Chronologic and physiologic age affect replicative life-span of fibroblasts from diabetic, prediabetic, and normal donors.

Cultured skin fibroblasts from subjects with clinically apparent diabetes mellitus and from subjects genetically predisposed to diabetes have a replicative lifespan that is inversely related to donor age. Fibroblasts from carefully defined normal subjects not predisposed to diabetes fail to show this correlation. The data support the idea that physiologic status of the tissue donor is a more precise determinant of fibroblast replicative lifespan than chronologic age.

Insulin release is glucose anomeric specific in the human.

The alpha-glucose anomer produces a greater insulin release than beta-glucose in various animal models. These glucose anomers were dissolved rapidly and administered intravenously to human volunteers at a high dose (0.5 g/kg) over a 3-min period or a low dose (3.5 g) over a 20-s period. Blood samples were obtained at frequent time intervals for measurement of whole blood glucose (ferricyanide), plasma glucose (beta-glucose oxidase) and serum immunoreactive insulin. The high-dose infusion test showed no differences between the anomers of either blood glucose or serum insulin levels. However, at the lower dose, the alpha-glucose anomer stimulated a significantly greater insulin release than did beta-glucose. It is concluded that the alpha-glucose anomer stimulates a greater insulin release than the beta-glucose anomer in human subjects at low but not at high doses intravenously and that this response is not apparently related to approximations of the degree of mutarotation. These results suggest that a steric specific glucose receptor site exists on the beta-cell as a rapid insulin release trigger, although the alpha-anomer does not exclusively produce this stimulation.

Diffusion of glucose, insulin, inulin, and Evans blue protein into thoracic duct lymph of man.

Immunoreactive insulin, like inulin, quickly equilibrates with interstitial fluid, as evidenced by recovery in thoracic duct lymph in man. Insulin-like activity not accounted for by immunoreactive insulin behaves as a large protein and is confined to the vascular compartment.

Elevated serum human growth hormone and decreased serum insulin in prediabetic males after intravenous tolbutamide and glucose.

Serum human growth hormone (HGH), serum immunoreactive insulin (IRI), plasma free fatty acids, and blood glucose were measured during intravenous glucose and intravenous tolbutamide tolerance tests in 13 normal and 13 prediabetic (offspring of two diabetic parents) males, closely matched for weight and age. Only prediabetics with normal glucose tolerance during oral, intravenous, and cortisone-primed glucose tolerance tests were evaluated. Mean serum HGH levels were significantly higher in prediabetics in response to intravenous tolbutamide and at the end of the 3-hr intravenous glucose tolerance tests (IVGTT). This is interpreted as a hyperresponsiveness of the growth hormone-releasing mechanisms in prediabetic subjects. The insulin response during the first 10 min of an IVGTT was significantly reduced in prediabetic males as compared to normal controls, whereas the insulin response to intravenous tolbutamide was not significantly different at the same time intervals in the same subjects.It appears, therefore, that measuring IRI during an IVGTT can be valuable in detecting the earliest signs of diabetes even before any disturbance of blood glucose homeostasis is seen. The possibility that growth hormone hypersecretion in prediabetics might play a role in the pathogenesis of human diabetes mellitus is discussed.

Amino acid balance across tissues of the forearm in postabsorptive man. Effects of insulin at two dose levels.

Amino acid balance across skeletal muscle and across subcutaneous adipose tissue plus skin of the forearm has been quantified in postabsorptive man before and after insulin infusion into the brachial artery. Skeletal muscle released significant amounts of alpha amino nitrogen after an overnight fast. Most individual amino acids were released. Alanine output was by far the greatest. The pattern of release probably reflects both the composition of muscle protein undergoing degradation and de novo synthesis of alanine by transamination. A significant correlation was observed between the extent of release of each amino acid and its ambient arterial concentration. Elevation of forearm insulin in eight subjects from postabsorptive (12 muU/ml) to high physiologic levels (157 muU/ml) in addition to stimulating muscle glucose uptake blocked muscle alpha amino nitrogen release by 74%. Consistent declines in output were seen for leucine, isoleucine, tyrosine, phenylalanine, threonine, glycine, and alpha-aminobutyric acid. Alanine output was insignificantly affected. Doubling forearm insulin levels (from 10 to 20 muU/ml) in eight subjects increased muscle glucose uptake in three and blocked alpha amino nitrogen output in two although both effects were seen concurrently in only one subject. Changes in net amino acid balance after insulin could be accounted for by increased transport of amino acids into muscle cells or retardation of their exit. It is likely that ambient arterial amino acid concentrations are established and maintained primarily by the extent of muscle amino acid release. The individual amino acids whose outputs from forearm muscle decline after forearm insulinization correspond well with those whose levels fall systematically after systemic insulinization. This suggests that declines in amino acid levels after systemic insulinization are due to inhibition of muscle release. Doubling basal insulin approaches the threshold both for blockade of muscle amino acid output and stimulation of glucose uptake, effects which appear to occur independently.

Insulin sensitivity of forearm tissues in prediabetic man.

In genetic prediabetic subjects (the glucose tolerant offspring of two diabetic parents or the identical twin of a known diabetic) serum insulin concentrations after glucose administration are subnormal. Maintenance of glucose tolerance in this setting is apparently paradoxical, suggesting increased tissue insulin sensitivity. Accordingly, forearm tissue insulin sensitivity in nine genetic prediabetic males was compared with that of seven males without familial diabetes. Diabetes was excluded in all subjects by preliminary oral glucose tolerance testing. On the preliminary 3 h oral glucose tolerance test (OGTT) the sum of increments in blood glucose above fasting was greater in prediabetic than in control subjects. Conversely, the sum of increments in serum insulin was subnormal for the first 2 h. The insulin index (the sum of increments in insulin divided by the sum of increments in glucose) was significantly lower in prediabetics throughout the test. High physiologic levels of insulin were produced in the forearm by intrabrachial arterial insulin infusion (100 muU/kg per min for 26 min). Balances of glucose and amino acids across forearm muscle became more positive, as did balances of glucose and free fatty acids across adipose tissue plus skin. There were no differences in response between prediabetic and normal subjects.Hence, the insulin sensitivity of peripheral tissues is normal in genetic prediabetes. Increased tissue insulin sensitivity is not essential to explain coexisting euglycemia and insulinopenia in prediabetes because blood glucose values on the OGTT are, in fact, elevated although still within the range considered normal.

Diabetes mellitus and genetic prediabetes. Decreased replicative capacity of cultured skin fibroblasts.

The idea that the gene(s) that cause diabetes mellitus can be expressed in extrapancreatic cells has been examined by tissue culture techniques. Skin biopsies were obtained from 25 normal subjects (N), 26 overt diabetics (D), 16 of juvenile onset (JOD) and 9 of maturity onset (MOD), and 21 subjects genetically predisposed to diabetes (P) on the basis of maturity-onset diabetes in both parents. Each biopsy was subdivided, multiple skin fragments were explanted in vitro, and several parameters of cellular outgrowth were monitored in primary and secondary cultures until cell division ceased because of senescence. In general, the rank order of growth vigor was N greater than P greater than D although differences were often marginal and statistically significant between N and JOD and(or) MOD. Outgrowth of epithelial cells was more vigorous in N explants in early stages, but later, JOD and MOD cells grew better than those of N. Outgrowth of fibroblast cells from N explants was more vigorous both at early and later stages and required less time to achieve maximum percent outgrowth. In secondary cultures, N cells grew faster than the other three groups so that fewer days elapsed between subcultures but significant differences were only seen between N and one or two of the other groups over some of the first seven subcultures. The onset of cellular senescence occurred earlier in P and JOD cultures both in mean population doublings and calendar time. N cultures had a higher percent surviving clones after picking than MOD, and a shorter recloning time than clones of JOD. The replicative life-spans of cultures (mean population doublings +/- SE) were N = 52.54 +/- 2.24, P = 47.84 +/- 2.43, JOD = 47.12 +/- 2.99, and MOD = 46.40 +/- 4.04, but differences did not reach significance for N vs the other three groups. The data demonstrate that cellular growth is impaired in both JOD and MOD types of cultures and to a generally lesser extent in P cultures. This is consistent with intrinsic genetic defects but the possibility that persistent deleterious effects of in vivo pathophysiology contribute alone or in combination cannot be ruled out. Therefore, the diabetic defect(s) can be expressed in extrapancreatic cells of mesenchymal origin. This system should prove useful in exploring the interplay between genetic and environmental factors in diabetes, the mechanisms(s) of hyperglycemia and other metabolic derangements, and the propensity that affected individuals have to develop degenerative diseases.

Hormone-fuel concentrations in anephric subjects. Effect of hemodialysis (with special reference to amino acids).

Arterial blood concentrations of insulin, glucagon, and various substrates were determined in six anephric subjects in the postabsorptive state and immediately after hemodialysis. Plasma glucose and serum insulin concentrations were normal, and declined during dialysis. Plasma glucagon was elevated and remained unchanged. There was moderate hypertriglyceridemia before dialysis, but this decreased significantly after administration of heparin just before the start of dialysis, and at the end of dialysis was lowered further into the normal range. Comparison of postabsorptive whole blood concentrations of amino acids with those in normal, healthy adults revealed striking differences. Glutamine, proline, citrulline, glycine and both 1- and 3-methyl-histidines were increased, while serine, glutamate, tyrosine, lysine, and branched-chain amino acids were decreased. The glycine/serine ratio was elevated to 300% and tyrosine/phenylalanine ratio was lowered to 60% of normal. To investigate the potential role of blood cells in amino acid transport, the distribution of individual amino acids in plasma and blood cell compartments was studied. Despite a markedly diminished blood cell mass (mean hematocrit, 20.6 +/- 1.4%), there was no significant decrease in the fraction of most amino acids present in the cell compartment, and this was explained by increases of several amino acids in cellular water. None were decreased. Furthermore, during dialysis, whole blood and plasma amino acids declined by approximately 30% and 40%, respectively, whereas no significant change was observed in the cell compartment. Alanine was the only amino acid whose concentration declined in the cells as well as in plasma. The results indicate (a) significant alterations in the concentrations of hormones and substrates in patients on chronic, intermittent hemodialysis; (b) removal of amino acids during hemodialysis, predominantly from the plasma compartment, with no significant change in cell content; and (c) a redistribution of amino acids in plasma and blood cell compartments with increased gradients of most of the amino acids per unit cell water, by mechanism(s) as yet undetermined.

Regulation of hemoglobin AIc formation in human erythrocytes in vitro. Effects of physiologic factors other than glucose.

The formation of hemoglobin AIc was studied in intact human erythrocytes in vitro. Satisfactory methods were developed for maintaining erythrocytes under physiologic conditions for greater than 8 d with less than 10% hemolysis. Hemoglobin AIc levels were determined chromatographically on erythrocyte hemolysates after removal of reversible components by incubation for 6 h at 37 degree C. Hemoglobin AIc concentration was found to increase linearly with time during 8 d of incubation. The rate of formation of hemoglobin AIc increased linearly as glucose concentration was increased from 40 to 1,000 mg/dl. Deoxyhemoglobin was glycosylated twice as rapidly as oxyhemoglobin. The rate of hemoglobin AIc formation was further increased by elevated 2,3-diphosphoglycerate levels, an effect that was most marked with deoxyhemoglobin. We conclude that the nonenzymatic glycosylation of hemoglobin is influenced by factors other than glucose, including oxygen tension and 2,3-diphosphoglycerate levels.

Glucagon levels and metabolic effects in fasting man.

The role of glucagon in the metabolic adaptation to prolonged fasting in man has been examined. Plasma immunoreactive glucagon was determined during 6-wk fasts and during infusion of exogenous glucagon using an assay which minimized nonpancreatic immunoreactivity. Plasma glucagon concentrations rose twofold to a peak on the 3rd day of fasting and then declined thereafter to a level maintained at or above postabsorptive. Insulin concentration declined to a plateau by the 3rd day. Thus a persisting altered relationship of glucagon and insulin concentrations characterized the fasted state. A synergism of low insulin and relative or absolute elevation of glucagon levels is viewed as a hormonal mechanism controlling the rate of hepatic substrate extraction for gluconeogenesis. Glucagon was infused systemically into 4-6 wk fasted subjects at three dose levels. A marked sensitivity of individual plasma free amino acids to the induced elevations of plasma glucagon within the physiologic range was demonstrated. At higher concentrations, equivalent to those present in the portal vein, stimulation of hepatic gluconeogenesis occurred, and the effects on glucose, insulin, and growth hormone levels and on ketone metabolism were induced.

Antibodies to a 64,000 Mr human islet cell antigen precede the clinical onset of insulin-dependent diabetes.

Antibodies in sera from newly diagnosed insulin-dependent diabetes mellitus (IDDM) patients are directed to a human islet cell protein of relative molecular mass (Mr) 64,000. Since IDDM seems to develop after a prodromal period of beta-cell autoimmunity, this study has examined whether 64,000 Mr antibodies could be detected in 14 individuals who subsequently developed IDDM and five first degree relatives who have indications of altered beta-cell function. Sera were screened by immunoprecipitation on total detergent lysates of human islets and positive sera retested on membrane protein preparations. Antibodies to the 64,000 Mr membrane protein were consistently detected in 11/14 IDDM patients, and in all 5 first degree relatives. 10 IDDM patients were already positive in the first samples, obtained 4-91 mo before the clinical onset of IDDM, whereas 1 patient progressed to a high 64,000 Mr immunoreactivity, at a time where a commencement of a decline in beta-cell function was detected. 64,000 Mr antibodies were detected before islet cell cytoplasmic antibodies (ICCA) in two patients. In the control groups of 21 healthy individuals, 36 patients with diseases of the thyroid and 5 SLE patients, the 64,000 Mr antibodies were detected in only one individual, who was a healthy sibling to an IDDM patient. These results suggest that antibodies against the Mr 64,000 human islet protein are an early marker of beta-cell autoimmunity and may be useful to predict a later development of IDDM.

Resting and exercise hyperemic pulsatile arterial blood flow in insulin-dependent diabetic subjects.

Pulsatile arterial blood flow was studied in 20 normal (N), 20 short-term (STIDDM; mean: 5.17 yr), and 20 long-term insulin-dependent diabetic patients (LTIDDM; mean: 14.76 yr) between the ages of 18 and 30 yr with no clinically detectable peripheral vascular disease. Measurements were taken from waveforms obtained noninvasively using an electromagnetic flowmeter at rest and immediately after a 3-min isometric exercise challenge of the right leg. At rest, both groups of diabetics exhibited minute flow values similar to those in the normal group. This was achieved, however, by increased vasodilation in peripheral tissues as indicated by a difference in waveform configuration. Diabetic subjects showed a significantly smaller peak flow, a less steep ascending and descending slope, and a higher minute heart rate than normal controls. After 3 min of isometric exercise, the diabetic groups exhibited significantly less minute flow, flow/pulse, and a more vasodilated flow pattern similar to that recorded at rest. In addition, the LTIDDM group showed significantly less arterial elasticity than N or STIDDM groups as indicated by a shorter propagation time. These findings imply that apparent functional changes in pulsatile arterial blood flow occur early in the time course of diabetes and are independent of duration.

Mean retinal circulation time as determined by fluorescein angiography in normal, prediabetic, and chemical-diabetic subjects.

A preliminary survey has been completed using manual densitometric technics to determine the mean retinal circulation times in groups of normal controls, offspring to two diabetic parents with normal glucose tolerance (prediabetics), and offspring of two diabetic parents with abnormal glucose tolerance (chemical diabetics). Comparisons of the mean retinal circulation time showed differences between the left eye and right eye in prediabetic and chemical diabetic groups and a sex difference in both normals and prediabetics. In addition, both age and per cent ideal body weight were inversely related to the mean retinal circulation time. The levels of fasting serum cholesterol, triglyceride, and growth hormone, in many instances, also appeared to be inversely related to the mean retinal circulation time. Similarly, the degree of glucose tolerance (determined by the area under the glucose curve above baseline) was significantly inversely related to the mean retinal circulation time. The mean retinal circulation time adjusted for per cent ideal weight was analyzed separately for both right eye and left eye, and a significantly shorter mean retinal circulation time was noted, particularly in males, for prediabetics than for normal controls and for chemical diabetics than for both prediabetics and normals. Analysis of the mean retinal circulation time adjusted for age showed similar differences. It is postulated that the genetic prediabetic state with or without glucose intolerance might be associated with significant alterations of mean retinal circulation time independent of age and per cent ideal weight. It is also suggested that a number of potentially meaningful interrelationships between the degree of glucose intolerance and/or hyperlipidemia might exist and should be further quantified.

Dynamics of tolbutamide, glucose, and insulin interrelationships following varying doses of intravenous tolbutamide in normal subjects.

Four healthy adult subjects received intravenous tolbutamide (TOL) at six different doses (twenty-four tests): 0.0625 gm., 0.125 gm., 0.25 gm., 0.5 gm., 1.0 gm. and 1.5 gm. Blood glucose (BG), serum immunoreacctive insulin (IRI) and serum TOL levels were determined before and for 180 minutes after TOL. There was a highly significant correlation of the dose of TOL with the peak IRI (p less than .01), zero to ten minute IRI area (p less than .001), and zero to sixty minute IRI area (p less than .001) and with the decline in BG expressed as zero to sixty minute BG area (p less than .001). Similar significant correlations were observed between levels of TOL and both IRI and BG. At each dose level the IRI response correlated significantly with the BG fall. An additional eighteen subjects received the 1.0 gm. dose. In these, serum TOL levels did not correlate with either BG or IRI. These subjects also received intravenous glucose (0.5 gm. per kilogram body weight). BG levels did not correlate with IRI. However, there were striking correlations between TOL and glucose-stimulated peak IRI (p less than .001), zero to ten minute IRI area (p less than .05). The mean (plus or minus SEM) space of distribution for glucose (G.S.) and tolbutamide (TLS.) was found to be 13.45 plus or minus 0.71 and 6.34 plus or minus 0.31 L., respectively. There was a significant dose-response relationship exists between TOL and IRI. TOL- and glucose-induced IRI secretion dynamics suggest strong similarities between mechanisms of rapid IRI release and/or size of available IRI storage pools.

Serum insulin response to slow-rise glucose infusion in "genetic prediabetics" (offspring of two diabetic parents).

The insulin response to a programed slow-rise glucose infusion designed to mimic the postprandial rise in glucose was studied in five offspring of two diabetic parents (ODP) and seven normal subjects. The ODP had higher mean blood glucose levels towards the end of the infusion and during the postinfusion period. The rates of glucose disappearance calculate during the postinfusion period were comparable in the two groups. Despite the apparent similarity of serum insulin levels of ODP and normal subjects, the amount of insulin secreted per unit of glycemic stimulus was lower in the ODP group. When the glucose infusion test was preceded by an acute load of glucose, similar findings in the insulin secretory dynamics were found in the ODP group. These data suggest that an impairment in insulin secretion exists in ODP when they are challenged by the slow rise of blood glucose achieved by this type of an intravenous glucose infusion.

Autoimmunity to insulin, beta cell dysfunction, and development of insulin-dependent diabetes mellitus.

Circulating insulin autoantibodies (INSAAb) were measured in discordant monozygotic twins, first-degree relatives, and other groups at "high risk" for the development of insulin-dependent diabetes mellitus (IDDM), and these results correlated with both islet cell antibody (ICAb) status and beta cell function. INSAAb were positive in 31.6% (12 of 38) ICAb-positive subjects but in only 3.1% (3 of 97) ICAb-negative subjects (X2 = 22.4; P less than 0.001). Elevated levels of INSAAb tended to correlate with younger age and were observed in individuals irrespective of the prevailing degree of their beta cell function. Eight of 15 subjects detected to be INSAAb positive have thus far progressed to clinical IDDM (X2 = 18.3; P less than 0.001). Thus, autoantibodies reactive with the insulin molecule (1) appear to constitute an additional serologic marker of ongoing autoimmunity and development of IDDM, and (2) may reflect heterogeneity in the pathogenesis of IDDM.

Familial clustering of insulin sensitivity.

This study's objective was to determine whether there is familial clustering of insulin sensitivity (SI) or insulin-independent glucose uptake (SG), which would be evidence that they are genetically determined traits. Outpatients had a 3-h intravenous glucose tolerance test. Nondiabetic individuals (n = 183), ranging in age from 16 to 60 yr, were from 105 families that had 2 parents with non-insulin-dependent diabetes mellitus. Of these families, 62 contributed 1 offspring, 21 contributed 2, 13 contributed 3, 6 contributed 4, and 2 and 1 contributed 5 and 6, respectively. The minimal model of glucose disposal and the glucose and insulin values from the intravenous glucose tolerance tests were used to estimate SI and SG. The intraclass correlation coefficient was used to compare the within-family variability of SI and SG with the respective between-family distributions. The intraclass correlation coefficients were 0.26 (P = 0.008) for SI and 0.081 (P = 0.45) for SG. SI and SG were uncorrelated (r = -0.059, P = 0.42). The intraclass correlation of SI could not be explained by familial clustering of fasting insulin or ideal body weight. Finally, the 10 families with the lowest values of SI had a significantly higher within-sibship variability of SI than the other 33 families (P less than 0.001, F test). SI but not SG showed familial clustering, which is consistent with a polygenic determinant of SI. In addition, a large within-family variability of SI in some families is compatible with a major gene effect with a dominant mode of inheritance.(ABSTRACT TRUNCATED AT 250 WORDS)

Life-table analysis of progression to diabetes of anti-insulin autoantibody-positive relatives of individuals with type I diabetes.

Cytoplasmic islet cell antibody-negative (ICA-; less than 20 Juvenile Diabetes Foundation units, n = 1670) and ICA+ (n = 42) first-degree relatives of type I (insulin-dependent) diabetic individuals were studied for competitive insulin autoantibodies (CIAAs) with a radioassay. Overall, 3.7% of first-degree relatives (64 of 1712) were CIAA+. Of ICA- relatives, 2.7% (45 of 1670) exceeded the upper limit of our normal CIAA range (greater than 39 nU/ml), and 45% (19 of 42) of ICA+ relatives exceeded this normal range. Follow-up serums for repeat CIAA determination have been obtained from 16 of the nondiabetic CIAA+/ICA- individuals (time between samples, 0.4-5.8 yr). Fourteen of these 16 (87%) CIAA+/ICA- relatives were found to still be positive on follow-up, and 2 of the relatives who were positive on the first determination were negative on their follow-up test. With a mean follow-up of approximately 2 yr, 4 of 45 (9%) of the CIAA+/ICA- relatives, 5 of 23 (22%) of the ICA+/CIAA- relatives, and 12 of 19 (63%) of the CIAA+/ICA+ relatives developed diabetes. Life-table analysis indicated that, overall, 53% of CIAA+ relatives become diabetic after 5 yr of follow-up versus 65% of ICA+ relatives. Also by life-table analysis, the predicted risk after 5 yr of follow-up for progression to diabetes is 17% for CIAA+/ICA- relatives, 42% for ICA+/CIAA- relatives, and 77% for CIAA+/ICA+ relatives. The highest rate of progression to diabetes was found in ICA+ relatives with CIAA levels greater than 150 nU/ml (100% projected to be diabetic within 5 yr, P less than .008 vs. ICA+/CIAA- relatives).

Reproducibility and comparative analysis of repeated intravenous and oral glucose tolerance tests.

We have developed a methodology for measuring the reproducibility of the oral glucose tolerance test (OGTT) and the intravenous glucose tolerance test (IVGTT) in normal subjects and in offspring of conjugal diabetic parents. Both groups of subjects revealed more striking correlations of several parameters of blood glucose and insulin secretion between two IVGTTs than between two OGTTs. Employing arbitrary criteria, we calculated a "reproducibility index" as a quantitative measure of blood glucose variability in each subject. No significant difference was found in the reproducibility of OGTT versus IVGTT, nor in normals versus the offspring. Only about 50 per cent of the tests in normals and in the offspring could be considered to be "reproducible." The offspring revealed greater correlations of several parameters, particularly insulin secretion, between the two IVGTTs and between the two OGTTs as compared with the normal group. However, the blood glucose variations tended to be considerably greater in the offspring from one to the other test.

Pre-type I diabetes. Linear loss of beta cell response to intravenous glucose.

Twenty-one intravenous (i.v.) glucose tolerance tests were performed on nine subjects before the onset of overt type I diabetes mellitus. Islet cell antibodies (6 of 9 subjects) and elevated levels of Ia-positive T-lymphocytes (3 of 3 subjects studied) were detected during the prediabetic period. Elevations of fasting blood glucose and peak glucose during oral glucose tolerance tests were not observed until the year before onset of clinically overt diabetes. During the prediabetic period, there was a progressive loss of early-phase insulin release to i.v. glucose (rate of decline, 20-40 microU/ml insulin release/yr; correlation coefficient, 0.9).