owen, J.: Effect of synalbumin on lipolysis. Lancet 2: 1390. 1969. J.: 68. Jervell, J., and Valiance-Owen, In-vivo effects of the synalbumin insulin antagonist and of B-chain albumin. Lancet 2:21. 1967. 69. Young, D. A. B.: A serum inhibitor

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of insulin action on muscle. I. Its detection and properties. Diabetologia 3 : 287, 1967. 69a. Ditschuneit, H.: Hormonal regulation of lipogenesis and hyperinsulinism in prediabetes and obesity. Acta Diabet. Latina 5: 364, 1968. 70. Davidson, M. B., and Goodner, C. J.: Failure of synalbumin to exhibit insulin antagonism in vivo. Diabetes 16:386, 1967. 71. Kammerer, L., and Valiance-Owen, J.: In vivo effects of the synalbumin insulin antagonist. Diabetologia 4:336, 1968. 72. Valiance-Owen, J., and Lukens, F. D. W.: Studies on insulin antagonism in plasma. Endocrinology 60:625, 1957. 73. Alp, H., Chaplin, H., and Recant, L.: Partial purification of an insulin inhibitor from human albumin fractions. J. Clin. Endocr. 26: 340, 1966. 74. Rubinstein, A. H.: Evidence for proinsulin in human urine and serum. Lancet 1: 1353, 1968. 75. Sonksen, P. H., Ellis, J. P., Lowy, C., Rutherford, A., and Narbarro, J. D.: A quantitative evaluation of the relative efficiency of gelatin and albumin in preventing insulin absorption to glass. Diabetologia 1: 208, 1965. 76. Poffenbarger, P. L., Ensinck, J. W., Hepp, D. K., and Williams, R. H.: The nature of serum insulin-like activity (ILA): Characterization of ILA in serum and serum fractions obtained by acid-ethanol extraction and adsorption chromatography. J. Clin. Invest. 47:301, 1968. 77. Lilley, M. D., and Valiance-Owen, J.: A factor associated with “Visking” tubing capable of antagonizing insulin. Nature 190: 1196, 1961. 78. Holcomb, G. N., and Dulin, W. E.: Studies on the nature of the synalbumin insulin antagonist. Proc. Sot. Exp. Biol. Med. 128:154, 1968. 79. Stadie, W. C., Haugaard, N., Hills, A. G., and Marsh, J. B.: Hormonal influences on the chemical combination of insulin with rat muscle (diaphragm). Amer. J. Med. Sci. 218:275, 1949. 80. Valiance-Owen, J., and McMaster, D. : Serum-albumin and insulin antagonism. Lancet 2: 1192, 1968. 81. Katzen, H. M., and Glitzer, M. S.: Insulin antagonists and disturbances in carbohydrate metabolism. In Dickens, F., Randle, P. J., and Whelan, W. J. (Eds.) : Carbohydrate Metabolism and Its Disorders,

685 Vol. 2. London, Academic, 1968, pp. 279283. 82. Glitzer, M. S.: Personal communication. 83. Ensinck, J.: Personal communication. 84. Ensinck, J. W., Mahler, R. J., and Valiance-Owen, J.: Antagonism of insulin action on muscle by the albumin-bound B-chain of insulin. Biochem. J. 94: 150, 1965. 85. Ensinck, J. W., Coombs, C. J., Williams, R. H., and Valiance-Owen, J.: Studies in vitro of the transport of the A and B chains of insulin in serum. J. Biol. Chem. 239: 3377, 1964. 86. Fenichel, R. L., Bechmann, W. H., and Alburn, H. E.: Pituitary and adrenal influence on reduced insuhn B-chain induced hyperglycemia. Diabetes 17:67, 1968. 87. Mahler, R. J., Szabo, O., and Penhos, J. C.: Antagonism to insulin action on the perfused hind limb of the rat by a reduced insulin B-chain-albumin complex. Diabetes 17:1, 1968. 88. Fenichel, R. L., Bechmann, W. H., and Alburn, H. E.: Inhibition of insulin activity in mitochondrial systems and in normal rats by reduced insulin B-chainalbumin complex. Biochemistry 5:461, 1966. 89. Meek, J. C., and Bolinger, R. E.: The biologic activity of insulin A & B chains as determined by the rat diaphragm and epididymal fat pad. Biochemistry 5: 3198, 1966. 90. Meek, J. C., Doffing, K. M., and Bolinger, R. E.: Radioimmunoassay of insulin A and B chains in normal and diabetic human plasma. Diabetes 17:61, 1968. 91. Varandani, P. T.: Plasma concentrations of A and B chains of insulin in nondiabetic, diabetic and high risk potential diabetic subjects. Diabetes 17:547, 1968. 92. Jervell, .I., and Valiance-Owen, J.: Inhibition of glutathione reductase in diabetics and non-diabetics. Diabetologia 3: 353, 1967. 93: Andres, R., and Zierler, K. L.: Spontaneous and insulin-induced resistance of peripheral tissues to insulin in diabetes. Clin. Res. 6:250, 1958. 94. Butterfield, W. J. H., and Whichelow, M. J.: Peripheral glucose metabolism in control subjects and diabetic patients during glucose, glucose-insulin, and insulin sensitivity tests. Diabetologia 1:43, 1965. 95. Berson, S. A., and Yalow, R. S.:

DAVIDSON

686 Some current controversies in diabetes research. The Banting Memorial Lecture, 1965. Diabetes 14:549, 1965. 96. Groen, J., Kamminga, C. E.. Willebrands, A. F., and Blackman, J. R.: Evidence for the presence of insulin in blood serum. A method for an approximate determination of the insulin content of blood. J. Clin. Invest. 31:97, 1952. 97. Wright, P. H.: Plasma-insulin estimation by the rat-diaphragm method. Lancet 2:621, 1957. 98. Unger, R. H., Siperstein. M. D., Madison, L. L., Eisentraut, A. M.. and Whissen, N.: Apparent growth hormone hyperresponsiveness in prediabetes. J. Lab. Clin. Med. 64:1013. 1964. 99. Merimee, T. J., Burgess, J. A., and Rabinowitz, D.: Arginine infusion in maturity-onset diabetes mellitus. Defective output of insulin and growth hormone. Lancet 1:1300, 1966. 100. Tchobroutsky. G., Rosselin, G., Assan, R., and Derot, M.: Arginine infusion in diabetes mellitus. Lancet 2:498, 1966. 101. Parker, M. L., Hammond, J. M., and Daughady, W. H.: The arginine provocative test: an aid in the diagnosis of hyposomatotropism. J. Clin. Endocr. 27: 1129. 1967. 102. Baker, L., Kaye, R., and Root, A. W.: The early partial remission of juveniIe diabetes mellitus. The roles of insulin and growth hormone. J. Pediat. 71: 825, 1967. 103. Drash. A., Field, J. B., Garces, L. Y., Kenny. F. M., Mintz, D., and Vazquez, A. M. V.: Endogenous insulin and growth hormone response in children with newly diagnosed diabetes mellitus. Pediat. Res. 2: 94, 1968. 104. Boden, G., Soeldner, J. S., Gleason, R. E., and Marble, A.: Elevated serum human growth hormone and decreased serum insulin in prediabetic males after intravenous tolbutamide and glucose. J. Clin. Invest. 47:729, 1968. 105. Burday, S. Z., Fine, P. H., and

AND

POFFENBARGER

Schlach, D. S.: Growth hormone secretion in response to arginine infusion in normal and diabetic subjects: relationship to blood glucose levels. J. Lab. Clin. Med. 71:897, 1968. 106. Baker, L., Root. A., Hague, N., and Kaye, R.: Metabolic homeostasis in juvenile diabetes mellitus. 1. Role of growth hormone. Metabolism 18: 110, 1969. 107. Yalow, R. S.. Click. S. M.. Roth. J., and Berson. S. A.: Plasma insulin and growth hormone levels in obesity and diabetes. Ann. N.Y. Acad. Sci. 131:357. 1965. 108. Sabeh, G., Corredor, D. G.. Mendelsohn. L. V., Morgan, C. R.. Sieracki. J. C.. Sunder, J. H.. Wingert, J. P., and Danowski. T. S.: Growth hormone and insulin levels in newly discovered glucose intolerance. Metabolism 18:741. 1969. 109. Bagdade, J. D.. Porte, D., and Bierman. E. L.: The interaction of diabetes and obesity on the regulation of fat mobilization in man. Diabetes 18:759, 1969. 110. Kipnis, D. M.: Growth hormone and insulin antagonism. In Leibel, B. S.. and Wrenshall. G. A. (Eds.) : On the Nature and Treatment of Diabetes. Amsterdam, Excerpta Medica Foundation. 1965. pp. 258287. 111. Yalow. R. S.. Goldsmith. S. J., and Berson, S. A.: Influence of physiologic fluctuations in plasma growth hormone on glucose tolerance. Diabetes 181402, 1969. 112. Cotes, P. M., Reid, E.. and Young. F. G.: Diabetogenic action of pure anterior pituitary growth hormone. Nature (London) 1641204. 1949. 113. Schalch, D. S.. and Kipnis. D. M.: Abnormalities in carbohydrate tolerance associated with elevated plasma monesterified fatty acids. J. Clin. Invest. 44:2010. 1965. 114. Wright. P. H.: The production of experimental diabetes by means of insulin antibodies. Amer. I. Med. 31:392. 1961. 115. Nestel. P. J.. Carroll, K. F., and Silverstein, A. S.: Influence of free-fattyacid metabolism on glucose tolerance. Lancet 2:115,

1964.