Diabetic regulation and its relationship to microangiopathy

PROGRESS

IN ENDOCRINOLOGY

Diabetic

This

review

addresses

effectiveness blood the

of

glucose vascular

in

rigid

Regulation and Its Relationship To Microangiopathy

the question regulation

delaying

or

complications

mellitus.

Morphometric,

chemical,

and

clinical

AND METABOLISM

Philip

Raskin

of the

sides

of

the

preventing of

diabetes

pathologic, evidence

on

bio-

of this

concluded adequate glucose betics,

question that

are

because

methods homeortasis

the question

presented. of

present

of completely to remains

normal

It is in-

returning in

dia-

moot.

both

D

IABETES MELLITUS is an interesting and complex disease that affects over 10 million Americans. Its manifestations are protean and affect all organ systems of the body. The metabolic disturbances in diabetes, which prior to the advent of insulin therapy more than 50 yr ago were responsible for almost 759,, of the mortality of the disease, now account for only a small percentage of deaths. These disturbances include hyperglycemia and ketonemia and, although this point is somewhat controversial, seem to be related to a bihormonal abnormality of the islets of Langerhans, that of insulin deficiency coupled with glucagon excess.’ Today almost all of the morbidity and mortality from diabetes mellitus results from a peculiar type of small blood vessel disease, commonly referred to as diabetic microangiopathy. The issue of whether or not the microangiopathy is secondary to the metabolic disturbances has been argued for decades and is no closer to being answered today than it ever has been. It is the purpose of this review to critically examine the question of whether rigid control of the blood glucose in diabetic patients would be beneficial in delaying or preventing the vascular complications of their disease. CLINICAL

AND

PATHOLOGIC

CORRELATIONS

Diabetic microangiopathy is usually thought of as the well-known triad of retinopathy, neuropathy, and nephropathy. Although diabetics also suffer from macroangiopathy, this review will only consider the characteristic small blood vessel disease associated with diabetes mellitus. Diabetic retinopathy is classified as either nonproliferative or proliferative. Nonproliferative diabetic retinopathy includes venous dilation, retinal microaneurysms, capillary closure, retinal hemorrhages, and exudates. Proliferative changes include neovascularization (new blood vessel formation), preretinal hemorrhages, fibrous tissue formation, and retinal detachments. Typically, retinopathy is unusual in patients whose diabetes is diagnosed before the age of From the Vererans Administration Hospiral and the Departmenr of Internal Medicine. Universitv of Texas Southwestern Medical School, Dallas. Tex. Receivedfor publication August 10. 1977. Supported by NIH Grams I-ROI-AM18/79-03 and I-MOI-RR0633 and by the American Diabetes Association, Norlh Texas Afiliate. Reprim requests should be addressed to Phirip Raskin. M.D., Department of Internal Medicine. University of Texas Health Science Center. 5323 Harry Hines Blvd., Dallas, Tex. 75235. @ 1978 bv Grune & Stratton. Inc. 0026-0495/78/2702~00l2~~2.~/~ Metabolism, Vol. 27, No. 2 (February), 1978

235

236

PHILIP RASKIN

30 until their disease has been present for more than 5 yr. When the diabetes is diagnosed between ages 30 and 50, the frequency of retinopathy occurring within the first 5 yr is higher. The prevalence of retinopathy in both groups increases with the duration of the disease.2 Interestingly, the capillary microaneurysm, which is considered a characteristic feature of diabetic retinopathy, occurs in other diseases such as sickle cell anemia, retinal venous thrombosis, and macroglobulemia. Histopathologic examination reveals that capillary microaneurysms occur only in the retina and they show exactly the same lipid and hyaline infiltration and PAS-positive staining as is present in other tissues in diabetes.3 Diabetic neuropathy is an important complication of diabetes because of its relative frequency and the fact that it involves every system of the body. Diabetic neuropathy includes peripheral nervous system syndromes such as radiculopathy, autonomic visceral symmetric and asymmetric neuropathy, neuropathy, and cranial neuropathy as well as various spinal cord syndromes including myelopathy, pseudotabes, diabetic amyotrophy, and acute vascular syndromes.’ The term “diabetic nephropathy” should be reserved for diabetic glomerulosclerosis, which results in the characteristic syndrome of proteinuria, edema, hypertension, and azotemia, which eventually leads to chronic renal failure. Although pyelonephritis and renal papillary necrosis occur with a greater frequency in the diabetic and present special problems, these will not be discussed further here. As is true with the other forms of diabetic microangiopathy. the prevalence of nephropathy increases with the duration of diabetes. Knowles patients with diabetes for et al. noted proteinuria in 33”,, of 108 “juvenile” more than 15 yr.5 In patients whose diabetes develops later in life, nephropathy often appears shortly after the diagnosis of the disease. NATURAL HISTORY OF DIABETES MELLITUS Although the vascular complications of diabetes are frequently encountered, they are certainly not inevitable, or if they do occur, they may cause only minor problems. There have been two major reviews of the natural history of this disease, the data of which is combined in Table I. Both studies reveal an astonishingly low complication rate. These data are, of course, biased to include only those patients who survived for 40 yr with their disease. Diabetics who died earlier are not included, so that this group of patients may have a milder form of the disease. Seventy-three patients whose diabetes was discovered before the age of 15 were followed for 40 or more years in the Joslin Clinic.6 All had been treated with insulin. Although 75.3”,, had retinopathy, only half of them had the severe proliferative type; 40”,, had nephropathy, yet only 8”,, had progressed to renal failure. Forty-eight percent of the patients had neuropathy. Ninety-two patients were followed in London’ for the same period of time. They too all required insulin therapy and only 11 patients were over 30 yr of age at the time of diagnosis. The same low complication rate was observed in this group. Only 6 I “(, had retinopathy with more than two-thirds of the patients having the nonproliferative type. Nephropathy was only present in 9”,, and neuropathy in

DIABETIC

REGULATION

AND MICROANGIOPATHY

237

Table 1. Vascular Complications

in Diabetes After 40 yr &ton6

Patients Therapy Complications

Nonproliferotive

type

Proliferative

type

Proliferative

type

(blind)

Nephropathy Proteinurio

only

Proteinuria

and

renal

failure

Neuropothy lschemic

heart

16.3”,,. In neither fared so well.

92 (32 M, 60 F)

Insulin

Insulin

75.3

60.8

45.0

43.4

17.8

10.8

12.3

6.5

(%)

Retinopathy

Peripheral

London7

73 (32 M, 41 F)

disease

vascular

disease

series were factors

CHEMISTRY

41.0

8.4

28.7

6.5

8.2

2.1

48.0

16.3

20.5

45.6

40.0

44.5

identifiable

OF NORMAL

to explain

BASEMENT

why these patients

MEMBRANE

Glomerular basement membrane is a collagenlike glycoprotein material that is rich in hydroxyproline, hydroxylysine, and cystine. Furthermore, it contains carbohydrate (10% by weight), which is present in the form of two distinct units.* One is a disaccharide (2-0-cY-D-glucosyl-D-galactose) that is attached in B-glycosidic linkage to the hydroxyl groups of about 80% of the membrane’s hydroxylysine residues, while the other is a branched heteropolysaccharide consisting of sialic acid, fucose, galactose, and N-acetylglycosamine. There are about 10 disaccharides for every heteropolysaccharide (Fig. 1). Levels of the glucosyltransferases and galactosyltransferases involved in the synthesis of the hydroxylysine-linked disaccharide units of the basement membrane appear to reflect the activity of the synthesizing machinery of the kidney basement membrane.‘.” CHEMISTRY

OF DIABETIC

BASEMENT

MEMBRANE

There is considerable disagreement regarding changes in the glomerular basement membrane in diabetes. Beisswenger and Spiro” have reported marked increases in hydroxylysine and in the number of glucosyllgalactose disaccharide units in glomerular basement membranes from human diabetics. They also

1 Fig. 1. Portion of the glomerular basement membrane. Jagged lines, helical (collagenlike) portions of the peptide chains; straight lines, more polar segments; Di, disaccharide; Poly, heteropolysaccharide. (From Spiro: Diabetologia 12:1-14, 1976)

238

PHILIP

Fig.

2.

therapy ferase

Effects on

activity.

controls; (From DURATION

OF DIABETES

(weeks)

648,

rat

I, Spiro

of

diabetes

kidney

cortex

D, diabetes; insulin-treated and

Spiro:

ond

RASKIN

insulin

glucosyltransN,

age-matched

diabetic Diabetes

rates. 20:641-

1971)

showed a reciprocal decrease in the number of lysine residues and significant differences in the hydroxyproline, glycine, valine, and tyrosine content of the diabetic and nondiabettc membranes. No changes in cystine or other components were noted. Measurement of glucosyltransferase activity in kidneys from alloxan-diabetic rats have shown a highly significant increase in the enzyme’s activity as compared to age-matched controls. This difference increased with the duration of the diabetes and could be prevented by meticulous insulin therapy instituted early in the course of the disease (Fig. 2).” Spiro concludes that in the diabetic glomerular basement membrane there is increased hydroxylation of lysine residues with subsequent glycosylation of the newly by Khalifa and formed hydroxylysine attachment sites.13 The demonstration Coheni of increased glomerular lysine hydroxylase activity in streptozotocin diabetes supports this conclusion. Kefalides” and Westberg16 have been unable to confirm Spiro’s observations in human diabetic glomerular membranes. They found only slight increases in the amounts of hydroxylysine, hydroxyproline, and the glucosyl-galactose disaccharide units but found significant decreases in cystine. Since it is the disulfide bonds that form the structural crosslinkages in basement membranes (Fig. 1). decreases in cystine could explain the observed changes in permeability of diabetic basement membranes.‘” ROLE OF HYPERGLYCEMIA OF DIABETIC

IN PATHOGENESIS

MICROANGIOPATHY

Morphonletric

The strongest piece of evidence against the hypothesis that diabetic microangiopathy is the consequence of the metabolic abnormalities of the disease has come from the work of Siperstein et al.” These investigators, using a simple morphometric method for measuring the basement membrane thickness of quadricep muscle capillaries, clearly showed (Table 2) that greater than 904,) of adults (over 19 yr of age) with diabetes had thickened capillary basement membranes as compared to a group of normoglycemic controls who had a negative family history for diabetes. In addition, they showed that in 30 genetic, pre-

DIABETIC

REGULATION

AND

MICROANGIOPATHY

Table 2.

239

Quadricep Capillary Basement Membrane Width

in Normal, Diabetic, and Prediabetic Subjects Average Basement Membray Width (A)

Subjects Normal

(SO)

1080

+ 27

Diabetic

(51) (30)

2403

f

1373

f 44

Prediobetic Data from Siperstein

119

Prevalence of Basement Membrane Thickening (Oh) 8 98 53

et al.”

diabetic adults (offspring of two overt diabetics) whose glucose tolerance was normal, 539; had thickened capillary basement membranes as compared to the control group. In these studies there was no correlation between the severity of duration of the diabetes and the thickness of the basement membrane. Mean capillary basement membrane widths dn 29 “mild” diabetics treated with diet and/or tolbutamide was 2410 f 167 A, whereas in 22 “severe” diabetics requiring insulin therapy it was 2395 + 173 A,. No differences were seen in basement membrane widths when the patients were divided into those whose disease began before or after age 21. There was also no correlation between the width of the basement membrane and the duration of the disease. Lastly, they showed that in 8 patients with “secondary” diabetes due to chronic pancreatitis, some of whom had long-standing fasting hyperglycemia, only 1 patient (2O”;J had a thickened basement membrane. Over the years this work has come under serious criticism. The main antagonist has been Williamson, whose data may be summarized as follows. He finds a much lower prevalence of basement membrane thickening in diabetes than did Siperstein with only 25”,, of patients under 50 yr of age and 63”: over the age of 50 showing the lesion. He also shows a very strong correlation of duration of the diabetes with the basement membrane width.” The differences between the two groups are probably related to differences in methodology. Siperstein’s method uses osmic acid for primary fixation and an average basement membrane measurement, whereas Williamson fixes in glutaraldehyde and uses a minimum measurement technique. Siperstein et a1.19 compared both methods of fixation and measurement in 20 diabetic patients and 20 nondiabetic controls. The results of this study clearly showed that the osmic acid fixation and an average measurement of the basement membrane was a much more specific way to identify the diabetic lesion than was glutaraldehyde fixation and a minimal measurement. As is outlined in Table 3, all diabetic patients showed thickened basement membrane widths using Siperstein’s method, whereas only 65”,, showed the lesion when Williamson’s techniques were used. These findings were independently confirmed by Danowski Williamson et al. were unable to do et a1.,20 whereas, in a recent publication, ~0.~’ The reasons for these differences are not readily apparent. In addition to the work of Siperstein and Williamson there are data from others which reflect on this issue. Pardo et al.‘* studied 26 patients with juvenile onset diabetes and 26 age-matched nondiabetic controls. They found that the diabetics had significantly increased basement membrane thickness irrespective

240

PHILIP

Table

3.

Prevalence

of Diabetic

Comparison

Fixation Method Osmic

acid

Glutoraldehyde Data

from

Siperstein

RASKIN

Microongiopathy:

of Methods

Meall 20/20

(100%)

15/20

(75%)

Minimum 13/20

(65%)

9/20

(45%)

et ctl.”

of whether an average basement membrane measurement (Siperstein) or a minimum measurement (Williamson) was used. They also found a positive correlation between the basement membrane width and the duration of the diabetes. However, there was so much overlap between values obtained from the diabetics and nondiabetics that the authors concluded that neither technique was sensitive enough to use to identify individual diabetics. Danowski et al.” measured the capillary basement membrane width in 15 patients with juvenile-onset diabetes. The minimal basement membrane thickness was less than that of age-matched controls in 7 of the 15 patients. Furthermore, the thickness of the basement membrane tended to correlate with the duration of the disease in that the minimum basement membrane thickness was 933 A or greater in those 7 patients who had diabetes for 6 yr or more and was 545 ,& or less in 7 of the 8 remaining patients whose disease was present for less than 6 yr. These authors concluded that hypertrophy of the capillary basement membrane generally appears toward the end of the first decade following the diagnosis. There seemed to be no good relationship to the severity of the hyperglycemia. only its duration. These data must be interpreted with respect to the recent work of Raskin et a1.,24 who showed that the characteristic thickening of skeletal muscle capillary basement membrane is not consistently seen in children with diabetes. In 48 diabetic children who were 19 yr of age or less at the time of study, thickening of the basement membrane was present in only 40”,,. The prevalence of blood vessel abnormalities seemed to be related to the age of the patient at the time of biopsy and not to the duration of the disease. In Danowski’s study23 all patients who were 20 yr old or older at the time of biopsy had a thickened capillary basement membrane width. One Ibyr-old patient whose disease had been present for 6 yr and one 13-yr-old patient who had diabetes for only 1 yr also had abnormal blood vessels. Since the number of patients in this study was small, it is possible that the results reflect only the older age of those diabetics with abnormal blood vessels rather than the duration of their hyperglycemia. Recently Aronoff et al.*’ compared both Siperstein’s and Williamson’s methods in a large double-blind study on the Pima Indiana. The results clearly confirm the original observation made by Siperstein and demonstrate the increased sensitivity of the techniques using osmic acid fixation and an average basement membrane measurement. In short, when Siperstein’s methods were embasement membrane ployed, greater than SO”, of adult Pimas had capiliary hypertrophy. A “prediabetic lesion” was seen in 22”;, of the genetic prediabetic

DIABETIC

REGULATION

AND MICROANGIOPATHY

241

Indians. Williamson’s methods were much less sensitive, identifying only SO’?:, of the diabetic and 37; of the prediabetic Indians. Both methods showed a striking correlation between the duration of the diabetes and the thickness of the capillary basement membrane. Since Siperstein’s original publication, we have studied 19 patients in whom the diagnosis of “pancreatic” diabetes seemed secure. Only 3 of the 19 (169,) had a thickened capillary basement membrane, confirming the much lower prevalence of this lesion in secondary diabetes.26 Recently we have had the opportunity to restudy 8 patients with pancreatic diabetes, 4 of whom were original patients of Siperstein’si7 The 8 patients were restudied after a mean interval of 5 yr (range 1.5-9 yr). All but 1 patient showed a significant increase in capillary basement membrane width over the years. Whereas only 1 of 8 was abnormal originally, this number had increased to 5 of 8 by the time of the second biopsy.26 These data are suggestive that prolonged hyperglycemia may play a role in the development of quadriceps capillary basement membrane thickening. However, an alternative explanation is also possible, i.e., only those patients who are genetically predisposed to diabetes will develop fasting hyperglycemia secondary to chronic pancreatitis. The reason those patients developed thickened basement membranes was that they were indeed genetic diabetics and their hyperglycemia was more a reflection of their predisposition to diabetes rather than the pancreatitis. Interpretation of the morphometric data presents a dilemma. Clearly, the presence of a “prediabetic lesion,” i.e., basement membrane thickening in normoglycemic genetic prediabetics, suggests that diabetic control could in no way affect the progression of the microangiopathy. However, the absence of significant basement membrane hypertrophy in children with diabetes and the subsequent development of such changes in patients with “pancreatic diabetes” leads one to the opposite conclusion. Perhaps further study will help resolve the issue. Pathologic The pathology of diabetes is complex and involves most organ systems. However, much of the important information regarding the question under discussion here revolves around the pathologic changes seen in the diabetic kidney. 1 would like to describe in some detail the pathology of the kidney in diabetes, for much of what follows will be dependent upon it. There are four glomerular lesions seen in the diabetic kidney:27 This is the classic Kimmelstiel-Wilson lesion.28 Nodufar glomerulosclerosis. The nodular glomerular lesion consists of a round hyaline mass centrally located in a peripheral glomerular lobule with a patent, often dilated capillary running over its surface. The nodule is strongly PAS-positive. With electron microscopy the lesions consist of nodular accumulations of basement membrane-like material in the mesangium.27 This lesion is thought to appear only in diabetes. This lesion appears as linear hyaline thickening Difuse glomerulosclerosis. of the basement membrane and mesangium.*’ This type, rather than the nodu-

242

PHILIP

RASKIN

lar type, is more closely associated with the clinical syndrome of proteinuria and renal failure.30 This lesion alone is not specific for diabetes as it is seen in benign nephrosclerosis, membrane glomerulonephritis, and systemic lupus erythematosis. Capsular-drop lesion. This lesion typically consists of homogenous deposits of eosinophilic, glossy material having a spherical or droplet shape. The deposit may be attached to the inner side of the parietal layer of Bowman’s capsule between the basement membrane and the parietal epithelial cells or may lie within the capillary lumen surrounded by endothelial cells. This lesion was noted in Kimmelstiel and Wilson’s original paper2* but little attention was paid to it. Kimmelstiel now attaches diagnostic significance” to it, especially when seen in its early stages. Exudative lesion. This lesion, also called the “fibrin cap,” appears as a highly eosinophilic, smooth, homogenous crescent structure lying within the concavity of the capillary. Although not considered specific for diabetes, as they appear in other types of renal disease, similar lesions are often seen in glomeruli of animals with spontaneous or experimental diabetes. In addition to glomerular lesions, characteristic changes in the renal vasculature and tubules may be identified in tissue from diabetics. The renal vascular lesions in the diabetic kidney consist of segmented hyaline thickening of both the afferent and efferent arterioles, which may progress to occlusion of the vessels. In essential hypertension hyaline thickening is seen only in the afferent arteriole. This lesion is considered very specific for diabetes.‘* The renal tubular lesion in the diabetic kidney consists of thickening of the basement membrane of the renal tubules. This is often seen in conjunction with one of the glomerular lesions mentioned above. In evaluating reports of diabetic microangiopathy involving the kidney, care must be taken to establish the specificity of the observed lesion for diabetics. Heptinstall has outlined specific criteria for the pathologic diagnosis of diabetic glomerulosclerosis.27 The nodular lesion is a very distinct histologic entity and should not be confused with other conditions, especially when seen in combination with hyaline arteriosclerosis. The diffuse glomerular lesion, when fully developed, is very suggestive of diabetes but not entirely diagnostic unless it is accompanied by hyaline arteriosclerosis. The capsular drop, when seen in its early form, is likewise diagnostic. However, it is rarely seen alone. Lastly, the exudative lesion, although seen in diabetes, is not helpful. as it is present in many other types of renal disease. One issue central to working out any relationship between the metabolic abnormalities in diabetes and its vascular complications relates to the blood vessel lesions observed in experimental diabetes. Foglia et al.-” described changes in the renal glomerulus in rats subjected to pancreatectomy after 2- 12 mo of sustained hyperglycemia. Mann et a1.34 made similar observations in alloxan-diabetic rats whose hyperglycemia was present for as long as 30 mo. Although similar lesions were never seen in normoglycemic rats, Mann was unable to correlate the lesions with the severity or the duration of the diabetes. The lesions observed in the renal glomerulus of the diabetic rats, although similar in some regards to the lesions seen in diabetic man, lacked both the

DIABETIC

REGULATION

AND MICROANGIOPATHY

243

characteristic nodule formation and hyaline arteriolar changes. A similar observation has been made by @rskov.35 Gibbs et al.36 found glomerular abnormalities in a group of monkeys made diabetic with alloxan and maintained hyperglycemic for l-4 yr. Although mesangial thickening occurred, these lesions in no way resembled the human lesion. Bloodworth noted the diffuse glomerular lesions of diabetes in 10 dogs made diabetic by administration of alloxan or growth hormone. In 7 dogs nodules developed that were identical to those seen in man. Arteriosclerosis was also seen. Recent studies using immunohistochemical techniques have added considerable information about renal lesions in diabetes. Miller and Michae13* carried out a comprehensive immunofluorescent analysis on renal tissue from three groups of patients. Group I consisted of 24 living normal renal allograft donors and 2 infants less than 1 wk of age. Group II included 24 patients, 2047 yr of age, with severe nephropathy that had progressed to chronic renal failure, who had juvenile-onset diabetes mellitus for 16-30 yr. The last group (group III) consisted of 33 patients, 5-63 yr old, with chronic renal failure of diverse cause other than diabetes. Renal sections from patients with diabetes were easily distinguished from those of the other patients and normals by the intense linear staining of extracellular membranes. The most specific reaction was the presence of IgG and albumin lining the tubular basement membrane. Note in Fig. 3 the relative specificity of the immunofluorescence in the renal sections from patients with diabetes. Save for some minimal staining of the

Fig. 3. Immunofluorescence for IgG and albumin in tubular basement membranes (TBM), glomerular basement membranes (GBM), and Bowman’s capsule in kidneys from normal subject and patients with chronic renal failure due to diabetic nephropathy and other renal disease. (From Miller and Michael: Diabetes 25:701-708. 1976)

244

PHILIP

RASKIN

glomerular basement membrane from the normal kidneys, there is practically no overlap among these three groups. The findings are consistent with the observation that in basement membranes obtained from human diabetics the decrease in the cystine content may reflect an alteration in the disulfide cross-linkage and thus make diabetic basement membranes more permeable to large macromolecules.” Similar but not identical observations have been made in alloxan-diabetic rats. Immunofluorescent staining for rat IgG and complement (C,) occurred in a mesangial pattern after 4- 6 mo of hyperglycemia.” Although age-matched control animals had some positive mesangial staining for IgG and C, with advancing age, it was observed in much smaller quantities. Renal transplantation of kidneys from experimentally diabetic rats into nondiabetic recipients results, within 2 mo, in the disappearance of IgG, IgM, and C3 from the mesangium and the arrest or reversal of mesangial thickening. Conversely, transplantations of normal kidneys into diabetic recipients result in deposition of immunoglobulin and complement in the mesangium and visible successful islet cell transmesangial thickening within 2 mo. Jo Furthermore, plantations in streptozotocin-diabetic rats whose diabetes had been present for 668 mo resulted in a significant reduction in mesangial thickening and in the mesangial staining for IgG, IgM, and Cj. These changes were apparent 2 wk after the restoration of normal glucose and insulin levels. Complete regression of the light microscopic changes had not occurred within 9 wk.“’ In man, experiments of a similar nature have given results remarkably like those obtained in experimental diabetes. Mauer et al.” examined kidney tissue obtained from I2 diabetic and I7 nondiabetic patients 2 I2 yr following renal transplantation. The frequency and intensity of IgG and albumin staining of the tubular and glomerular basement membrane and Bowman’s capsule was significantly greater in the diabetic than in the nondiabetic patients. Except for some staining of the glomerular basement membranes in the nondiabetic kidneys, there was practically no overlap between the two groups (Fig. 4). Although 9 of the I2 diabetic patients received their kidneys from a living related donor, no immunofluorescence was observed in 7 kidneys studied at the time of their transplantation into diabetic recipients. The same group studied renal transplant tissue from 12 diabetic and 28 nondiabetic patients who had had a renal graft for at least 2 yr.43 Of the 12 kidneys studied from diabetic patients, IO showed arteriolar hyalinosis: in 6 of the IO, the hyaline change involved both the afferent and efferent limb of the glomerular arterioles. One diabetic patient developed typical nodular glomerulosclerosis 35 mo after transplantation. Of the 28 kidneys studied from the nondiabetic transplant recipients, 3 had hyaline vascular changes. This occurred only in rare vessels, did not appear until 5 yr posttransplantation, and never involved both afferent and efferent arterioles. None of the blood vessel changes were present in the kidneys transplanted into the diabetic recipients at the time of transplantation, although IO of the I2 received living related donor grafts. The pathologic data are perhaps the most convincing that control of hyperglycemia in diabetes might alter the course of the vascular complications. The

DIABETIC

REGULATION

AND MICROANGIOPATHY

245

QiMArV~ CAPSU E

Fig. 4. lmmunofluorescence for IgG and albumin in tubular basement membranes (TM), gtomerolar basement membranes (GBM), and Bowman‘s capsule of kidneys transplanted into diabetic and nondiabetic potients. (From Mover et al: Diabetes 25:709-712, 1976)

development of typical diabetic immunofluorescent and pathologic changes in previously normal kidneys transplanted into diabetic subjects and reversal of similar lesions when experimental diabetes is reversed by islet-cell transplantation strongly supports this conclusion. Biochemical Hemoglobfin Al,. In 1968, Rahba? noted the presence of a fast moving hemoglobin on agar gel electrophoresis in 2 patients of 1200 who were studied. Both of these patients had diabetes mellitus. Further study of an additional 47 patients with diabetes showed the abnormal hemoglobin to be present in all patients. Rahbar et al.45 later identified this unusual hemoglobin to be HbAl,, which had been previously shown to comprise about 6”,, of the HbA of normal people.46 Since its initial identification, numerous studies have shown that the concentration of HbAl, is elevated 2-3-fold in human diabetes.47-49 The mouse also has HbAl, that is structurally analogous to that of man and it is present in larger amounts in the genetically diabetic (db/db) mouse or in rodents made diabetic by either alloxan or streptozotocin. The rise in concentration of HbAl, occurs 3-4 wk after the onset of the hyperglycemia but does not seem to be related to the severity or the duration of the hyperglycemia.” The increase in HbAI, is apparently not an inherited phenomenon as it is present only in the hyperglycemic member of identical twin pairs discordant for juvenile-onset diabetes mellitus. Four pairs of identical twins concordant

246

PHILIP

RASKIN

for adult-onset diabetes showed lower values for HbAI, and could not be separated from the nondiabetic controls.5’ The level of HbAl, is not related to the fasting plasma glucose concentration nor to the thickness of the skeletal muscle capillary basement membrane but appears to correlate with the degree of postprandial glucose intolerance present in diabetics. 52Institution of rigid blood sugar control in five hospitalized diabetics with a resultant decrease in the mean fasting plasma glucose concentration from 343 to 84 mg/dl caused HbAl, levels to decrease from 9.8”,, to 5.8”,,. The degree of control, assessed by a combination of both pre- and postprandial blood glucose levels, gave the best correlation with HbAI, levels.5J Thus, the periodic monitoring of HbAI, levels may provide a better index as to the adequacy of blood glucose control over the previous weeks or months. a parameter that is presently extremely difficult to evaluate accurately. The formation of HbAI, appears to be a postsynthetic modification of HbA that occurs throughout the life of the red cell. This was shown first in micejJ and more recently in man.55 HbAI, is the product of the chemical condensation of hemoglobin and glucose. It differs from HbA in the glucose moiety that is attached to the N-terminal amino group of the beta chain by a unique ketoamine linkage formed by a rearrangement of the Schiff base.56 It appears from the kinetic studies of Bunn et al.55 that the rate of formation of HbAl, should be proportional to the time-averaged concentration of glucose within the erythrocyte. Thus, the level of HbAI. would be a reflection of the adequacy of control over a sustained period of time. Furthermore, the postsynthetic glycosylation of HbA in the diabetic may be representative of similar changes in other proteins that may occur during periods of sustained hyperglycemia. M,oinosirol. Sciatic motor nerve conduction velocity is definitely impaired 2 wk after the initiation of diabetes in rats by the administration of streptozotocin. Although this abnormality is not associated with pathologic changes in the nerve, it does seem to be associated with persistent hyperglycemia in nerve conduction velocity was not corrected (Table 4).57 This impairment by insulin therapy, which produced normal weight gain in the diabetic animals and resulted in a marked reduction in plasma glucose levels. albeit not quite to normal. Only when insulin was administered twice daily in doses adjusted on the basis of blood glucose determination (group III) could the defect in nerve conduction velocity be corrected. However, this type of therapy resulted in lower mean glucose levels than in nondiabetic controls and 7 of the 24 rats

Table

4.

Effects Sciatic

of Streptozotocin-Diabetes Motor

Nerve Weight

Group

Conduction

and

Insulin

Velocity

Plasma Glvcore

(9)

on

MNCV (m/=)

(v/4

6

Treatment

(MNCV)

NOWd

247 i

167 * 9

64.6 f 0.9

Diabetic

178 f 9

539 * 23

50.1 * 0.9

Insulin Treated

I

235 i 6

214 f 40

51.4*

Group II

234 f 6

213 f

18

51.1 * 1.7

Group Ill

280 + 5

75+

18

62.9 i 0.9

Group

1.1

DIABETIC

REGULATION

AND MICROANGIOPATHY

247

in this group died from hypoglycemia. Thus, prevention of even mild hyperglycemia was necessary to prevent the nerve conduction defect. The pathologic lesion in diabetic neuropathy is segmental myelin degeneration.58 In experimental diabetes in rats there is a reduced free myoinositol (a major component of peripheral nerves) concentration in nerves which correlates with impaired motor nerve conduction velocity. In glycosuric patients, 409: of the dietary intake of myoinositol is excreted in the urine as compared to only a small fraction in nondiabetics. Supplementation of commercial rat chow with lo;, myoinositol prevented the impairment in nerve conduction velocity in diabetic rats despite persistent hyperglycemia.57 Thus, the effect of insulin deficiency and its resulting hyperglycemia on peripheral nerve conduction velocity appears to be mediated by a defect in myoinositol metabolism which can be modified by increasing dietary and, thereby, plasma levels of myoinositol.60 Clinical If the metabolic abnormalities of diabetes are in some way responsible for the development of the microangiopathy, then it should be possible to demonstrate that correction of these metabolic abnormalities prevents or delays the development of the vascular complications. There have been many attempts to prove this is the case, but as yet no adequate study has been reported. There are many anecdotal reports put forward as proof that good diabetic as exemplified by the report of control prevents the microangiopathy, Shepherd.6’ Dr. Shepherd, a 50-yr-old physician, developed diabetes when he was 6 yr old. After 43 yr of diabetes he had only a few microaneurysms and proteinuria of less than 500 mg/day. For 27 yr he used regular insulin three times a day; finally, because of midafternoon hypoglycemia, he omitted his prelunch insulin injection. Other studies, such as those of Keiding et a1.62 and in juvenile Hardin et a1.,63 conclude that the rigid control of the hyperglycemia diabetes can delay and minimize the development of vascular complications. However, these studies are scientifically unsound. They are retrospective, the assessment as to the degree of control is based more or less on the presence or absence of glycosuria, and, most importantly, there is no real control group. analysis in evalThe report by Dolger,” which used the same retrospective uating the effect of control on the vascular complications in 200 diabetics whose diabetes began at less than 50 yr of age, came to the opposite conclusion. Vascular complications occurred in his patients at the same rate and severity regardless of the level of control (again judged by the amount and frequency of fashion glycosuria). Knowles et al., in a classic paper,5 followed in a prospective for a IO-yr period 108 juvenile-onset diabetic patients who were allowed a free diet. Hyperglycemia and glycosuria were common; in fact, 81”,, had fasting blood sugars (Folin-Wu) greater than 130 mg/dl when measured at a routine clinic visit. At best, control would be rated as poor. The prevalence of diabetic vascular complications in this group of patients was similar to other groups of diabetics in whom more rigid control was attempted. Perhaps the best study of this type is that of Johnsson. He reviewed all diabetic patients in Malmo, Sweden, whose diabetes was diagnosed between 1922

248

PHILIP

RASKIN

and 1945 and who were less than 40 yr of age at the onset of their disease. They were divided into two groups. Series I consisted of 54 patients diagnosed from 1922 to 1935. All these patients were initially treated with strict diet control and multiple injections of insulin in an attempt to keep the urine as free of glucose as possible. Series II consisted of 105 patients whose disease was diagnosed from 1935 to 1945. These patients were treated with a single injection of longacting insulin and a much less regimented diet. The patients in series 11 had a much higher incidence of vascular complications than did those of series I. Whereas the mean duration of the diabetes for the patients in series I was 10 yr longer than for those in series II, only 327, had nephropathy, as compared with 56”,, in series II. The difference is even more striking when one looks at the incidence of nephropathy in patients who have had their disease for more than 15 yr. Only 9”,, of patients in series I had nephropathy as compared to 61”,, of series II. Similar striking differences were seen between the groups when the incidence of retinopathy was compared. However. hypoglycemia was far more common in series I patients, reflecting attempts to achieve normoglycemia and the use of multiple daily insulin injections. The recent study by Job et al. has been frequently cited as proof that good diabetic control can delay the development of retinopathy.66 This study was designed in a prospective fashion and diabetic patients were randomly assigned to either a single insulin injection group or a multiple (three times daily) insulin injection group. Both groups were followed for a mean duration of 3 yr. The progression of retinopathy was evaluated by fluorescein angiography and funduscopic examination. They reported a significantly greater increase in the number of microaneurysms in the single-injection group as compared to the multiple-injection group. Unfortunately, this study suffers from an important defect that makes the results difficult to evaluate. Of those 2 1 patients whose data were analyzed from the single-injection group, only 16 received one daily insulin injection for the entire period of the study; the other 5 were changed either to two or three injections per day after the study began. In the multiple-injection group, only 5 of the 21 patients analyzed had three insulin injections daily for the entire period of the study. The others either initially accepted only two injections (4 patients) or some only one (4 patients). Of the 13 who originally accepted three injections daily, 9 were reduced to two injections after 1 yr. The small number of patients initially studied coupled with the many crossovers in protocol make this study impossible to interpret. It seems a travesty that now, more than 50 yr after the discovery of insulin, this very important question remains unanswered. There are several reasons for this dilemma. First of all, the proper clinical experiment has yet to be conducted. What is needed is a long-term prospective study in which patients are randomly assigned to either a “tight” or “loose” control treatment group. This study should continue for decades rather than years, as. clearly, many of the complications of this disease take decades to develop. However, this question remains moot because it is impossible with presently available modes of therapy to completely restore the diabetic’s blood glucose to normal levels throughout the day.

249

DIABETIC REGULATION AND MICROANGIOPATHY

Fig. 5. Glucose, insulin, and glucagon concentrations in plasma obtained at 2-hr intervals in nine normal subjects. Meals are marked by the arrows. (From Unger et al: Recent Prog Horm Rer 33:477-5 17, 1977)

6 DAY

AM

12 6

12 6 12 6 12 6

I

,

PM

AM

;

PM

,

AM

Figure 5 shows the bihourly plasma glucose concentration in nine nondiabetic subjects studied for 48 hr in the Clinical Research Unit at Parkland Memorial Hospital. The plasma glucose concentration rarely exceeded 150 mg/ dl at any time during the day, even when blood samples were taken 1 hr after breakfast and lunch.67 Figure 6 shows the plasma glucose levels measured bihourly for 6 days in a newly discovered 42-yr-old diabetic patient whose diabetes was eventually brought under what would be considered excellent control using two injections of insulin daily. Despite the fact that glycosuria was eliminated and fasting plasma glucose levels were well within the normal range, the postprandial plasma glucose concentration often exceeded 200 mg/dl. We have repeated this experiment in some 20 diabetic patients of both juvenile- and adult-onset types with similar results.@ It is a relatively simple task in the controlled environment of a clinical research unit to eliminate glycosuria and re-

Fig. 6. Glucose, insulin, and glucagon concentrations in plasma obtained at 2-hr intervals and the daily 24-hr glucose excretion in an adult-onset diabetic of recent onset, during 6 days of aggressive insulin therapy. limes of meals and insulin administration are marked by the arrows. (From Unger et al: Recent Prog Harm Res 33:477-5 17, 1977)

250

PHILIP

RASKIN

store fasting glucose values to normal, but postprandial hyperglycemia often continues despite large doses of insulin. Others have had similar experiences.6Y If one is unable to completely normalize the blood glucose throughout the day in a clinical research unit, imagine how impossible it must be to do so in a reallife situation. There has been little convincing clinical evidence for the view that rigid control of the metabolic abnormalities will prevent the vascular complications of the disease because few diabetics have ever been rigidly controlled. However, there is no overwhelming evidence to the contrary, i.e., that the vascular complications are independent of the hyperglycemia. Perhaps with new advances such as pancreatic islet cell transplantation” or perhaps even partial pancreatic transplantation,” or the use of new drugs such as somatostatin,72~73 rigid diabetic control will be possible and the answer to this 50-yr-old question will be found. ACKNOWLEDGMENT The secretarial

assistance

of Denise Wernet

is gratefully

acknoaledged.

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