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Purpose: We seek to identify pathogenic mechanisms for diabetic retinopathy that can become therapeutic targets beyond hyperglycemia and hypertension. We investigated if a defective myogenic response of retinal arteries to increased perfusion pressure, which exposes capillaries to increased pressure and flow, is associated with the onset of clinical retinopathy., Methods: We examined prospectively the incidence of retinopathy in type 1 diabetic individuals tested 4 years earlier for the retinal arterial myogenic response, and in a cross-sectional study the prevalence of defective myogenic response in type 1 patients who had diabetic retinopathy. Among these, we contrasted early-onset (after 15 ± 2 years of diabetes, E-DR; n = 5) to late-onset (after 26 ± 3 years of diabetes, L-DR; n = 7) retinopathy. We measured the myogenic response using a laser Doppler blood flowmeter after a change in posture from sitting to reclining, which increases retinal perfusion pressure., Results: Five of seven participants who 4 years prior had a defective myogenic response had now developed clinical retinopathy; as compared with only one of six participants who 4 years prior had a normal response (P = 0.10). In the cross-sectional study, all participants had normal retinal hemodynamics at steady state. In response to the postural change, only the E-DR group showed defective myogenic response (P = 0.005 versus controls, P = 0.02 versus L-DR) and abnormally high retinal blood flow (P = 0.016 versus controls)., Conclusions: In type 1 diabetic patients, a defective myogenic response of retinal arteries to pressure is not required for the development of clinical retinopathy, but is prominently associated with an accelerated onset of retinopathy.

Endothelial progenitor cells (EPCs) enter the systemic circulation in response to cues related to vascular damage and need for neovascularization. Thus, EPCs could become readily accessible informers of vascular status and enable the survey of vascular pathologies during preclinical stages. To identify EPC changes with biomarker potential, we investigated whether discrete EPC abnormalities were associated with early nonproliferative diabetic retinopathy (NPDR). Two EPC subtypes with different functions have been characterized to date-one solely committed to the endothelial lineage and the other carrying both endothelial and monocytic markers. We found that only the latter, colony-forming units (CFU)-Hill cells, manifested abnormalities in type 1 diabetic patients with NPDR compared with control subjects. The abnormalities consisted in an increased number of colonies formed in vitro and downregulation of the molecules that facilitate homing at sites of vascular injury. The abnormalities were absent in type 1 diabetic patients free of retinopathy and other complications, despite long diabetes duration, but were detected in some of the patients without clinical retinopathy after short diabetes duration. CFU-Hill cells are potential informers of diabetic microangiopathy but may be preempted from carrying out reparative functions if the molecular abnormalities compromise interactions with the damaged vascular wall.

Aims/hypothesis: Impaired activity of the pentose phosphate pathway of glucose metabolism caused by hereditary deficiency of its key regulatory enzyme glucose-6-phosphate dehydrogenase (G6PD) has consequences that may worsen or attenuate the course of diabetic complications. Decreased availability of NADPH can predispose to oxidative stress and endothelial dysfunction, but can also limit the activity of the polyol pathway and cholesterol synthesis. Reduced availability of pentose phosphates for nucleic acid synthesis could impair cell proliferation. We sought to learn in which direction G6PD deficiency affects diabetic retinopathy., Methods: We enrolled patients who were G6PD-deficient or -sufficient with type 1 diabetes of duration 15 years or longer for whom HbA(1c) records were available for at least the previous 3 years. Renal failure and smoking were exclusion criteria. For each participant seven standard field colour photographs were obtained of each eye, and retinopathy was graded in a masked fashion., Results: The clinical characteristics of the 19 G6PD-deficient patients studied (age 42 ± 9 years, diabetes duration 24 ± 6 years, average HbA(1c) over 3 years 6.7 ± 0.8%) were similar to those of the 35 G6PD-sufficient patients. Almost 90% of patients in both groups had retinopathy; however, proliferative retinopathy was noted solely among G6PD-deficient patients (28%, p = 0.0036 vs G6PD-sufficient). The G6PD-deficient patients also showed a trend for increased frequency of microalbuminuria., Conclusions/interpretation: The data suggest that G6PD deficiency accelerates the microvascular complications of diabetes, and that among the consequences of G6PD deficiency those that can enhance the damage caused by diabetes outweigh those that could be protective.

Purpose: The current approach to the prevention of diabetic retinopathy relies on intensive anti-diabetes treatment and is only partially successful. A marker of retinopathy risk would enable strategies of surveillance, screening of adjunct drugs, and targeted drug interventions. The authors sought to identify early abnormalities of retinal vessels that are not prevented by the current therapeutic approach., Methods: Retinal thickness (an informer of vascular permeability) and hemodynamic parameters at baseline and longitudinally were measured in 27 subjects (age, 32 ± 9 years [mean ± SD]) with well-controlled type 1 diabetes of 12.4 ± 6.4 years' duration and no retinopathy, and in 27 control subjects. In a subset of 17 patients and 11 controls, the hemodynamic response to reclining, a postural change that increases retinal perfusion pressure, was measured., Results: Baseline foveal thickness and hemodynamic parameters were similar in the diabetic and control subjects. Foveal thickness increased over 12 months in the diabetic subjects, from 217 ± 22 μm to 222 ± 20 μm (P = 0.0036), remaining however within the normal range. Reclining uncovered in 47% of diabetic subjects (P = 0.016 compared with controls) an absent myogenic response (i.e., unchanged or increased arterial diameter instead of the normal decrease). The patterns were repeatable. Only the diabetic group with defective vasoconstriction showed widening arterial diameter over 12 months, a change presaging vascular dilatation in diabetic retinopathy., Conclusions: Defective myogenic response to pressure was the first detectable abnormality of retinal vessels in subjects with well-controlled type 1 diabetes. Because of its selective occurrence, interpretability in individual patients, and pathogenic potential, the abnormality deserves evaluation as a risk marker for retinopathy.

Aims/hypothesis: Abnormalities in retinal haemodynamics have been reported in patients with type 1 diabetes in advance of clinical retinopathy. These abnormalities could therefore be useful as early markers or surrogate endpoints for studying the microangiopathy. Since the DCCT, the increased focus on good glycaemic control is changing the natural history of diabetic retinopathy. Based on this, the aim of this study was to investigate whether patients with type 1 diabetes treated entirely or mostly in the post-DCCT era and tested in the absence of confounding factors show retinal haemodynamic abnormalities., Methods: We measured retinal haemodynamics by laser Doppler flowmetry in 33 type 1 diabetic individuals with no or minimal retinopathy (age 30+/-7 years, duration of diabetes 8.8+/-4.6 years, 9% showing microaneurysms), and 31 age- and sex-matched non-diabetic controls. The study participants were not taking vasoactive medications, and blood glucose at the time of haemodynamic measurements was required to be between 3.8 and 11.1 mmol/l., Results: HbA1c was 7.5+/-1.2% and blood glucose 7.7+/-2.8 mmol/l in these type 1 diabetic individuals, indicating relatively good glycaemic control. Retinal blood speed, arterial diameter and blood flow were not different between the diabetic individuals and the matched controls., Conclusions/interpretation: Type 1 diabetic patients with no or minimal retinopathy who maintain relatively good glycaemic control do not show abnormalities of the retinal circulation at steady state, even after several years of diabetes. In such patients it may be necessary to test the vascular response to challenges to uncover any subtle abnormalities of the retinal vessels.

To test the hypothesis that cerebral capillaries, which share the embroyologic and morphologic characteristics of retinal capillaries, might have the same abnormal permeability in diabetic patients, we investigated the growth hormone response to a small amount of peripherally administered dopamine (1.5 microgram/kg.min). Consistent with the known exclusion of systemic dopamine from brain parenchyma, no rise was observed in 12 normal subjects. In 10 of 12 juvenile-onset, insulin-dependent diabetic patients, however, a substantial growth hormone rise occurred (peak value, 19.2 +/- 3.0 ng/ml [mean +/- SE]). Comparision of metabolic and cardiovascular responses to the infusion in both groups did not suggest that higher circulating levels of dopamine had been achieved in the diabetics. Other growth hormone stimuli (apomorphine in decreasing amounts, glucagon, and graded physical exercise) failed to indicate that hypothalamic hypersensitivity could account for the consistent rise. We postulate that an abnormal permeability of the blood-brain barrier in the diabetic patients permitted exposure of the hypothalamic structures regulating growth hormone secretion to a greater fraction of the infused dopamine.

We have studied the epidemiologic characteristics of insulin-dependent (Type 1) diabetic patients aged 0-19 in a city (San Diego, southern California, USA) characterized by an impressive racial diversity and especially mild and constant climatic conditions. Ascertainment was through retrospective review of medical records in 19 hospitals. For the 3 years 1978-1981 the mean annual incidence of diabetes was 7.3 cases/100,000, with no statistical difference between the sexes. The observed incidence rates in the various ethnic groups was significantly different from expected (p less than 0.03), with an excess of cases among Caucasians and fewer than expected cases among Mexicans, Blacks and Orientals. There was no identifiable seasonal trend. Some of the clinical characteristics at diagnosis differed between the sexes: males were slightly older (9.3 +/- 5.2 years versus 8.8 +/- 3.9 for females), had a shorter duration of diabetes-related symptoms and a higher frequency of infections both at the time of diabetes diagnosis and in preceding months. Females tended to have a higher frequency of Type 1 diabetes in first-degree relatives. This study documents for the first time that, among multiple racial groups living in the same environment, Caucasians are at the highest risk of developing juvenile-onset Type 1 diabetes.

To assess further the role of insulin in the abnormal alpha-cell dysfunction found in human diabetes mellitus, the effects of acute insulin withdrawal and administration on plasma glucagon responses to intravenous arginine were studied in eight insulin-dependent diabetic subjects. Arginine infusions (30 gm. over 30 minutes) were performed during and at one and four hours after discontinuation of a 14-hour insulin infusion (1.5 U. per hour), which had rendered the subjects euglycemic, and on another occasion before and one and four hours into a five-hour infusion of insulin (1.5 U. per hour). During the last hour of the 14-hour infusion, glucagon responses to arginine (area under the curve, nanograms per milliliter per minute) were similar to those found in normal subjects (10.3 +/- 0.8 vs. 9.0 +/- 0.8, respectively). After discontinuation of the insulin infusions, glucagon responses increased progressively (p less than 0.01) to values (16.8 +/- 1.2) that exceeded those of normal subjects by four hours (p less than 0.01). These were similar to results found in the same subjects studied when their diabetes was in less than optimal control (14.9 +/- 1.3). Infusion of insulin under these conditions progressively decreased glucagon responses to arginine to values (9.6 +/- 0.8; p less than 0.01) that, at four hours, were similar to those of normal subjects and to values found at the end of the 14-hour infusion of insulin in the same diabetic individuals. These results demonstrate a rapid effect of insulin on glucagon responses to arginine and suggest that the abnormal responses seen in diabetes mellitus are the immediate result of insulin deficiency. Since abnormal glucagon responses to glucose in diabetes are not as readily corrected by insulin, the mechanisms underlying the abnormal responses to these two stimuli may differ.

Plasma glucagon levels rose within 1 hr after withdrawal of insulin in 7 juvenile-type diabetics previously kept normoglycemic by prolonged intravenous infusions of insulin. These changes preceded subsequent elevations in plasma alanine levels. Individual rises in plasma glucagon were correlated with elevations in plasma glucose, beta-hydroxybutyrate, free fatty acid, and glycerol levels, suggesting that glucagon may play an important role in the development of diabetic ketoacidosis in man.

Plasma from both type I and type II patients with diabetes mellitus displayed elevated alpha 2-macroglobulin trypsin-binding activity relative to normals. Column isoelectric focusing indicated that the average isoelectric point (pI) of the major form of crude alpha 2-macroglobulin was 6.6, 5.9, and 6.2 for normal, type I and type II subjects, respectively. Focused crude diabetic alpha 2-macroglobulin displayed significantly less recovered trypsin-binding activity relative to controls, particularly above a pI value of 6.0. Following preincubation with trypsin, the crude diabetic alpha 2-macroglobulin displayed a single isoelectric form (pI value of 5.3-5.5) and a substantial increase in recovered activity which were both comparable to normal alpha 2-macroglobulin. Purified alpha 2-macroglobulin from both normal and diabetic subjects displayed similar focusing profiles having a distribution of three forms with a pI value of 5.3-5.4 for the principal form. Trypsin-preincubated samples of purified alpha 2-macroglobulin displayed a single form with a pI value of 5.5-6.0. Preincubation of purified normal or diabetic alpha 2-macroglobulin with a plasma preparation devoid of alpha 2-macroglobulin from normal or diabetic plasma resulted in significantly lowered recovery of activity. All of the above studies suggest that the altered focusing properties observed for crude diabetic alpha 2-macroglobulin are due to components of the plasma rather than to alpha 2-macroglobulin itself.

In man, epinephrine induces increases in plasma levels of glucagon, a lipolytic and hyperglycemic hormone. To determine glucagon's contribution to this hyperglycemia and lipolysis, the effects of inhibition of pancreatic alpha-cell responses to epinephrine were investigated with somatostatin and adrenergic receptor blockade. To avoid ambiguities that might result from concomitant changes in endogenous insulin secretion, these studies were performed in juvenile-type, insulin-deficient diabetic subjects. Compared with normal subjects, the diabetics had excessive glucagon responses to epinephrine, which had been infused to attain circulating levels within the range found in man in severe stress. Both somatostatin and propranolol completely prevented glucagon responses and diminished the glycemic response to epinephrine by 40 to 50 per cent. Free fatty acid responses to epinephrine were completely prevented by propranolol but unaffected with somatostatin. Phentolamine had no effect on glucose, free fatty acid, or glucagon responses to epinephrine. These studies demonstrate that epinephrine, via a beta-adrenergic receptor mechanism, causes excessive plasma glucagon elevation in human diabetes mellitus and indicate that this hyperglucagonemia participates in the hyperglycemic, but not the lipolytic, response to epinephrine. Catecholamine-induced hyperglucagonemia may thus provide an additional explantation for the deterioration in carbohydrate tolerance associated with stress.

The glucagon response to hypoglycemia, which fulfills a primary role toward restoring the plasma glucose level, is blunted or absent in most patients with type I diabetes. To identify predictive factors for this abnormality and for the capability of glycemic counterregulation, we investigated the relationship between the duration of diabetes and glucagon and glucose responses to insulin-induced hypoglycemia. In 18 type I diabetic patients with 1 through 28 years of disease who had no detectable autonomic neuropathy, individual glucagon increments after insulin hypoglycemia were inversely correlated with the duration of disease (r = -.53, P < .025). Patients with disease for ten or fewer years showed a glucagon rise that was lower than in controls but significantly higher than in patients with a duration of more than ten years. The plasma glucose rise after the nadir correlated with peak glucagon increments (r = .60, P < .01); eight of the nine patients with glycemic increments comparable to normals had had diabetes for ten years or less. Thus, having diabetes for more than ten years implied that not only were glucagon responses to insulin hypoglycemia severely compromised but also that the abrupt restoration of plasma glucose levels was impaired. These findings should be taken into account when establishing goals and modalities for tight metabolic control.

In patients with juvenile-onset diabetes, plasma concentrations of 3',5'-adenosine cyclic monophosphate (cAMP) were significantly lower than those of norman subjects [16 +/- 4 and 24 +/- 7 pmol per milliliter (p less than 0.025), respectively] as determined in this laboratory; whereas there were essentially no differences in plasma levels of 3',5'-guanosine cyclic monophosphate (cGMP). Because cAMP inhibits cell growth and cGMP stimulates it, these findings may represent an important factor in the atherosclerotic and obliterative angiopathies of diabetic individuals. We observed that cyclic nucleotide values were the same whether or not the subjects were receiving insulin. Those given insulin plus enough glucose to maintain hyperglycemia revealed modest elevations in cyclic nucleotide levels. Thus, the ratio of cAMP to cGMP, abnormally low in juvenile-onset diabetes, is relatively independent of short-term variations in plasma levels of either glucose of insulin.

Certain aspects of the chronic complications of diabetes suggest that, with time, the abnormal metabolic milieu leads to irreversible changes in some cell populations. Since we have previously observed that high glucose concentrations induce an increase in single strand breaks in the DNA of cultured human endothelial cells, we have investigated whether the same abnormality occurs in cells derived from the in vivo diabetic environment. Peripheral blood lymphocytes obtained from 21 type I diabetic patients and age- and sex-matched controls were tested for rate of unwinding in alkali (a measure of DNA single strand breaks). The patients were subdivided into two groups on the basis of glycohemoglobin values above or below 9%. The group with glycohemoglobin values of 12.9 +/- 2.4% (mean +/- SD), but not the group with glycohemoglobin values of 7.4 +/- 1.5%, showed accelerated unwinding of lymphocyte DNA when compared to controls (P less than 0.01). These studies suggest that poorly controlled diabetes may result in DNA lesions, whose impact on long-term complications deserves to be investigated.

In an attempt to elucidate the mechanism of action of phenformin, eleven juvenile-onset, insulin-requiring diabetic subjects underwent four different treatment regimens during standard breakfast tests. These four treatments were: control (no insulin or phenformin); insulin alone (15 U regular insulin administered subcutaneously one-half hour before breakfast); phenformin alone (50 mg of the timed-release capsule given twice daily for three days before the study and two and one-half hours before breakfast on the day of study); and phenformin plus insulin (in the amounts and at the times stated above). Phenformin was found to decrease postprandial hyperglycaemia significantly when compared with control values, and its addition to insulin further decreased the postprandial glucose rise below that found with insulin alone (p less than 0.005). These effects were associated with a reduction in early (30-min) postprandial hyperglucagonaemia (p less than 0.05). Triglyceride levels, gastrin secretion, growth hormone levels, and increments of alpha-amino nitrogen were not affected by phenformin. Thls, suppression of postprandial hyperglucagonaemia may be an additional mechanism in the reduction of postprandial hyperglycaemia after phenformin.