Effect of intensive glycemic control on microalbuminuria in type 2 diabetes. Veterans Affairs Cooperative Study on Glycemic Control and Complications in Type 2 Diabetes Feasibility Trial Investigators.

OBJECTIVE: Microalbuminuria can reflect the progress of microvascular complications and may be predictive of macrovascular disease in type 2 diabetes. The effect of intensive glycemic control on microalbuminuria in patients in the U.S. who have had type 2 diabetes for several years has not previously been evaluated. RESEARCH DESIGN AND METHODS: We randomly assigned 153 male patients to either intensive treatment (INT) (goal HbA(1c) 7.1%) or to standard treatment (ST) (goal HbA(1c) 9.1%; P = 0.001), and data were obtained during a 2-year period. Mean duration of known diabetes was 8 years, mean age of the patients was 60 years, and patients were well matched at baseline. We obtained 3-h urine samples for each patient at baseline and annually and defined microalbuminuria as an albumin:creatinine ratio of 0.03-0.30. All patients were treated with insulin and received instructions regarding diet and exercise. Hypertension and dyslipidemia were treated with similar goals in each group. RESULTS: A total of 38% of patients had microalbuminuria at entry and were evenly assigned to both treatment groups. INT retarded the progression of microalbuminuria during the 2-year period: the changes in albumin:creatinine ratio from baseline to 2 years of INT versus ST were 0.045 vs. 0.141, respectively (P = 0.046). Retardation of progressive urinary albumin excretion was most pronounced in those patients who entered the study with microalbuminuria and were randomized to INT. Patients entering with microalbuminuria had a deterioration in creatinine clearance at 2 years regardless of the intensity of glycemic control. In the group entering without microalbuminuria, the subgroup receiving ST had a lower percentage of patients with a macrovascular event (17%) than the subgroup receiving INT (36%) (P = 0.03). Use of ACE inhibitors or calcium-channel blockers was similarly distributed among the groups. CONCLUSIONS: Intensive glycemic control retards microalbuminuria in patients who have had type 2 diabetes for several years but may not lessen the progressive deterioration of glomerular function. Increases in macrovascular event rates in the subgroup entering without albuminuria who received INT remain unexplained but could reflect early worsening, as observed with microvascular disease in the Diabetes Control and Complications Trial.

Two years of intensive glycemic control and left ventricular function in the Veterans Affairs Cooperative Study in Type 2 Diabetes Mellitus (VA CSDM).

OBJECTIVE: The Veterans Affairs Cooperative Study in Type 2 Diabetes Mellitus (VA CSDM) was a multicenter randomized prospective study of 153 male type 2 diabetic patients to assess the ability to sustain clinically significant glycemic separation between intensive and standard treatment arms. A trend toward an excess of combined cardiovascular events in the intensive treatment arm of this trial was reported earlier. The present analysis was done to evaluate the effect of 2 years of intensive glycemic control on the left ventricular (LV) function. RESEARCH DESIGN AND METHODS: The patients were randomized to intensive step treatment with insulin alone or with sulfonylurea (intensive treatment arm [INT], n = 75) or to standard once-daily insulin injection (standard treatment arm [STD], n = 78) treatment. A total of 136 patients (standard treatment arm [STD], n = 70; INT, n = 66) had radionuclide ventriculography at entry and at 24 months for the assessment of LV function. RESULTS: There was no difference in the mean LV ejection fraction (at entry: STD 57.1+/-9.51%; INT 58.1+/-8.7%; at 24 months: STD 57.3+/-10.8%, INT 59.5+/-10.7%), peak filling rate (at entry: STD 2.6+/-0.7 end diastolic volume per second, INT 2.4+/-0.8 end diastolic volume per second; at 24 months: STD 2.7+/-1.0 end diastolic volume per second, INT 2.5+/-0.7 end diastolic volume per second), or time to peak filling rate (at entry: STD 195.3+/-69.5 ms, INT 185.6 +/-62.4 ms; at 24 months: STD 182.6+/-64.8 ms, INT 179.2+/-61.2 ms) between the 2 treatment arms. A subgroup analysis of 104 patients (STD, n = 53; INT, n = 51) that omitted individuals with intervening cardiac events/revascularization or a change in cardioactive medications also showed no difference in the LV function at entry and at 24 months between the 2 groups. Abnormal LV ejection fraction at baseline predicted cardiac events (interval between cardiac beats [RR] = 2.5). CONCLUSIONS: Two years of intensive glycemic control does not affect the LV systolic or diastolic function in patients with type 2 diabetes.

Ethnic differences in the glycemic response to exogenous insulin treatment in the Veterans Affairs Cooperative Study in Type 2 Diabetes Mellitus (VA CSDM).

OBJECTIVE: The Veterans Affairs Cooperative Study in Type 2 Diabetes Mellitus was conducted in NIDDM patients to determine if a significant difference in HbA1c could be achieved between groups receiving standard and intensive treatment. We observed differences in the response to exogenous insulin between African-Americans and other intensively treated patients. Therefore, we assessed the variations of response and correlated factors that might explain such differences. RESEARCH DESIGN AND METHODS: One hundred fifty-three men aged 40-69 years with NIDDM for < or = 15 years were randomized to either the standard therapy (n = 78) or the intensive therapy (n = 75) arm. Of the 75 patients in the intensive therapy group, 57 completed the study on insulin therapy alone. Of these, 18 were African-Americans and 39 were non-African-Americans. We conducted an analysis of the data collected to determine differences in baseline characteristics, glycemic response, insulin requirement, body weight, exercise, and basal C-peptide level, factors that may explain a difference in response to insulin therapy. RESULTS: Glycemic control improved in all patients with intensive insulin therapy. African-Americans achieved a greater improvement in HbA1c compared with non-African-Americans with a similar increment in insulin. This difference could not be explained by differences in body weight, activity, concomitant use of other medicines, or insulin-secretory capacity of the pancreas. CONCLUSIONS: We conclude that ethnic differences may exist in the response to insulin therapy. A knowledge of such differences may aid in achieving good glycemic control, especially since minorities have a greater prevalence of and burden from the microvascular complications of diabetes.

Response to intensive therapy steps and to glipizide dose in combination with insulin in type 2 diabetes. VA feasibility study on glycemic control and complications (VA CSDM).

OBJECTIVE: The feasibility study for the VA Cooperative Study on Glycemic Control and Complications in Type 2 Diabetes (VA CSDM) prospectively studied 153 insulin-requiring type 2 diabetes patients, randomized between an intensively treated arm and a standard treatment arm during a mean follow-up of 27 months. The glycemic response to each of the progressive, sequential phases of insulin treatment was assessed, along with the incidence of hypoglycemic reactions and the relative efficacy of different doses of glipizide in combination with fixed doses of insulin. RESEARCH DESIGN AND METHODS: Five medical centers participated; half of the patients were assigned to the intensive treatment arm aiming for normal HbA1c levels. Age of patients was 60 +/- 6 years, duration of diabetes 8 +/- 3 years, and BMI 30.7 +/- 4 kg/m2. A four-step management technique was used, with patients moving to the next step if the operational goals were not met: Phase I, evening intermediate or long-acting insulin; phase II, added day-time glipizide; phase III, two injections of insulin alone; and phase IV, multiple daily insulin injections. Home glucose monitoring measurements were done twice daily and at 3:00 A.M. once a week. Hypoglycemic reactions and home glucose monitoring results were recorded and counted in each of the treatment phases. RESULTS: Baseline HbA1c was 9.3 +/- 1.8%, and fasting plus serum glucose was 11.4 +/- 3.3 mmol/1. Fasting serum glucose fell to near normal in phase I, and remained so in the other treatment phases. An HbA1c separation of 2.1% between the arms was maintained during the course of the study, while the intensive arm kept HbA1c levels below 7.3% (P = 0.001). Most of the decrease in HbA1c occurred with one injection of insulin alone (phase I, -1.4%) or adding day-time glipizide (phase II, -1.9% compared with baseline). HbA1c did not decrease further after substituting two injections of insulin alone, with twice the insulin dose. Multiple daily injections resulted in an additional HbA1c fall (-2.4% compared with baseline). However, two-thirds of the patients were still on one or two injections a day at the end of the study. Changes in home glucose monitoring levels paralleled those of the HbA1c, as did the increments in number of reported hypoglycemic reactions, virtually all either "mild" or "moderate" in character. For the combination of glipizide and insulin (phase II), the only significant effect was obtained with daily doses up to 10 mg a day; there were no significant additional benefits with up to fourfold higher daily doses, and HbA1c levels had an upward trend with doses > 20 mg/day. CONCLUSIONS: A simple regime of a single injection of insulin, alone or with glipizide, seemed sufficient to obtain clinically acceptable levels of HbA1c for most obese, insulin-requiring type 2 diabetes patients. Further decrease of HbA1c demanded multiple daily injections at the expense of doubling the insulin dose and the rate of hypoglycemic events. In combination therapy, doses of glipizide > 20 mg/day offered no additional benefit.

Effects of sulfate deprivation on the production of chondroitin/dermatan sulfate by cultures of skin fibroblasts from normal and diabetic individuals.

Human skin fibroblast monolayer cultures from two normal men, three Type I diabetic men, and one Type I diabetic woman were incubated with [3H]glucosamine in the presence of diminished concentrations of sulfate. Although total synthesis of [3H]chondroitin/dermatan glycosaminoglycans varied somewhat between cell lines, glycosaminoglycan production was not affected within any line when sulfate levels were decreased from 0.3 mM to 0.06 mM to 0.01 mM to 0 added sulfate. Lowering of sulfate concentrations resulted in diminished sulfation of chondroitin/dermatan in a progressive manner, so that overall sulfation dropped to as low as 19% for one of the lines. Sulfation of chondroitin to form chondroitin 4-sulfate and chondroitin 6-sulfate was progressively and equally affected by decreasing the sulfate concentration in the culture medium. However, sulfation to form dermatan sulfate was preserved to a greater degree, so that the relative proportion of dermatan sulfate to chondroitin sulfate increased. Essentially all the nonsulfated residues were susceptible to chondroitin AC lyase, indicating that little epimerization of glucuronic acid residues to iduronic acid had occurred in the absence of sulfation. These results confirm the previously described dependency of glucuronic/iduronic epimerization on sulfation, and indicate that sulfation of the iduronic acid-containing disaccharide residues of dermatan can take place with sulfate concentrations lower than those needed for 6-sulfation and 4-sulfation of the glucuronic acid-containing disaccharide residues of chondroitin. There were considerable differences among the six fibroblast lines in susceptibility to low sulfate medium and in the proportion of chondroitin 6-sulfate, chondroitin 4-sulfate, and dermatan sulfate. However, there was no pattern of differences between normals and diabetics.

Glycosaminoglycans of bovine aorta endothelial cells: identification and localization by use of a platelet factor 4-fluorescein probe.

We utilized platelet factor 4 (PF4) conjugated to fluorescein to stain the proteoglycans of permeabilized fixed bovine aorta endothelial cells in monolayer culture. Treatment of the monolayers with chondroitin ABC lyase and/or a preparation from Flavobacterium heparinum was used to remove chondroitin sulfate and/or heparan sulfate before staining, with resultant separate identification and partial localization of these glycosaminoglycans. When PF4-fluorescein was utilized with untreated control monolayers, fairly uniform reticular, perinuclear, and cell surface fluorescence was seen. After treatment with chondroitin ABC lyase, fluorescence was retained only on the cell surface. In contrast, treatment with the F. heparinum preparation resulted in the loss of all cell surface fluorescence. Use of both glycosaminoglycan lyases together resulted in loss of essentially all the fluorescence. The cell surface heparan sulfate observed by fluorescence after removal of cell surface chondroitin sulfate appeared to be unevenly distributed, with a heavier accumulation at one pole of each cell. This technique offers a specific method for identification and partial localization of cell surface heparan sulfate.

Production of [3H]hexosamine-labeled proteoglycans by cultures of normal and diabetic skin fibroblasts: dilution of exogenous [3H]glucosamine by endogenous hexosamine from glucose and other sources.

Human skin fibroblast monolayer cultures from two normal men, three Type I diabetic men, and one Type I diabetic woman were incubated with [3H]glucosamine and [35S]-sulfate for varying periods of time. Incorporation of 3H into macromolecules appearing in the medium was linear after approximately 45 min, and incorporation of 35S was linear after approximately 30 min. The amounts of 35S-proteoglycan formed by each of the cultures during 5-h incubations were compared and were found to be fairly similar for the six lines, varying from 0.08 to 0.14 nmol sulfate/microgram DNA. Isolated 3H,35S-glycosaminoglycans were then treated with chondroitin ABC lyase to characterize the location and degree of sulfation. Results indicated a considerable variation in completeness of chondroitin/dermatan sulfation and in proportions of 6-sulfation to 4-sulfation among the various lines. However these variations did not seem to be related to whether the cells were from normals or diabetics. 3H,35S-Labeled disaccharides were isolated and ratios of 3H to 35S determined in order to calculate the [3H]glucosamine dilution by endogenous glucosamine derived from glucose or other sources during the period of incubation. Dilutions varied widely from 160- to 635-fold among the different cell lines, but the variations did not seem to be related to whether the cells were from normals or diabetics.

Sulphation by cultured cells. Cysteine, cysteinesulphinic acid and sulphite as sources for proteoglycan sulphate.

Bovine aortic smooth-muscle cells, bovine aortic endothelial cells, and IMR-90 human embryonic lung fibroblasts were tested to determine their ability to use cysteine or cysteine metabolites as a source of sulphate (SO4). Cells were incubated in SO4-depleted medium containing [3H]glucosamine plus 0.2 mM-cystine, 0.3 mM-cysteinesulphinic acid or 0.3 mM-sulphite (SO3). The [3H]chondroitin sulphate produced by the different cells was found to vary considerably in degree of sulphation under these conditions. One line of smooth-muscle cells utilized cysteine effectively as a SO4 source and thus produced chondroitin sulphate which was highly sulphated. IMR-90 fibroblasts produced partly sulphated chondroitin sulphate under these conditions, while another smooth-muscle cell line could not utilize cysteine, but could utilize cysteinesulphinic acid as a partial SO4 source. In contrast with the above cells, endothelial cells could not use cysteine or cysteinesulphinic acid as a source of SO4 and produced chondroitin with almost no SO4. All of the cells were able to utilize SO3. Incubation of the cells in the SO4-depleted medium containing [35S]cysteine confirmed that only the first line of smooth-muscle cells could convert significant amounts of [35S]cysteine to 35SO4. Furthermore, the addition of 0.4 mM inorganic SO4 did not inhibit the production of SO4 from cysteine by these cells.

Formation of dermatan sulfate by cultured human skin fibroblasts. Effects of sulfate concentration on proportions of dermatan/chondroitin.

[3H,35S]Dermatan/chondroitin sulfate glycosaminoglycans produced during culture of fibroblasts in medium containing varying concentrations of sulfate were tested for their susceptibility to chondroitin ABC lyase and chondroitin AC lyase. Chondroitin ABC lyase completely degraded [3H]hexosamine-labeled and [35S] sulfate-labeled dermatan/chondroitin sulfate to disaccharides. Chondroitin AC lyase treatment of the labeled glycosaminoglycans produced different results. With this enzyme, dermatan/chondroitin sulfate formed at high concentrations of sulfate yielded small glycosaminoglycans and larger oligosaccharides but almost no disaccharide. This indicated that the dermatan/chondroitin sulfate co-polymer contained mostly iduronic acid with only an occasional glucuronic acid. As the medium sulfate concentration was progressively lowered, there was a concomitant increase in the susceptibility to degradation by chondroitin AC lyase. Thus, the labeled glycosaminoglycans formed at the lowest concentration of sulfate yielded small oligosaccharides including substantial amounts of disaccharide. The smaller chondroitin AC lyase-resistant [3H,35S]dermatan/chondroitin sulfate oligosaccharides were analyzed by gel filtration. Results indicated that, in general, the iduronic acid-containing disaccharide residues present in the undersulfated [3H,35S]glycosaminoglycan were sulfated, whereas the glucuronic acid-containing disaccharide residues were non-sulfated. This work confirms earlier reports that there is a relationship between epimerization and sulfation. Moreover, it demonstrates that medium sulfate concentration is critical in determining the proportions of dermatan to chondroitin (iduronic/glucuronic acid) produced by cultured cells.

Glycosaminoglycan production by bovine aortic endothelial cells cultured in sulfate-depleted medium.

Bovine aortic endothelial cells were cultured in medium containing [3H]glucosamine and concentrations of [35S]sulfate ranging from 0.01 to 0.31 mM. While the amount of [3H]hexosamine incorporated into chondroitin sulfate and heparan sulfate was constant, decreasing concentrations of sulfate resulted in lower [35S]sulfate incorporation. Sulfate concentrations greater than 0.11 mM were required for maximal [35S]sulfate incorporation. Chondroitin sulfate was particularly affected so that the sulfate to hexosamine ratio in [3H]chondroitin [35S]sulfate dropped considerably more than the sulfate to hexosamine ratio in [3H] heparan [35S]sulfate. Sulfate concentration had no effect on the ratio of chondroitin 4-sulfate to chondroitin 6-sulfate. The ratios of sulfate to hexosamine in cell-associated glycosaminoglycans were essentially identical with the ratios in media glycosaminoglycans at all sulfate concentrations. DEAE-cellulose chromatography confirmed that sulfation of chondroitin sulfate was particularly sensitive to low sulfate concentrations. While cells incubated in medium containing 0.31 mM sulfate produced chondroitin sulfate which eluted later than heparan sulfate, cells incubated in medium containing less than 0.04 mM sulfate produced chondroitin sulfate which eluted before heparan sulfate and near hyaluronic acid, indicating that many chains were essentially unsulfated. At intermediate concentrations of sulfate, chondroitin sulfate was found in very broad elution patterns suggesting that most did not fit an "all or nothing" mechanism. Heparan sulfate produced at low concentrations of sulfate eluted with narrower elution patterns than chondroitin sulfate, and there was no indication of any "all or nothing" sulfation.

Relationship of fat distribution to glucose tolerance. Results of computed tomography in male participants of the Normative Aging Study.

Computed tomography (CT) scanning was used to assess the relationship of glucose tolerance to fat distribution in men. Three cross sections [chest (including upper arms), abdomen, and thigh] were scanned in 41 men randomly selected from the Normative Aging Study, a longitudinal study of aging. Greater amounts of fat in the upper body and greater ratios of upper-body fat to lower-body fat were significantly correlated with higher 2-h serum glucose levels after adjustment for age and body mass index. In particular, intra-abdominal fat, a feature uniquely measured by CT, was a significant correlate of 2-h glucose. Largely parallel results were obtained when we compared a sample of male diabetic subjects (N = 8) with the male normal subjects from our random sample. This investigation demonstrates that body fat distribution, adjusted for overall degree of obesity, is a significant correlate of glucose tolerance even in a sample unselected for extremes of physique.

Effects of heparan sulfate removal on attachment and reattachment of fibroblasts and endothelial cells.

Human skin fibroblasts and calf aorta endothelial cells were grown as tissue culture monolayers in the presence of [35S]sulfate in order to label the glycosaminoglycan portions of proteoglycans for investigation of their role in cell attachment. The [35S]glycosaminoglycans were then selectively removed from the cell monolayers by the addition of various glycosaminoglycan-degrading enzymes. As previously described, in contrast to trypsin treatment none of these enzymes removed any cells from the culture plates. Incubation with a preparation from Flavobacterium heparinum left only small stubs of [35S]glycosaminoglycans on the cell monolayers, indicating that all the cell-surface proteoheparan [35S]sulfate and proteochondroitin [35S]sulfate was accessible to this enzyme preparation. The treatment did not change the amount or time of incubation with trypsin necessary for release of the cells from the monolayers. Thus, cell attachment was not weakened by removal of heparan sulfate or chondroitin sulfate. In contrast, neither fibroblasts nor endothelial cells in suspension would reattach in the presence of the F. heparinum preparation while reattachment occurred readily in the presence of chondroitin ABC lyase. This provides evidence that heparan sulfate, but not chondroitin sulfate, is involved in the process of cell attachment even though neither is necessary for maintaining attachment.

Age changes in body composition revealed by computed tomography.

Computed tomography scans were taken of 21 middle-aged men (M age 46.3 years) and 20 older men (M age 69.4 years) to measure differences in body composition with age. Overall, the older men weighed 8.2 kg less than the middle-aged men, and this difference was primarily the result of their having less lean tissue. Although fat mass was only slightly less in older men, there were clear distributional differences in fat between the age groups. Total abdomen fat area was similar in both groups, although the subcutaneous portion of the abdomen fat was less in the older men, and they had correspondingly greater intra-abdominal fat. Muscle areas of the leg and arm were significantly less in the older men, as were all lean tissues of the abdomen and chest. Analysis of fat accumulation between muscles of the abdomen and leg indicated fat infiltration into lean tissue in the older men. Causes of this apparent fat redistribution and lean body mass decline with age are presently unknown.

Assessment of abdominal fat content by computed tomography.

Computed tomography (CT) produces thin cross-sectional radiographs that may prove very useful in body composition research. CT images of the abdomen allow computerized measurement of total fat area, and also enable the differentiation of subcutaneous fat from intraabdominal fat. The preset investigation examines whether a single CT scan of the abdomen provides an accurate indication of overall abdominal adiposity. Graphs of measurements from seven sequential scans of the abdomen in eight patients showed that rankings of total abdominal area, total fat area, subcutaneous and intraabdominal fat area are relatively consistent no matter which abdominal level is chosen. Correlations of 0.89 to 0.99 between single scans and the average values for all scans show that a single CT image contains the same information on adiposity as a series of scans. These results suggest that future CT studies of body composition can limit radiation exposure by using single scans at different anatomical sites. If only a single scan at one site can be obtained, the level of the umbilicus may be the most useful, because it contains the largest percentage of fat in the body, and best allows differentiation of intraabdominal from subcutaneous fat.

Removal of glycosaminoglycans from cultures of human skin fibroblasts.

Early-passage human skin fibroblasts were grown as monolayers for 2-3 days in minimum essential medium containing [35S]sulphate, [3H]glucosamine, [3H]fucose, [3H]proline or [3H]leucine to label proteoglycans, glycoproteins or collagen and other proteins. A crude enzyme preparation obtained from a supernatant from sonicated freeze-dried Flavobacter heparinum was added to the cell monolayers. This treatment removed most of the 35S-labelled glycosaminoglycans, with no appreciable removal of the 3H-labelled proteins or 3H-labelled glycoproteins. The cells remained attached and viable as a monolayer. The formation of 35S-labelled glycosaminoglycans was examined after pretreating cultures with crude F. heparinum enzyme, followed by addition of fresh growth medium containing [35S]sulphate. The F. heparinum enzyme did not significantly alter the amount or type of 35S-labelled glycosaminoglycans produced. Thus F. heparinum enzyme can be used to provide cultured-cell monolayers depleted of surface glycosaminoglycans. These cells remain attached, viable and subsequently synthesize normal amounts and type of glycosaminoglycans.

Studies of cultured human fibroblasts in diabetes mellitus: changes in heparan sulfate.

The incorporation of [35S]sulfate into glycosaminoglycans was studied in cultures of normal and diabetic skin fibroblasts. Heparan sulfate was determined by column chromatography after enzymatic degradation of chondroitin sulfates and dermatan sulfate by chondroitinase ABE. Cultured skin fibroblasts from both insulin-dependent and noninsulin-dependent diabetics were found to have increased proportions of heparan sulfate in the media relative to the other sulfated glycosaminoglycans.

Tumor hypoglycemia: deficient splanchnic glucose output and deficient glucagon secretion.

Fasting hypoglycemia occurred in a patient with a histologically benign mesothelioma; the serum insulin was low (2-4 muU./ml.), as was the glucose utilization rate. Splanchnic glucose output was markedly decreased on direct measurement (21 mg./min.; normal: 108-180 mg./min.). Splanchnic uptake of gluconeogenic substrates plasma glucagon was low normal during hypoglycemia and responded poorly to oral and intravenous alanine. The nonsuppressible insulin-like (NSILA-s) and somatomedin-like activities of the serum were not elevated, and the tumor did not release insulin-like activity on incubation nor did it contain somatostatin. The marked decrease in splanchnic glucose output was the principal cause of hypoglycemia, was associated with an apparent decrease in glycogenolysis, and was at least partly due to deficient glucagon secretion. The relationship of the tumor to these defects is unclear. The tumor may have secreted an unknown insulin-like material affecting primarily the liver and/or pancreatic alpha cell. The approach used here may serve as a paradigm for the analysis of hypoglycemia not caused by excessive insulin.