1. Whether an alteration in cell membrane cation transport after exposure to insulin and angiotensin II (two important growth promoters that have been shown to be involved in the pathogenesis of atherosclerosis and hypertension) is present in cells from non-insulin-dependent diabetes patients with microalbuminuria, a known risk factor for cardiovascular and renal disease, is unknown. We therefore examined intracellular pH and calcium changes after acute exposure to insulin and angiotensin II in cultured skin fibroblasts from eight non-insulin-dependent diabetes patients with and eight others without microalbuminuria and from a group of seven matched, normal control subjects. 2. Cultured fibroblasts were loaded with 2′,7′-bis (2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester or fura 2-acetoxymethyl ester for continuous monitoring of intracellular pH and free calcium concentrations respectively. 3. In quiescent growth-arrested cells, both intracellular pH and free calcium concentrations were similar in the three groups of subjects. Acutely, insulin induced a gradual alkalinization in all groups of patients. The pH increase was significantly greater in non-insulin-dependent diabetes mellitus patients with microalbuminuria (ΔpH +0.24 ± 0.04 pH units) than in normoalbuminuric patients with non-insulin-dependent diabetes mellitus (0.08 ± 0.02; P < 0.01) and normal control subjects (0.05 ± 0.01; P < 0.01). Although the alkalinizing effect of angiotensin II was smaller than that obtained by insulin, intracellular pH increase after angiotensin addition was more pronounced in non-insulin-dependent diabetes mellitus patients with microalbuminuria (ΔpH ± 0.14 ± 0.04 pH units) than in those without (0.08 ± 0.02; P < 0.01) and in normal control subjects (0.02 ± 0.02; P < 0.01). That the increase in intracellular pH was mediated by the sodium—hydrogen antiport was demonstrated by its dependence on the presence of sodium in the medium and its inhibition by amiloride. Whereas insulin addition did not evoke any significant increase in intracellular free calcium levels in fibroblasts from the three groups studied, angiotensin II evoked a fast and transient rise in intracellular free calcium that was higher in fibroblasts from microalbuminuric patients with non-insulin-dependent diabetes mellitus than in cells from normoalbuminuric patients with non-insulin-dependent diabetes mellitus and control subjects. In the whole population of patients with non-insulin-dependent diabetes mellitus, the increase in intracellular pH after exposure to angiotensin II was positively correlated with intracellular free calcium increase ( r = 0.53; P < 0.05), suggesting a possible role of intracellular free calcium levels in the activation of the sodium—hydrogen antiport. 4. In conclusion, we have described an association between increased agonist-induced responsiveness of sodium—hydrogen antiport activity and the presence of microalbuminuria in patients with non-insulin-dependent diabetes mellitus. This increased responsiveness, persisting in cultured fibroblasts after several passages in vitro , suggests that in vitro phenotypic characteristics of fibroblasts are likely to be genetically determined and to be, at least in part, independent of the degree of metabolic control in vivo.

1. To evaluate tubular damage in diabetic patients, we measured the 24 h urinary excretion of five enzymes ( N -acetyl-β-D-glucosaminidase, γ-glutamyl transpeptidase, dipeptidyl aminopeptidase IV, alanine aminopeptidase and alkaline phosphatase) that originate in renal proximal tubular cells. 2. Studies were performed on 118 non-insulin-dependent diabetic patients, 59 non-diabetic patients with chronic renal disease and 47 normal control subjects. First, the correlation between renal function, glycaemic control and urinary enzyme excretion was investigated. Secondly, the subjects were treated by controlled diet therapy to assess the effects of better glycaemic control on urinary enzyme excretion. 3. Regardless of a diabetic or non-diabetic cause of renal dysfunction, all of the five enzymes showed abnormal urinary excretion in patients with renal insufficiency (serum creatinine concentration < 2.0 mg/dl). In diabetic patients, however, an increase in N -acetyl-β-D-glucosaminidase excretion and a decrease in γ-glutamyl transpeptidase excretion were noted even in those who had no signs of renal dysfunction, including microalbuminuria. Moreover, the excretion of these two enzymes had a higher degree of correlation with glycaemic control and renal function than did that of the other three enzymes. Multiple regression analysis revealed that excretion of N -acetyl-β-D-glucosamindase is best correlated with urinary protein ( r 2 = 0.35), whereas excretion of γ-glutamyl transpeptidase is closely associated with glomerular filtration rate ( r 2 = 0.33). 4. In diabetic patients, diet therapy improved glycaemic control but had no effects on renal function, microalbumin excretion and β 2 -microglobulin excretion. In contrast, abnormal N -acetyl-β-D-glucos-aminidase and γ-glutamyl transpeptidase excretion were both significantly improved in diabetic patients without overt renal dysfunction. However, in non-diabetic patients and diabetic patients with renal insufficiency, there were no changes in urinary enzyme excretion. 5. These results suggest that there is reversible tubular damage in the early stages of diabetic nephropathy. Measurement of urinary excretion of N -acetyl-β-D-glucosaminidase and γ-glutamyl transpeptidase may be especially useful in the detection of this tubular dysfunction.

1. Disturbances of sodium and water homoeostasis may contribute to the close association between diabetes, hypertension and proteinuria. We therefore studied the patterns of two natriuretic hormones, plasma atrial natriuretic peptide and urinary dopamine, in 165 Chinese patients with non-insulin-dependent diabetes mellitus controlled by diet or oral hypoglycaemic agents on two occasions over a 6-week period. Patients were divided into three groups based on the mean value of two 24 h urinary albumin excretion measurements. In group 1, 88 patients had normoalbuminuria (urinary albumin excretion ≤ 30 mg/day), in group 2, 48 patients had microalbuminuria (urinary albumin excretion between 30 and 300 mg/day), and in group 3, 29 patients had macroalbuminuria (urinary albumin excretion ≥ 300 mg/day). 2. The supine systolic blood pressure (mean ± sd ) was higher in patients with abnormal albuminuria (group 1: 140.9 ± 27.4 mmHg; group 2: 158.1 ± 26.4 mmHg; group 3: 166.7 ± 23.9 mmHg; F = 13.1, P <0.001, analysis of variance). Urinary sodium output was similar in these three groups of patients. The geometric means (anti-logarithm of 95% confidence interval logarithm) of plasma atrial natriuretic peptide concentrations increased with increasing proteinuria [group 1: 33.3 (29.9–37.1) pg/ml; group 2: 39.1 (34.2–44.6) pg/ml; group 3: 50 (38.6–54.7) pg/ml; F = 4.24, P <0.01; analysis of variance], whereas those of urinary dopamine output were related inversely to proteinuria [group 1: 1291.7 (1167.2–1437.0) nmol/day; group 2: 1142.3 (975.9–1337.2) nmol/day; group 3: 982.7 (775.7–1245) nmol/day; F = 3.10, P <0.05, analysis of variance]. Using stepwise multiple regression analysis, plasma atrial natriuretic peptide concentration ( r 2 = 0.31, F = 56.2, P <0.001) was associated with supine systolic blood pressure (β = 0.56, P <0.001), which was ( r 2 = 0.38, P <0.001) related to urinary albumin excretion (β = 0.23, P <0.001). In patients with urinary albumin excretion > 30 mg/day, plasma atrial natriuretic peptide concentration was negatively correlated with urinary dopamine output ( r = −0.25, P <0.02). 3. Based on these observations, we hypothesize that, in patients with non-insulin-dependent diabetes mellitus, defective dopamine mobilization in the renal tubules may cause impaired natriuresis with increased blood pressure. A compensatory rise in the plasma atrial natriuretic peptide concentration may then contribute to the development of abnormal albuminuria.

1. Some non-insulin-dependent (type II) diabetic patients show albuminuria without arterial hypertension. In these patients, angiotensin-converting enzyme inhibitors reduce urinary albumin excretion without producing any changes in systemic blood pressure and renal haemodynamics. However, up to now it has not been clear whether these favourable renal effects are specifically related to angiotensin-converting enzyme inhibition or not. 2. Twelve type II diabetic outpatients with persistent macroalbuminuria (greater than 300 mg/daily on at least three consecutive occasions), without any other signs of renal disease and whose blood pressure was persistently below 140/90 mmHg, were studied. 3. In a randomized sequence and in a double-blind fashion, after a 2-month run-in period, patients were allocated to receive 5 mg of enalapril or 50 mg of atenolol daily for the next 6 months. At the end of this first period and after 6 months on placebo in a cross-over fashion, active treatment was replicated. Blood pressure and urinary albumin excretion were measured every 2 months, whereas the other variables studied were determined at the end of each period. 4. Kidney function and blood pressure did not change significantly, whereas albuminuria decreased significantly, after both of the drugs. 5. These data suggest that the inhibition of tissue angiotensin formation and the consequent reduction in glomerular permeability, rather than changes in renal and systemic haemodynamics, are the common mechanisms by which both enalapril and atenolol decreased albuminuria in our patients.

1. A study on seven Caucasian glucose-tolerant women with previous gestational diabetes and seven matched control subjects is presented. The insulin response to oral glucose, insulin sensitivity and fasting glucose production rates were measured by using a 75 g oral glucose tolerance test, an insulin tolerance test and a non-radioactive tracer, [6,6- 2 H]glucose, respectively. 2. Fasting plasma glucose levels were similar between the women with previous gestational diabetes and the control subjects (4.8 ± 0.3 versus 4.7 ± 0.2 mmol/l), as were fasting plasma insulin levels (median 4 m-units/l, range 1–13 m-units/l versus median 4 m-units/l, range 1–24 m-units/l). After oral glucose the 60 min plasma glucose levels in the women with previous gestational diabetes were significantly higher (8.5 ± 0.6 versus 6.7 ± 0.8 mmol/l, P < 0.05), whereas the plasma insulin level was significantly lower at both 30 min (median 23 m-units/l, range 4–47 m-units/l versus median 55 m-units/l, range 23–100 m-units/l, P < 0.02) and at 60 min (median 23 m-units/l, range 4–43 m-units/l versus median 60 m-units/l, range 16–126 m-units/l, P < 0.02). 3. Insulin sensitivity, expressed as the slope of the regression line of plasma glucose level against time after intravenous infusion of insulin (0.05 unit/kg), was similar in the women with previous gestational diabetes and the control subjects (mean slope, −0.17 ± 0.01 versus −0.17 ± 0.01). 4. Fasting glucose production rates were similar in the women with previous gestational diabetes and the control subjects (2.2 ± 0.3 versus 1.9 ± 0.1 mg min −1 kg −1 ). 5. Women with previous gestational diabetes, a group at risk of future non-insulin-dependent diabetes, have abnormalities of insulin release at a time when insulin sensitivity and fasting glucose production are normal.

1. DNA insertions in the 5′-flanking region of the insulin gene have been studied by Southern-blot-hybridization techniques using a cloned gene probe in 159 unrelated Caucasians. 2. Subjects homozygous for a large DNA insertion at this locus were found to have a higher risk of concurrence of non-insulin-dependent diabetes (NIDD) than controls ( P < 0.01). 3. Analysis of a single large NIDD pedigree ( n = 67 in four generations; 11 diabetic subjects) showed no linkage between the size of DNA inserts and diabetes (Lod score = −5.7), suggesting a separate diabetogenic locus (or loci) in this family.