ENZYME CHANGES IN DIABETES MELLITUS

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C l i n i c a l L a b o r a t o r y S c i e n c e , Vol. 3 , 1 9 7 3 , Institute for Clinical Science

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No.

5

Enzym e Changes in Diabetes Mellitus DONALD T. FORMAN, Ph.D.* AND KAREN WIRINGA, B.S. Department of Pathology, Evanston Hospital, Evanston, IL 60201and Departments of Pathology and Biochemistrfe Northwestern University Medical School, Chicago, IL 60611

ABSTRACT Derangement of metabolic processes in disease is often associated with alter­ ation in serum enzymatic activities, and the assay of serum enzymes has be­ come an important diagnostic procedure. However, there has been some ques­ tion concerning changes in serum enzyme patterns in diabetes mellitus. A large number of enzyme changes can be related to diabetes mellitus and these abnormal findings are complicated by concomitant coronary sclerosis, renal, retinal, neurologic disorders and other idiopathies. The complexity of the problem is illustrated by considering early versus late onset diabetes, acute versus chronic diabetes and the possible genetic basis of diabetes. Early-or juvenile and late-onset diabetes present very different patterns which suggest different enzyme pathology. The antagonistic actions of insulin and adrenocorticoid hormones on the biosynthesis of glycolytic and gluconeogenic en­ zymes are described. The role of enzymes in metabolic pathways (TCA cycle, glycogen deposition, pentose pathway, fatty acid metabolism, energy transfer, gluconic acid formation) and the pathology of diabetes are discussed.

disorders. Many subjects with premature arterial disease show signs of preclinical or subclinical diabetes when tested for fasting blood sugar concentration or glucose tol­ erance and it seems necessary to consider how many of these protean manifestations are common to “basic” diabetes. Are there underlying enzyme changes responsible for these complications, which are found in all * Address reprint requests to Donald T. For­ diabetics but are generally not well ex­ man, Ph.D., Division of Biochemistry, Department pressed because of the possibility of fairly of Pathology, Evanston Hospital, Evanston, IL good control? Or is it necessary to break 60201.

In tro d u ctio n The large number of enzyme changes which can be related to diabetes mellitus point to the basic problem inherent in study of the disease. Herman and Gorlin1 6 describe diabetes as a disease with protean manifestations, which may include coro­ nary sclerosis, coronary artery disease, idi­ opathies, and renal, retinal and neurologic

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diabetes down into a number of separate entities? The complexity of the problem can be illustrated by a consideration of early onset versus late-onset diabetes, the generally recognized clinical stages of diabetes, acute versus chronic diabetes and the possible genetic basis of diabetes. Early-onset or juvenile-onset and late-onset diabetes preent very different patterns, which suggest afferent enzyme changes. Early-onset di­ abetes appears during the growth period, has an associated weight loss, presents the probem of severe ketoacidosis when insulin is withdrawn, has normal insulin sensitivity and shows decreased pancreatic and plasma insulin content. 38 Late-onset dia­ betes appears during adult life, is associ­ ated with obesity, shows absence of severe ketoacidosis on withdrawal of insulin, has low insulin sensitivity and presents little evidence for decreased plasma or pan­ creatic insulin content. Many clinicians divide late-onset diabet­ ics into four successive and progressively more severe stages: ( 1 ) prediabetics who are essentially normal but have a high probability of later manifesting the disease because they are either the identical twin of a diabetic or the offspring of two dia­ betic parents; ( 2 ) subclinical diabetics who show abnormal glucose tolerance tests only in conjunction with cortisone usage; (3 ) latent diabetics with normal fasting blood sugar but abnormal glucose toler­ ance tests; (4 ) latent diabetics with ele­ vated fasting blood sugar as well as ab­ normal glucose tolerance tests. A diabetic can move up or down the classification scale, and it is interesting to speculate on the probable enzyme differences between these stages. At just what point do the enzyme changes correlated with the nu­ merous clinical manifestations appear? Do they appear only with increasing severity of the diabetes or are they related to the onset as Herman and Gorlin’s1 6 observa­ tions suggest?

375

Renold and Cahill3 8 discuss the differ­ ence between acute and chronic diabetes, and this is certainly an important classifi­ cation to keep in mind when considering enzyme changes. This classification may not be enzymatically valid and needs fur­ ther study. The acute diabetic syndrome is related to the hyperglycemia found in uncontrolled, overt diabetes. There is ex­ cess glucose in the urine (glycosuria) and excess urination (polyuria) which leads to excessive hunger and thirst. The glucose loss is related to excessive catabolic mobil­ ization of proteins and fats, and this results in proteinuria and excess ketone bodies and accompanying ketonuria. Presence of excess ketone bodies and overloading of the Krebs cycle leads to production of ex­ cess H+ from the breakdown of oxybutyrate. This can lead to ketoacidosis cora and, in many cases, death. The chronic diabetic syndrome includes any, and possibly all, of the numerous clinical manifestations (such as the various angiopathic or retinal problems) as well as the acute manifesta­ tions. Another problem associated with en­ zyme studies of diabetes is the nature of its genetic basis. There is evidence that it is frequently inherited as a simple Mendelian autosomal recessive, 3 8 but this is cer­ tainly not the only basis of the disease. Renold and Cahill3 8 suggest the disease may represent a genetically conditioned susceptibility with variable penetrance de­ pendent on non-genetic factors. Although these authors state that there is no really good evidence of etiologically distinct forms, this possibility should not be ruled out in view of the numerous clinical man­ ifestations and the wide-ranging enzyme activity changes involved. The possible genetic basis of diabetes will be further considered with the changes in gluconeo­ genic and glycolytic enzymes and the sug­ gestion that these two enzyme groups rep­ resent two distinct genomes. 45

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F O R M A N AND W IR IN G A

The diversity of the classifications con­ sidered above suggest that further enzyme studies are necessary to determine the na­ ture of the “basic” disease or the possibility of a number of genetically and/or sympto­ matically distinct forms which account for the wide variety of changes. It can be seen that any clear and inclusive definition of the disease is very difficult. However, hy­ perglycemia is still a good starting point in any attempt to understand this disease; it explains many of the symptoms. 3 8 Also, it is valuable to consider the activity of the circulating insulin, its effects, its antago­ nists and its inhibitors. E xperim en tal A pproaches Studies on enzyme changes in diabetes have been carried out primarily with rats, although there has been some work with other vertebrates and some invertebrates as well. In most cases these studies involved drug-induced diabetes, although diabetic strains of mice and Chinese hamsters are sometimes used. 38 The most widely used drug is alloxan, and it induces a condition similar to di­ abetes. However, in addition to destroying the /3-cells of the pancreas it also causes kidney and liver damage. 28 Other common ways used to induce diabetes include use of a mixture of dehydroascorbic acid and alloxan28 or streptozotocin, 2 0 which are both reported to have more gentle effects than alloxan alone. Anti-insulin-serums, 28 pancreatectomies, prolonged administra­ tion of anterior pituitary extracts or pitui­ tary growth hormones and prolonged ad­ ministration of glucose38 are also used to produce diabetic-like conditions in experi­ mental animals. The last two methods ap­ parently exhaust the secretory capacities of the /?-cells. A common feature of all these induced forms of diabetes is the decrease in circulatory insulin. It is important to remember that these conditions are only diabetic mimics and not thoroughly under­ stood. They cannot be expected to illus­

trate all the complicating clinical manifes­ tations, and thus severely limit the range of the studies and conclusions and the cor­ relations possible with human diabetes. Inducers like alloxan present special prob­ lems because of the liver and kidney dam­ age, and additional unrelated enzyme changes, particularly in serum determina­ tions, can be expected. Studies on enzyme changes in humans are rather scanty. In general, the authors are very careful to try and rule out patients with complicating factors such as liver or pancreatic disease, but the problem of “basic” diabetes again presents itself and one wonders if all the various clinical man­ ifestations should be separated from the simple acute form. The degree of control of the disease also presents a problem; it seems important to determine how effec­ tive the treatment is at the time of mea­ surement. Of course, there is the problem of lack of uncontrolled subjects for studies. T h e R ole o f In su lin Insulin activity occupies a central posi­ tion in any study on diabetes. It is a reg­ ulator which promotes glucose metabolism, protein anabolism, fat disposition ( in­ creased lipogenesis) and, in general, it will reverse most of the basic changes of di­ abetes. It acts in opposition to many of the adrenal and pituitary hormones. How much of its effect is related to preferential glucose uptake in the various tissues, how much is due to a direct effect on enzyme synthesis4 5 and how much is just a “pull­ ing” effect through the interrelated met­ abolic pathways is not well understood. Bessman2 has suggested that one of the primary actions of insulin may be on the Mg++ dependent coupling of glucokinase to the electron transport chain at sites of ATP production. Here it would provide a means of dephosphorylating the ATP for the con­ tinuation of electron transport. Bessman2 has suggested that in the brain, insulin has no effect because all the hexokinase

EN ZYM E

CH AN GES IN D IA B E T E S M E L L IT U S

377

F i g u r e 1. The effects of insulin and adrenocorticoid hormones on the biosynthesis of glycolytic and gluconeogenic enzymes.

is already attached to mitochondria. This would account for the insulin-insensitivity of the brain. Ilyin 1 7 has suggested that in­ sulin reverses the inhibition of hexokinases brought about by their binding to mito­ chondria. There are numerous explanations for the changes in insulin activity observed in the various forms of diabetes; Renold and Cahill38 provide a good summary of them. Abnormal or inadequate numbers of cells are frequently found in early-onset dia­ betes; this accounts for the lack of insulin in this form. There may be an inability to store insulin. There is also speculation that there is a stored form and a circulatory form of insulin. The circulatory form has a half-life of about forty minutes and is rapidly inactivated. 4 6 There may be an in­ ability to release the stored insulin owing to membrane-passage problems or inade­ quate reaction in some peripheral tissue. There may be excessive binding to struc­ tural protein, insulin neutralization or de­ struction or excessive requirements for in­ sulin brought about by high circulating blood glucose levels, resulting in eventual “exhaustion” of the ^3-cells. Any one of these or any combination of these proposed situations may be the actual mechanism.

It is probable that early-onset and lateonset diabetes involve different patterns; chronic manifestations may also be tied in. Weber et al4 5 have considered the antag­ onistic actions of insulin and the adreno­ corticoid hormone on the biosynthesis of glycolytic and gluconeogenic enzymes. The interactions are summarized in figure 1. In general, insulin increases the synthesis of glycolytic enzymes and decreases the syn­ thesis of gluconeogenic enzymes. Adreno­ corticoid hormones increase the synthesis of gluconeogenic enzymes. It has been sug­ gested that the gluconeogenic and glyco­ lytic enzymes may be one genome which is repressed or derepressed as a whole. Ad­ ditional studies have been carried out using subjects who have undergone pancreatec­ tomy or who suffer from hemochromatosis which involves the deposition of iron pig­ ments in the pancreas with resulting sec­ ondary fibrosis and loss of function. These studies have been used primarily to deter­ mine the nature of insulin action. At present it is necessary to view most of the animal studies cautiously and to avoid excessive generalizations to human diabetes and to consider carefully the com­ plications involved in human studies. Again, the safest conclusions appear to be

F O R M A N AND W IR IN G A

378

GLYCOGEN-: 10, l«,4SN

oc-amylase POLYSACCHARIDES

phosphcrylasef |

AMYLOPECTIN MALTOSE LIMIT DEXTRIN GLUCURONIC ACID

I

glycogen synthase | kinase

amylo 1,6/ glucosidasei/ X -D-glycogen transferase'!’ (in WBC)

30 4

JÎ-glucuronidaseT

l7, PENTOSE GLUCOSE-1-© ^--------" 37 glucose 6-P dehydrogenase'*' PATHWAY y/Áphosphoglucor ^■ iv ^iv w y m iifM C A 6phosphogluconate a trehaloset “ /' » ¡ ÎS ÿ ---------------», r . . lrn t.p dehydrogenase* TREHALOSEE ---------------G LU C O S E ¿ ^ Í G L Ü C 0 S E - 6^ ) — phosphatase GLUCOSE

♦ dehydrogenase ÏE * 36

aldose reduchbse'J'

phosphohexose- isomerase

GLUCONIC ACID

SORBITOL FRUCTOSE- 6 - ® y' 4 phosphofructokinase fructose-1, 6-diphosphatasS^ ^/sorbitol dehydrogenasSî D-FRUCTOSE FRUCT0SÉ-1, 6-di ® aldolase GLYCEROL-

▲

PEP i 48-48! | pyruvate kinase’

45

■fPEP carbaxykinase

—- 7 * ------------------ -PYMJVATE / pyruvate,carboxylasef 'I'malic

enzyme

OXALOACETATE------ ^ malic I 49 dehydrogenase X c'/raf8 T cleavage MALATE

ACETYL CoA

serine dehydratase^ LACTATE LOrit? acet^P?_____ »-MALONYL CoA CoA carboxylase

+ 'i^5carnitine acetyl a ca mi tine^^transferase ^

\ x - KETOGLUTARATE F ig u r e

;

+

FATTY ACIDS

ACETYL CARNITINE + CoA

2. Enzyme activity changes in diabetes.

those involving the basic ramifications of hyperglycemia. E nzym e C hanges Most of the enzyme changes to be dis­ cussed are summarized in figure 2. Al­ though all the products and substrates are interconnected, several of the pathways or enzymes are found only in certain tissues. For instance, the glycogen transferase in­ terconversion occurs primarily in white

blood cells and the sorbitol dehydrogenase step is mentioned with reference to the eye. E n z y m e s A f f e c t in g I n s u l in D

ir e c t l y

Insulinase is an insulin-specific, hydro­ lytic, enzyme found only in the liver8 1 and is responsible for the short circulating life of insulin. 4 6 Its presence has been reported in rats, mice, rabbits, guinea pigs, cows, chickens, ducks, monkeys and men al­

EN ZYM E

CH AN GES IN D IA B E T E S

though the concentration among species varies. Its activity increases after a high carbohydrate meal and also after a high protein meal, although more slowly. After fasting its activity is diminished. Mirsky31 also postulates that there is an insulinaseinhibitor produced in the liver. He suggests that diabetes may be the result of a met­ abolic derangement, which interferes with the synthesis of this insulinase inhibitor, which results in a higher level of insulinase and a lower level of insulin. Glutathione-insulin transhydrogenase is found in the liver where it catalyzes the reduction of interchain disulfide bonds of insulin which results in its inactivation. 4 6 One can speculate that it may also be in­ volved in diabetic induced changes in inulin concentrations. Gl u c o n e o g e n ic E

nzym es

Changes in these enzymes have been dis­ cussed in the section on the role of insulin in diabetes. Glucose-6 -phosphatase, fructose-1,6 -diphosphatase, PEP carboxykinase and pyruvate carboxylase activities have all been shown to increase in the liver. 45 Prasannan and Subrahmanyan3 7 found a decrease in glucose-6 -phosphatase in the brains of alloxan-diabetic rats, which they felt accounted for the high glycogen levels found there. G l y c o l y t ic E n z y m e s

Glucokinase, phosphofructokinase and pyruvate kinase activities all decrease in diabetes. 4 5 The effects of insulin and adrenocorticoid hormones on the synthesis of these enzymes has already been discussed. Pyruvate kinase and glucokinase have isoenzymatic natures which warrant further discussion. Two pyruvate isoenzymes have been reported. 4 2 Type L is found exclu­ sively in the liver; its activity has been shown to decrease in diabetes, and this action is restored by insulin. It has been electrophoretically divided into three sub­ peaks of unknown significance. Type M,

M E L L IT U S

379

the muscle type, is found primarily in mus­ cle although it is also present in the liver. It is, however, unaffected by insulin or diabetes. Tanaka et al4 2 speculate that the L isoenzymes are related to the metabolic control of gluconeogenesis and the M iso­ enzyme is a less differentiated form. The hexokinase isoenzyme system is the one most widely studied in relation to di­ abetes. The majority of the work has been done with alloxan-diabetic rats, but the im­ plications for the mechanism of control of metabolism are still very significant and worth considering. Katzen2 0 and others have reported a system of five isoenzymes: Types I, II, III and IV in order of increas­ ing electrophoretic mobility toward the anode and the S.T. or sperm type which is found nearest to the cathode. Types I, II and III are hexokinases. They are not sub­ strate specific and are found in all tissues although in different proportions. All three have a molecular weight of 96,000 and seem to be localized in the non-parenchymal cells of the liver. 4 0 Type IV is found only in the liver, is known as glucokinase and is glucose-specific. It has a high Km for glucose in comparison to the other three hexokinases mentioned. Brown et al5 have suggested that this property would allow glucokinase to handle large glucose loads. Its activity drops sharply in diabetes. Sharma et al39 suggest that glucose and in­ sulin are crucial in its induction and high levels and normal activity must be main­ tained. 1 1 Borrebach and Spydevold4 sug­ gest that they accomplish this by acting on the net balance between synthesis and deg­ radation. The molecular weight of gluco­ kinase is 48,000,20 approximately half the molecular weight of the hexokinase isoen­ zyme. 4 0 Glucokinase is found in the paren­ chymal cells of the liver. Katzen and Shimke2 1 have reported on the tissue distribution of these isoenzymes. Liver contains all four types with a large amount of Type III, brain and kidney con­ tain mostly Type I, fat pad and muscle

380

F O R M A N AND W IR IN G A

contain primarily Type II and heart con­ tains approximately equal amounts of Types I and II. It is usually difficult to get data on Type III because it is substrate inhibited. Katzen et al22 have suggested that as the full complement of Types I, II and III in­ creases (particularly Type I ) , the insulin sensitivity of the tissue decreases. This ac­ counts nicely for the known insulin insensi­ tivity of the brain and kidneys. Katzen20 has reported a further breakdown of Type II when it is electrophoresed without mercaptoethanol. A Ha fast band appears, which decreases preferentially in diabetes, and there is also a relatively stable lib slow band. Katzen believes that mercaptoethanol catalyzes the conversion of lib to II, and this accounts for the inability of most investigators to find a change in band II in diabetic specimens. Katzen feels that Ila is a monomer and lib a dimer and inter­ conversion occurring through thiol-disulfide interchanges. In addition he has suggested that these forms may be involved in an insulin-stimulated glucose carrier system across membranes. McLean et al, 29 how­ ever, have found a decrease in band II in alloxan-diabetic rats even in the presence of mercaptoethanol. It is possible that there may be significant differences be­ tween the streptozotocin-diabetic rats used by Katzen20 and the alloxan-diabetic rats used by McLean et al. 2 9 Pilkes and Han­ sen35 have found that Type IV hexokinase from alloxan-diabetic rat liver breaks up into two bands, IVf (fast), which preferen­ tially disappears in diabetics, and IVs (slow), which is unchanged when it un­ dergoes electrophoresis in the absence of EDTA. The molecular weight of each band is 48,000, which suggests that they are not subunits of band IV. Although Kaplan and Beutler’s work1 9 does not relate directly to diabetes, it is interesting to note that they have found two bands of Type I in human red blood cells: Type If (fast) found only in fetal cells and Type la found only in

adult cells. This finding suggests that these multiple sub-bands may be quite wide­ spread in the hexokinase isoenzyme system. Although most of the work on this sys­ tem has been done on rats and Lauris and Cahill2 7 found no trace of glucokinase in the human as late as 1966, Grossbard et al1 5 have stressed the species similarities and the likelihood of finding similar systems in all the vertebrates. Brown et al5 have re­ cently reported a five isoenzyme hexokin­ ase system in men and dogs which is com­ parable to the rat isoenzyme system. These are really only the beginnings in the study of hexokinase, but it is already clear that this isoenzyme system is impor­ tant as a control mechanism in glucose metabolism and may have a very basic role in the changes occurring in the diabetic’s metabolic processes. The possible linkages with membrane transport systems2 0 and the reversible binding to microsomal fractions, including increased binding of the hexokinases to the mitochondria with increasing glucose or insulin concentrations, 2 ’4 could prove to be very important processes. T r i c a r b o x y l ic A c id C y c l e -B e l a t e d E

nzym es

The TCA cycle is overloaded and sup­ pressed in diabetes. McGilvery 28 reports decreases in the activities of citrate cleav­ age enzyme and malic dehydrogenase. Malic enzyme also decreases. 2 6 These changes are in line with the overall pattern of decrease in glucose-breakdown path­ ways and increase in glucose-forming path­ ways. G l y c o g e n -B e l a t e d E n z y m e s

Pathologic glycogen deposits have been implicated in many of the clinical manifes­ tations of chronic diabetes. 3 8 Spiro, 4 1 how­ ever, has reported a general decrease in glycogen levels in an alloxan-diabetic rat. It is generally recognized that the insulin deficiency of diabetes is accompanied by glycogen breakdown and hyperglycemia. 4 6

EN ZYM E

CH AN G ES IN D IA B E T E S M E L L IT U S

These findings are difficult to reconcile. Prasannan and Subrahmanyans work37 with cerebral cortex slices from normal and diabetic rats and guinea pigs suggests a possible answer. They have found there is a seven fold increase in brain glycogen in diabetic animals which is explained by a 640 percent increase in glycogen synthetase activity and a 133 percent increase in phosphoglucomutase activity. At the same time, however, phosphorylase activity is re­ ported to decrease and McGilvery2 8 has reported a decrease in amylo-l,6 -glucosidase activity in the diabetic. Although it is premature to form conclusions on the basis of work done on the brain alone, it is possi­ ble that these divergent findings can be accounted for by increases in both glyco­ gen synthesis and glycogen breakdown with synthesis exceeding breakdown, as it appears to happen in the brain. Serum amylase has been reported to in­ crease in diabetic coma complicated by acute pancreatitis, and Tully and Lowenthal4 3 suggest that the concentration is nor­ mal in diabetes when the pancreas is not involved. Finn and Cope, 1 0 however, report that its activity is decreased in simple di­ abetic coma. A change in glycogen transferase from a glucose-6 -phosphate dependent “D” form to a glucose-6 -phosphate independent “I” form in white blood cells has been reported by Esmann et al. 7 The conversion is medi­ ated by insulin, which also increases the rate of the conversion. The combined I and D activity is low in uncontrolled diabetes. These two enzymatically interconvertible forms have also been found in muscle and liver, but their significance is not discussed. P en to se P a th w ay E

nzym es

Ilyin 17 has reported a decrease in both glucose-6 -phosphate dehydrogenase and 6 phosphogluconate dehydrogenase activity in alloxan-diabetic rats. This would result in the increased production of glucose-6 phosphate and glucose.

F

atty

A c id M

e t a b o l is m

381

-R e l a t e d

E nzym es

In diabetes, there is a mobilization of free fatty acids to the liver and a decrease in lipogenesis. 3 8 McGilvery28 has reported a decrease in acetyl CoA carboxylase activ­ ity which would result in a decrease in fatty acid synthesis; this provides an ex­ ample of the general mechanism. P r o t e in

and

R elated E

A m i n o A c id M e t a b o l i s m -

nzym es

The glucose loss in diabetes results in a need for increased mobilization and catab­ olism of proteins. 3 8 Mullan32 has reported an increase in serum leucine aminopeptidase in men with poorly controlled dia­ betes and a spillage of the enzyme into the urine when gangrene is present. Heavy proteinuria was found along with a marked increase in LAP excretion. McGilvery2 8 has reported an increase in serine dehydratase. Goldberg et al1 4 have reported an increase in serum gamma-glutamyl transpeptidase activity in 30 out of 85 diabetic patients, but found liver dysfunction in all cases of greatly increased activity. Kohot and Kuska24 have reported no distinct patterns of change in serum gamma glutamyl trans­ peptidase although all the diabetic fractions differed somewhat from normal. E

n erg y -T r a n sfer -R el a t ed

E nzym es

Adelman and Weinhouse1 have reported an increase in adenylate kinase activity in alloxan-diabetic rats, but the significance of this change and the resultant increase in ATP is not well understood. Kupiecki25 has reported a decrease in phosphodiesterase activity in spontaneously-diabetic mice which should lead to a buildup of cyclic 3',5'-AMP which is known to stimulate in­ sulin release from the /3-cells of the pan­ creas. This may account for the high level of plasma insulin found in these mice. This does not result in increased glucose utiliza­ tion and could be due to the blockage of

F O R M A N AN D W IR IN G A

382

the pentose phosphate shunt by the accu­ mulation of cyclic AMP. G l u c u r o n i c A c id P a t h w a y E n z y m e s

Winegard and DePratti4 7 have found high fasting levels of serum L-xylulose in fasting diabetic patients and suggest that this may indicate greater usage of the glucuronic acid pathway. Miller et al3 0 have speculated that the increased usage of the pathway in diabetics may lead to an excessive deposition of mucopolysac­ charides and glycoproteins, which are im­ plicated in many vascular diseases. They have generally found increased serum /3glucuronidase in diabetics and a still higher average activity in diabetics with atherosclerosis. Miller et al suggest that the increase of this enzyme may be a way of preventing excessive buildup of polysac­ charides in the basement membranes of the capillaries. Therefore, this increase may serve a protective function. O ther E

nzym e

C hanges

In yiew of the high incidence of cata­ racts in diabetic patients, it is worthwhile to consider enzyme activity changes within the eye. Gabbay and O’Sullivan1 3 suggest that accumulations of sorbitol and fructose in lenses lead to diabetic cataracts. How­ ever, they found a 30 percent decrease in aldose reductase activity in the eyes of di­ abetic animals with cataracts. They suggest several explanations for this paradox: since Wallerian degeneration of the Schwann cells was used for enzyme localization, there may have been loss of aldose reduc­ tase activity owing to macrophage activity. This decrease in activity may occur only in certain Schwann cells while others con­ tinue to produce excessive amounts of sor­ bitol; or the reduction in activity may lead to localized accumulations of polyols, glu­ cose and other sugars and the formation of sugar cataracts owing to hypertonicity of the lens followed by bursting. Pottinger36 has also found increased activity of glucose

dehydrogenase and high levels of gluconic acid. Although Gabbay and O’Sullivan1 2 found no change in sorbitol dehydrogenase activity, the accumulations of fructose found in the eye of a diabetic animal with cataracts suggest that there may be an in­ crease in this enzyme. Clearly, more work is needed on this subject. Nilson et al3 4 have reported some interesting changes in MAO (monoamine oxidase) in serum in diabetic patients. This enzyme catalyzes the oxidative deamination of several mono­ amines found in nerve endings to alde­ hydes and ammonia. Its level has been reported to be already increased at the first clinical appearance of the disease and its activity appears to be independent of complications, duration of the disease, therapy used, age of the patient, sex of the patient, type of meals or occurrence of pregnancy. Only 14 out of 340 diabetic pa­ tients showed activities within the normal range. Its characteristics make it a likely candidate for future use in diagnosis. D ia g n o stic R ole o f E n zym e C hanges The diagnostic role of enzyme changes in diabetes is very limited at the present time. Most of the studies have dealt with changes in tissue homogenates rather than changes in serum levels. A major portion of the work to date has been done on the hexokinase isoenzymes, and these are found in the microsomal fragments. Some serum enzyme studies have been carried out, but the evidence is mostly inconclu­ sive. Serum alkaline phosphatase has been found to increase in human diabetes and shows no correlation with sex, age, body weight, duration of diabetes, type of treat­ ment, previous or present control, amount of insulin given, diet or blood cholesterol. 6 In another study of 161 diabetic patients no changes in serum enzymes were found. 31 Both papers are somewhat suspect because of the lack of screening for anything except recent coma and liver disease. Nakamura et al3 1 also report no changes in lactic de­

E N Z Y M E CH AN G ES IN D IA B E T E S M E L L IT U S

hydrogenase levels. Many changes have been reported in patients with ketoacidosis. Velez-Garcia44 and Coodley7 have reported increases in serum CPK during and after treatment for ketoacidosis. Normal diabetic patients showed little or no change in CPK levels and patients with ketoacidosis before treatment were normal. The levels in­ creased as treatment progressed and showed no correlation with insulin dose or degree of ketoacidosis. These authors sug­ gest that this could be the result of a direct effect of insulin on the membrane or due to the osmotic changes accompanying elec­ trolytic and fluid shifts, resulting in mem­ brane leakage. 7 The latter possibility seems more probable in view of the severe pH and osmotic changes occurring in keto­ acidosis. Janowitz and Dreiling18 have re­ ported lower levels of serum a-amylase in diabetes and higher levels in pancreatic disease. Tully and Lowenthal4 3 reported elevated serum amylase in diabetic coma with associated pancreatitis, and suggest that it is not clearly distinguished from uncomplicated diabetic coma. Finn and Cope1 0 found a general decrease in serum amylase in diabetic coma unless there was pancreatic involvement. This was generally accompanied by an increase in circulating amylase. Serum GOT and GPT are altern­ ately reported to be unchanged2 3 and slightly increased3 3 in newly diagnosed un­ controlled diabetes. Mullans studies32 on serum LAP have already been mentioned. There seem to be a variety of explanations for its increase. Its most valuable use could possibly be the indication of poor control in diabetic men, but the evidence seems very inconclusive. At the present time, serum MAO seems to offer the best possibility for diagnosis in view of its apparent increase before the onset of clinical diabetes and the lack of complicating factors. 3 4 In general, glucose tolerance tests with or without cortisone, fasting blood sugar levels and urinalysis still seem to be the

383

most useful diagnostic tools for diabetes. Most of the enzyme change studies to date have been more valuable in elucidating the nature and range of the disease than the underlying metabolic changes. F u tu re S tu d ies It is interesting to speculate on what di­ rection future research in this field will take. Most of the studies raise many more questions than they answer. A clearer def­ inition of just what constitutes diabetes is necessary. The possible connection with the coronary angiopathies are certainly sig­ nificant. 1 6 More general clinical studies will help to clarify how closely interrelated these conditions are and which come first chronologically. Winegard and De Pratti47 have found that L-xylulose is increased in the serum of the diabetic patient, and this would seem to point toward increased use of the glu­ curonic acid pathway, which would yield increased glycoprotein deposits. Excess glycoproteins are closely connected with many of the diabetic angiopathies and ret­ inal problems. The presence or absence of coronary vascular complications may be closely related to the level of the ^-glucu­ ronidase enzyme. 3 0 It may also be worth­ while to determine the frequency of changes in the pathways of the eye because of the connection with cataract formation. The evidence here is still rather contra­ dictory. In view of the probable appearance of serum MAO before clinical onset, it will undoubtedly receive much more attention and investigators are likely to search for other enzymes that show changes before clinical onset of the disease. More studies of the hexokinase isoen­ zymes and other isoenzyme systems may provide information on the control mecha­ nism involved in metabolism in normal and diabetic states. This work may give further insight into the genetic control about which Weber et al4 5 have speculated.

F O R M A N AND W IR IN G A

384

Although the differences between earlyonset and late-onset diabetes certainly seem to suggest that there would be differ­ ences in accompanying enzyme changes, there seems to be little work in this area. This is probably due to the fact that these conditions cannot be studied in experimen­ tal animals. A better understanding of the differences in these two forms of diabetes may help clarify the genetic basis of the disease. Much remains to be done. Much of the work done on rats must be verified in human studies before the conclusions can be considered valid. Before this can be successful, diabetes must be more clearly defined. Interplay between human and an­ imal studies will probably provide the answers. R eferences 1.

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