Increased Maternal Fasting Proinsulin as a Predictor of Insulin Requirement in Women with Gestational Diabetes

Circulating proinsulin was assessed during a 75g OGTT in 55 pregnant women who fulfilled WHO criteria for impaired glucose tolerance before the 32nd gestational week. Proinsulin was assayed retrospectively using a two-site immunoradiometric assay and immunoreactive insulin by radioimmunoassay. Of the 55 women, 19 required insulin treatment in addition to diet later in pregnancy. Fasting proinsulin concentrations were significantly higher in the 19 women who later required insulin treatment compared with the 36 women treated with diet alone (3.4 ± 0.7 vs 1.8 ± 0.2 pmol l^?1, p < 0.005). There was no difference between the treatment groups of 60 and 120 min proinsulin values during the OGTT. Fasting plasma glucose and immunoreactive insulin were similar in the insulin-treated and diet-treated women and remained similar during the OGTT. No women within the insulin-treated group had a fasting plasma proinsulin value < 1.1 pmol l^?1 in contrast with 12 women in the diet-treated group (p = 0.0123). Ten of the 19 insulin-treated women had a fasting plasma proinsulin > 2.5 pmol l^?1 compared with 8 of the 36 diet-treated women (p = 0.0346). Fasting proinsulin values early in pregnancy have prognostic implications in women with gestational diabetes.     

Insulin prevents adoptive cell transfer of diabetes in the autoimmune non-obese diabetic mouse

Summary Early intensive insulin treatment is thought to improve subsequent Beta-cell function in Type 1 (insulin-dependent) diabetic patients. Prophylactic insulin administration also reduced diabetes incidence in diabetes-prone animals. To study the mechanisms by which these effects occur, we tested the ability of insulin therapy in the model of non-obese-diabetic mice, to prevent the penetration of committed T cells into the islets and subsequent Beta-cell destruction. Sublethally irradiated non-obese-diabetic males of 8 weeks of age were adoptively transferred with splenocytes from diabetic donors and treated with the maximum tolerable dosage of fast-acting insulin (0.5 U, twice daily) until 30 days after cell transfer. Diabetes incidence was compared to control animals injected with the same concentration of insulin diluent. After one month of treatment, the cumulative diabetes frequency was significantly less within the insulin-treated group (4 of 15, 26.6%) than in the control group (15 of 18, 83.3%; p <0.01). Pancreatic histological analysis of insulin-treated animals revealed a lower severity of insulitis and Beta-cell necrosis and a higher percentage of normal islets (46.6±10% vs 2.3±2%, p < 0.01), including five (33%) mice with no lesions. Immunoperoxydase staining of pancreatic sections indicated similar insulin and ganglioside staining of Beta cells from insulin-treated mice and control animals. Insulin-treated mice had comparable pancreatic insulin content to normal mice. Flow cytometry analysis of spleen cell populations indicated that insulin increased the number of Thy1,2⁺ and Lyt-2⁺ T cells. Although an effect at the Beta cell level cannot be definitely excluded, several lines of evidence suggest that insulin may influence the capacity of effector T cells to invade the islets and cause Beta-cell destruction. These effects may have potential interest for future immunointervention trials in Type 1 diabetic patients of recent onset.     

Expression of glycogen synthase and phosphofructokinase in muscle from Type 1 (insulin-dependent) diabetic patients before and after intensive insulin treatment

Summary The aim of the present study was to determine whether short-term appropriate insulinization of Type 1 (insulin-dependent) diabetic patients in longterm poor glycaemic control (HbA_1C>9.5%) was associated with an adaptive regulation of the activity and gene expression of key proteins in muscle glycogen storage and glycolysis: glycogen synthase and phosphofructokinase, respectively. In nine diabetic patients biopsies of quadriceps muscle were taken before and 24-h after intensified insulin therapy and compared to findings in eight control subjects. Subcutaneous injections of rapid acting insulin were given at 3-h intervals to improve glycaemic control in diabetic patients (fasting plasma glucose decreased from 20.8±0.8 to 8.7±0.8 mmol/l whereas fasting serum insulin increased from 59±8 to 173±3 pmol/l). Before intensified insulin therapy, analysis of muscle biopsies from diabetic patients showed a normal total glycogen synthase ctivity but a 48% decrease (p=0.006) in glycogen synthase fractional velocity (0.1 mmol/l glucose 6-phosphate) (FV_0.1) and a 45% increase (p=0.01) in the half-maximal activation constant of glycogen synthase (A_0.5). The activity of phosphofructokinase and the specific mRNA and immunoreactive protein levels of both glycogen synthase and phosphofructokinase were similar in the two groups. The 2.8-fold increase in serum insulin levels and the halving of the plasma glucose level for at least 15 h were associated with a normalization of glycogen synthase fractional activity (FV_0.1) and of the half-maximal activation constant (A_0.5) whereas the enzyme activity of phosphofructokinase and the mRNA and protein levels of both glycogen synthase and phosphofructokinase remained normal. In conclusion: 1) Reduced allosterical activation of glycogen synthase in muscle of Type 1 diabetic patients in poor metabolic control occurs in the presence of normal total activity as well as normal immunoreactive protein mass and mRNA level of glycogen synthase. 2) Changes in serum insulin within the physiological range play no role in the short-term regulation of glycogen synthase mRNA and protein abundance in muscle from Type 1 diabetic patients.     

Increased expression of low-affinity insulin receptor isoform and insulin/insulin-like growth factor-I hybrid receptors in term placenta from insulin-resistant women with gestational hypertension

Summary Gestational hypertension is associated with insulin-resistance; insulin and insulin-like growth factor-1 (IGF-1), acting through their receptors, play a role in the growth of the feto-placental unit. Since both receptors are exposed to the maternal circulation, it has been suggested that maternal metabolic abnormalities might affect placental insulin (HIR) and IGF-1 (IGF-1R) receptors. To clarify this issue, we characterized HIR and IGF-1R in placenta at term from normal women, normoinsulinaemic women with gestational hypertension (NGH), and hyperinsulinaemic women with gestational hypertension (HGH). Insulin binding was decreased in HGH women (B/T 0.12±0.03) compared to control and NGH women (B/T 0.18±0.07, p<0.036; and 0.22±0.5, p<0.009 respectively). Receptor affinity was lower in HGH women (ED₅₀ 0.95±0.32 nmol/l) than control and NGH women (ED₅₀ 0.42±0.19 nmol/l, p<0.01; and 0.40±0.1 nmol/l, p<0.007, respectively), whereas low-affinity Ex11⁺ isoform was higher in HGH women (Ex11⁺ 50±7,%) than in control and NGH women (Ex11⁺ 34±9%, p<0.001; and 39±4%, p<0.01, respectively). Increased expression of Ex11⁺ isoform was correlated with ED₅₀ (r=0.71; p<0.002) and insulinaemia (r=0.70, p<0.002). IGF-I binding was increased in HGH women (B/T 0.17±0.03) compared to control and NGH women (B/T 0.09±0.05, p<0.002; and 0.11±0.03, p<0.002, respectively). IGF-IR affinity was similar in the three groups. The percentage of insulin/IGF-I hybrid receptors was increased in HGH women (85±3%) compared to control and NGH women (68±7%, p<0.001; and 63±9%, p<0.001, respectively), and was positively correlated with insulinaemia (r=0.62, p<0.018), ED₅₀ of insulin binding (r=0.62, p<0.05), and maximal IGF-I binding (r=0.69, p<0.004); whereas it was inversely correlated with maximal insulin binding (r=–0.69, p<0.004). Results provide the first evidence for altered expression of insulin/IGF-I hybrids found in insulin-resistance states.Abbreviations IGF-1 Insulin-like growth factor-1 - IGF-1R IGF-1 receptor - HIR human insulin receptor - OGTT oral glucose tolerance test - HGH hyperinsulinaemia with gestational hypertension - NGH normoinsulinaemia with gestational hypertension - PA-C polyclonal antibody C Drs. H. Valensise, Y. Y. Liu, M. Federici have contributed equally to this work     

A controlled trial of a high fibre,low fat and low sodium diet for mild hypertension in type 2 (non-insulin-dependent) diabetic patients

Summary Fifty hypertensive Type 2 (non-insulin-dependent) diabetic patients were allocated, in a controlled trial, to a treatment diet of high fibre, low fat and low sodium composition, or to a control diet by the hospital dietitian. After 3 months treatment, the modified diet-treated group showed a highly significant reduction in mean systolic (180.5±19.0 to 165.0±20.7 mmHg) and diastolic blood pressure (96.6±9.3 to 88.0±10.5 mmHg), accompanied by significant reductions in urinary sodium excretion (183.0±62.1 to 121.7+ 65.8 mmol/day) glycosylated haemoglobin (12.4±3.1 to 10.5±2.9%), weight (74.6s±13.5 to 71.7±12.1 kg) and serum triglyceride levels (p<0.05). The mean values of diastolic pressure (p<0.01), urinary sodium/potassium ratio (p< 0.001), urinary potassium (p<0.01) was significantly reduced at 3 months compared to control. No changes in serum HDL-cholesterol levels were observed. The number of patients with normal blood pressure at 3 months was greater in the modified diet-treated group (ten versus five). Treatment of mild hypertension in diabetic subjects with this form of dietary regimen has a hypotensive response, with improvement in glycaemic control and no side effects. This modified diet may be an attractive alternative to anti-hypertensive drug therapy as a first line treatment.     

Renal insulin-like growth factor I and growth hormone receptor binding in experimental diabetes and after unilateral nephrectomy in the rat

Summary We have measured specific binding of insulin-like growth factor I and growth hormone to renal plasma membranes from control, streptozotocin-diabetic, insulin-treated diabetic, uninephrectomised and combined diabetic-uninephrectomised male Wistar rats. Control, insulin-treated and uninephrectomised rats had similar body weights after 7 days (243±2 g), whereas diabetic and diabetic-uninephrectomised animals were significantly lighter (219±4 and 203±4 g, p<0.05). Blood glucose concentrations were similar in the diabetic and diabetic-uninephrectomised animals (around 26 mmol/l) but significantly lower in the insulin-treated group. Right kidney weight increased by 14% in the control, insulin-treated and sham-nephrectomised animals, by 33% in the diabetic group, 38% in the nephrectomised animals and 60% in the diabetic-nephrectomised group. The renal content of insulin-like growth factor I was similar and stable in the control, insulin-treated and sham-nephrectomised animals (208±14 ng/g wet weight) but rose to a peak of 669±35 ng/g in the diabetic group (p<0.001), 871±34 ng/g in the nephrectomised animals (p<0.001) and 1012±43 ng/g in the diabetic-uninephrectomised group (p<0.001). Maximum binding of insulin-like growth factor I fell on day 1 in the diabetic group (8.3±1.4 vs 5.2±0.71× 10^– mol/l; p<0.01) but thereafter was identical to control animals. In the insulin-treated animals, maximum binding rose to 11.0±1.1×10^–11 mol/l, significantly different from control and diabetic animals (p<0.01). Growth hormone binding fell acutely in both the diabetic and diabetic-nephrectomised animals (3.13±0.58 and 2.83±0.21 vs 7.77±0.68×10^–12 mol/l; p<0.001 for both). Following uninephrectomy, maximum binding of insulin-like growth factor I and growth hormone was unchanged from control values. We conclude that the rise in renal content of insulin-like growth factor I which precedes the compensatory growth seen after induction of diabetes and uninephrectomy is not due to alterations in insulin-like growth factor I receptor binding and is independent of growth hormone binding.     

Immunoradiometric assay of insulin,intact proinsulin and 32–33 split proinsulin and radioimmunoassay of insulin in diet-treated Type 2 (non-insulin-dependent) diabetic subjects

Summary Plasma insulin, intact proinsulin and 32–33 split proinsulin measured by specific immunoradiometric assays and insulin and C-peptide measured by radioimmunoassay were measured during a constant infusion of glucose test in ten diet-treated subjects with a history of Type 2 (non-insulin-dependent) diabetes (termed diabetic subjects), mean fasting plasma glucose 6.0 ± 1.0 mmol/l (mean ± SD), and 12 non-diabetic control subjects. Immunoreactive insulin concentrations measured by radioimmunoassay were 33 higher than insulin and 16 % higher than the sum of insulin and its precursors by immunoradiometric assay. The diabetic and non-diabetic subjects had similar fasting concentrations of insulin, intact proinsulin and 32–33 split proinsulin. The ratio of fasting intact proinsulin to total insulin was greater in the diabetic than the non-diabetic group 12.0 % (6.8–21.0 %, 1 SD range) and 6.3 % (4.0–9.8 %), respectively,p < 0.01), though the groups overlapped substantially. After glucose infusion, diabetic and non-diabetic subjects had similar intact proinsulin concentrations (geometric mean 4.9 and 5.2 pmol/l, respectively), but the diabetic group had impaired insulin secretion by immunoradiometric assay (geometric means 55 and 101 pmol/1,p < 0.05) or by radioimmunoassay C-peptide (geometric means 935 and 1410 pmol/1,p < 0.05), though not by radioimmunoassay insulin (87 and 144 pmol/1,p = 0.12), respectively. Individual immunoradiometric assay insulin responses to glucose expressed in terms of obesity were subnormal in nine of ten diabetic subjects. Radioimmunoassay insulin and C-peptide gave less complete discrimination ( subnormal responses in six of ten and eight of ten, respectively). Thus, raised proinsulin and proinsulin:total insulin ratio are not necessarily a feature of mild diet-treated Type 2 diabetic patients with subnormal insulin responses to glucose.     

Amniotic fluid C-peptide in normal and insulin-dependent diabetic pregnancies

Summary Glucose, insulin and C-peptide were determined in amniotic fluid from 28 normal and 46 insulin-treated diabetic pregnant women. Glucose, insulin and C-peptide concentrations in amniotic fluid were higher in the diabetics than in the normal subjects. In diabetic women insulin levels did not correlate with birth weight or birth weight adjusted for gestational age, but C-peptide did. C-peptide correlated poorly with insulin (p<0.05) in diabetics but closely (p<0.002) in normal subjects. These results suggest that amniotic fluid investigations in insulintreated diabetic women should use C-peptide assays as these seem to reflect more closely the insulin production of the fetus than do insulin assays. There were no differences in amniotic fluid glucose, insulin and C-peptide concentrations where the amniotic fluid lecithin-sphingomyelin ratio indicated fetal pulmonary maturity or immaturity.     

Muscle triglycerides in diabetic subjects

Summary Muscle triglycerides and glycogen were measured in biopsy specimens of the vastus lateralis muscle before and after 1 h of ergometric exercise at 50 to 60% of maximal capacity (i. e. at a pulse rate during exercise of 180 minus age) in 3 groups of 19 to 35 year old, non-obese male subjects: 10 normals, 10 insulin dependent diabetic patients in relatively good control and 10 poorly controlled insulin dependent diabetic patients in whom insulin was withdrawn 24 h prior to examination. At rest in all subjects muscle triglyceride content was positively correlated with serum triglycerides (p<0.001) and blood glucose (p<0.05), resulting in elevated muscle triglyceride stores in the insulin deficient diabetic patients (17.9 ±1.8 mol/g protein vs. 13.4±1.3 and 9.4±1.2 in the normal subjects and the well controlled diabetic patients; p<0.05 and <0.001). During exercise, utilisation of muscle triglycerides and glycogen were directly related to content at rest (p<0.001), including the insulin-deprived patients with decreased glycogen. The decrease of muscle fat was associated with a rise in serum glycerol (p<0.001) and nonesterified fatty acids (p<0.001) during exercise.     

Insulin sensitivity,insulin secretion and glucose effectiveness in diabetic and non-diabetic cirrhotic patients

Summary In cirrhotic patients with normal fasting glucose levels both insulin insensitivity and a blunted early insulin response to oral glucose are important determinants of the degree of intolerance to oral glucose. It is not known whether the ability of hyperglycaemia per se to enhance glucose disposal (glucose effectiveness) is also impaired. It is also unclear whether overt diabetes is due to (1) more marked insulin insensitivity; (2) impaired insulin secretion; (3) reduced glucose effectiveness; or (4) a combination of these mechanisms. We used the minimal model to analyse the results of a 3-h intravenous glucose tolerance test to assess glucose effectiveness, insulin sensitivity and insulin responses in 12 non-diabetic cirrhotic patients, 8 diabetic cirrhotic patients and 10 normal control subjects. Fasting blood glucose levels were 4.8±0.2, 7.5±0.6 and 4.7±0.1 mmol/l, respectively. Fasting insulin and C-peptide levels were higher in both cirrhotic patient groups compared with control subjects. The glucose clearance between 6 and 19 min after i.v. glucose was lower in both cirrhotic groups (non-diabetic, 1.56±0.14, diabetic, 0.76±0.06, control subjects, 2.49±0.16 min^–1%, both p<0.001 vs control subjects). Serum insulin peaked at 3 and 23 min in the non-diabetic cirrhotic patients and control subjects; both peaks were higher in the non-diabetic cirrhotic patients and showed a delayed return to basal levels. In the diabetic cirrhotic patients, the first phase insulin and C-peptide response to i.v. glucose was absent; their early (22–27 min) incremental insulin response to i. v. tolbutamide was however similar to that of control subjects but 43% lower than in the non-diabetic cirrhotic patients (p<0.05). Insulin sensitivity was markedly reduced in both cirrhotic groups (non-diabetic, 1.11±0.24×10^–4, diabetic, 0.33±0.53×10^–4, control subjects, 4.37±0.53×10^–4 min^–1 per mU·l^–1, both p<0.001 vs controls). Glucose effectiveness was normal in the non-diabetic cirrhotic patients but 29% lower in the diabetic group. It would appear that overt diabetes develops in those cirrhotic patients who in addition to insulin insensitivity have a marked impairment of insulin secretion. An associated reduction in glucose effectiveness may be a contributory factor.     

Disproportionate elevation of immunoreactive proinsulin in Type 2 (non-insulin-dependent) diabetes mellitus and in experimental insulin resistance

Summary In this study, we found that the ratio of proinsulin to total immunoreactive insulin was much higher in 22 patients with Type 2 (non-insulin-dependent) diabetes mellitus than in 28 non-diabetic control subjects of similar age and adiposity (32±3 vs 15±1%, p<0.001). In addition, the arginine-induced acute proinsulin response to total immunoreactive insulin response ratio was greater in diabetic patients (n=10) than in control subjects (n=9) (8±2 vs 2±0.5%, p=0.009), suggesting that increased islet secretion per se accounted for the increased ratio of proinsulin to immunoreactive insulin. One explanation for these findings is that increased demand for insulin in the presence of islet dysfunction leads to a greater proportion of proinsulin secreted from the B cell. We tested this hypothesis by comparing proinsulin secretion before and during dexamethasone-induced insulin resistance in diabetic patients and control subjects. Dexamethasone treatment (6 mg/day for 3 days) raised the proinsulin to immunoreactive insulin ratio in control subjects from 13±2 to 21±2% (p<0.0001) and in diabetic patients from 29±5 to 52±7% (p<0.001). Dexamethasone also raised the ratio of the acute proinsulin response to the acute immunoreactive insulin response in control subjects from 2±0.5 to 5±2% (p=0.01) and in diabetic patients from 8±2 to 14±4% (p=NS), suggesting that the dexamethasone-induced increment in the basal ratio of proinsulin to immunoreactive insulin was also due to increased secretion. We conclude that: (1) The basal proinsulin to immunoreactive insulin ratio is increased in obese Type 2 diabetic patients. (2) An increase in tissue demand for insulin leads to a rise in the proinsulin to immunoreactive insulin ratio, which is exaggerated in Type 2 diabetic patients. (3) The increased proinsulin to immunoreactive insulin ratio in these diabetic patients in the basal state and in diabetic patients and control subjects during experimental insulin resistance is probably due to increased B-cell secretion of proinsulin.     

Measurement of glucose metabolism and insulin secretion during normal pregnancy and pregnancy complicated by gestational diabetes

Summary Gestational diabetes affects 2–3% of pregnant women and is associated with foetal complications including macrosomia and an increased likelihood of developing diabetes in later life. We have therefore studied seven women with gestational diabetes and five control women both during the third trimester of pregnancy and again 2–3 months post-partum, using the minimal model analysis of the frequently sampled labelled ([6, 6-²H₂]-glucose) intravenous glucose tolerance test. Glucose tolerance (glucose K_d) was significantly reduced in the women with gestational diabetes compared with the normal pregnant women both in pregnancy (1.16±0.11 vs 1.78±0.23%/min; p<0.05) and post-partum (1.47±0.22 vs 2.59±0.43%/min; p<0.05) and increased significantly in the control women after delivery (p<0.05). Glucose effectiveness was not significantly different between the women with gestational diabetes and the control group either during or after pregnancy. Insulin sensitivity was significantly lower during pregnancy than after delivery in the women with gestational diabetes (p<0.05). There was no significant difference in basal insulin secretion in the two groups during pregnancy or post-partum. However, during pregnancy the control subjects significantly increased (p<0.001) their insulin secretion over a period of 20 min in response to an intravenous glucose tolerance test (96.2±42.7 pmol/kg) compared with post-partum values (58.3±25.2 pmol/kg) while in the women with gestational diabetes insulin secretion was similar in pregnancy (65.5±9.3 pmol/kg) and after delivery (57.7±15.7 pmol/kg). These data suggest that the glucose intolerance in gestational diabetes compared to normal pregnancy is due to reduced insulin sensitivity and an impaired ability in gestational diabetes to increase insulin secretion in response to glucose.Abbreviations BMI Body mass index - GCMS gas chromatography mass spectrometry - GDM gestational diabetes mellitus - HOMA homeostasis model assessment - IVGTT intravenous glucose tolerance test - OGTT oral glucose tolerance test - Sab above basal insulin secretion - Sb basal insulin secretion - K_d glucose disappearance - CV coefficient of variation - AIR_glucose acute insulin response to glucose - SI insulin sensitivity index - SG glucose effectiveness     

Adipocyte insulin binding and insulin sensitivity in ‘brittle’ diabetes

Summary Adipocyte insulin binding and insulin sensitivity to stimulation of lipogenesis were assessed in a group of extremely brittle diabetic patients who were resistant to subcutaneous insulin therapy and had required frequent and prolonged hospital admission. These patients had significantly lower maximum adipocyte insulin binding (1.78±0.18%) than age-, sex- and weight-matched stable diabetic control subjects (2.57±0.36%; p<0.05). Scatchard analysis suggested that the decreased binding was secondary to reduced receptor affinity with no change in receptor number. Adipocytes from the brittle subjects displayed resistance to insulin stimulation of lipogenesis compared with those from diabetic or normal control groups (half-maximal stimulation at 34±4, 15±3 and 13±2 pmol/l respectively; p<0.01 between brittle and stable diabetic groups). In the one subject who was treated with intraperitoneal insulin, the changes in insulin binding and sensitivity were found to have reverted towards normal. The peripheral tissue abnormalities of brittle diabetes may exacerbate the clinical syndrome although the relationship of these changes to the primary cause of the syndrome is uncertain.     

Immunogenicity of recombinant DNA human insulin

Summary We postulated that human insulin of recombinant DNA origin would be a poor immunogen and might prove to be less immunogenic than purified pork insulin. Results are reported for 100 diabetic subjects not previously treated with insulin. Individuals completed the first 12 months of a clinical trial of human insulin of recombinant DNA origin. These patients are contrasted with 121 similar individuals who are taking part in a trial of purified pork insulin. Prior to therapy, species-specific binding of ¹²⁵I human insulin and pork insulins and insulin bound to antibody were undetectable in all individuals. In patients treated with human insulin of recombinant DNA origin, binding of ¹²⁵I human insulin increased to 10±1.2% at 12 months versus increases in binding of ¹²⁵I pork insulin in pork insulin-treated patients to 12.6±1.4% (NS). Mean percentages of species-specific binding tended to reach a plateau in the human insulin-treated group but continued to increase in the pork insulin group (p<0.001). Median bound values were nil throughout in patients treated with human insulin, but increased to 52 mU/l in the pork insulin group with significantly less bound insulin seen in the former group at all visits (p<0.001). The percentage of individuals who remained antibody free at 12 months, as indicated by bound insulin, was 56% in the human insulin-treated patients and 40% in the patients treated with pork insulin (p<0.01). In 11 out of 55 individuals who initially developed detectable insulin antibodies while being treated with human insulin, bound insulin levels later became undectable compared with three out of 77 individuals in the pork insulin-treatment group (p<0.005). Human insulin of recombinant DNA origin is less immunogenic than purified pork insulin. Level of antibodies in patients treated with human insulin of recombinant DNA origin reached a plateau after 6 months and antibody levels often tended subsequently to decrease below detection limits.     

Serum immunoreactive insulin responses to a glucose load in Asian Indian and European Type 2 (non-insulin-dependent) diabetic patients and control subjects

Summary The serum immunoreactive insulin response to an oral glucose load was estimated in 15 Asian Indian and 29 European non-diabetic subjects, and in 45 Asian Indian and 72 European Type 2 (non-insulin-dependent) diabetic patients. In the non-diabetic group, basal insulin values were higher in the Asian Indians than the Europeans (16.7 ± 3.0 vs. 6.9 ± 0.7 mU/l, p < 0.001), and remained higher throughout the glucose tolerance test. Total insulin response was also higher in the Asian Indians (p < 0.001), and linear regression analysis revealed basal insulin, body mass index and race to be important predictors of insulin response. Amongst the diabetic patients, basal insulin values were again higher in the Asian Indians compared with the Europeans (18.0±5.0 vs. 11.5±0.9 mU/l, p<0.05). Total insulin response was also greater (p < 0.01). Linear regression analysis revealed the basal insulin value to be the only significant predictor of insulin response. The results demonstrate higher insulin levels in Asian Indians than Europeans in both normal subjects and Type 2 diabetic subjects. The insulin response to a glucose load is also greater in the Asian Indians. In the control subjects, ethnic differences contribute to this response, whereas in the diabetic patients this is a function of the elevated basal insulin values of the Asian Indians.     

Reduced glycogen synthase activity in skeletal muscle from obese patients with and without Type 2 (non-insulin-dependent) diabetes mellitus

Summary In order to evaluate the importance of a defect in insulin mediated non-oxidative glucose metabolism and glycogen synthase activity in skeletal muscles in obese subjects with and without Type 2 (non-insulin-dependent) diabetes mellitus we studied: 10 lean and 10 obese control subjects and 12 obese diabetic patients using the euglycaemic hyperinsulinaemic clamp technique (basal, 20 mU · (m²)^–1 · min^–1, 80mU·(m²)^–1·min^–1) in combination with indirect calorimetry. Muscle biopsies were taken from m. vastus lateralis at each insulin level. We found that non-oxidative glucose metabolism could be stimulated by insulin in all three groups (p<0.01). The values obtained at the highest insulin levels (around 140 U/ml) were lower in both obese groups compared to the lean control subjects (118±21, 185±31, 249±14 mg·(m²)^–1·min^–1 (p< 0.01)). Insulin stimulation of the glycogen synthase activity at a glucose-6-phosphate concentration of 0.1 mmol/l was absent in both obese groups, while activities increased significantly in the lean control subjects (19.6±4.2% to 45.6±6.8%, p< 0.01). Glycogen synthase activities at the highest insulin concentrations only differed significantly between lean control subjects and obese diabetic patients (45±7% and 31±5%, p< 0.05). We conclude that insulin resistance in peripheral tissues in obese subjects with and without Type 2 diabetes may be partly explained by a reduced insulin mediated non-oxidative glucose metabolism and that this abnormality might be due to an absent insulin stimulation of glycogen synthase in skeletal muscles. This enzyme defect is correlated to obesity itself.     

Muscle enzyme activity and insulin sensitivity in Type 1 (insulin-dependent) diabetes mellitus

Summary The mechanisms of insulin insensitivity in diabetes are poorly understood. We have therefore assessed the relationship between glucose disposal during a euglycaemic clamp, muscle glycogen formation, and the activities of insulin regulated enzymes within skeletal muscle in five Type 1(insulin-dependent) diabetic patients, both on conventional injection therapy (HbA₁ 11.0±1.0 (SD) %) and after 6 weeks continuous subcutaneous insulin infusion (HbA₁ 7.6±1.4%,p < 0.01). On both regimens, overnight euglycaemia before the clamp was maintained with an intravenous insulin infusion. The increase in clamp glucose requirements (insulin 0.1 U kg^–1·h^–1) between injection therapy and continuous subcutaneous insulin infusion was significant (6.2±0.9 (SE) to 7.0 ± 0.9 mg·kg^–1·min^–1,p<0.05), but small compared to differences between subjects. Glucose requirement remained lower than in control subjects (10.4 ± 0.7 mg·kg^–1·min^–1,p < 0.05). The increase in muscle glycogen with the clamp was slightly higher on continuous subcutaneous insulin infusion (9.5 ± 2.5 mg/g protein) than on injection therapy (8.5 ± 2.4 mg/g,p < 0.05), but less than in control subjects (17.9 ± 2.1 mg/g,p < 0.05). The expressed activity of glycogen synthase and pyruvate dehydrogenase increased significantly between fasting and the end of the clamps in the patients (p < 0.001 and < 0.005), but was not significantly different between the two treatment regimens. Expressed glycogen synthase activity at the end of the clamp was lower on both treatments than in control subjects (p < 0.05). Both enzyme activities were, however, highly correlated with glucose requirement between patients, (r=0.89–0.94,p<0.05-0.02), and glycogen synthase was similarly correlated in the control subjects (r = 0.84,p < 0.05). Patients had significantly different enzyme activities, glucose requirement, and glycogen stored by analysis of variance (p < 0.05-0.01). Correlation of each enzyme activity between subjects on the two treatment regimens was also high (r=0.94–0.98,p < 0.02–0.01). At the end of the clamp the enzyme activities were themselves closely related (injectionsr = 0.99,p < 0.001; infusionr = 0.88,p < 0.05), and glycogen synthase activity predicted muscle glycogen deposition (r=0.94–0.97,p < 0.02–0.01). We suggest that: (1) preceding metabolic control has a relatively small influence on whole body insulin sensitivity measured immediately after careful overnight control; (2) insulin sensitivity derived from glucose clamp data is strongly related to skeletal muscle glycogen deposition and skeletal muscle enzyme activities.     

Strict insulin therapy normalises organ nitrogen contents and the capacity of urea nitrogen synthesis in experimental diabetes in rats

Summary Rats with experimental diabetes due to streptozotocin (75 mg/kg body weight) and free access to food were divided into two groups. One group (n=9) was optimally treated with insulin (glucosuria <4.0 mmol/24 h), using heat treated very long-acting ultralente insulin. The other group (n=10) was poorly treated with insulin (glucosuria 20–30 mmol/24 h).The nitrogen balance and energy balance of optimally treated diabetic rats was positive and not different from the control group (n=6). In the poorly treated diabetic rats the nitrogen balance was reduced whereas the energy balance was not different from that of control rats. After 4 weeks the fasting glucagon was: 50±21 ng/l (mean±SEM) in control rats, 62±18 ng/l in optimally treated diabetic rats and 249±58 ng/l in poorly treated diabetic rats (p<0.01). The capacity of urea nitrogen synthesis determined during alanine loading was: 9.6±1.0 umol/(min 100 g body weight) in control rats, 10.6±1.7 umol/(min 100 g body weight) in optimally treated diabetic rats and 17.3±1.3 umol/(min 100 g body weight) in poorly treated diabetic rats (p<0.01).Nitrogen contents of carcass, heart, intestines, liver, and kidneys as determined by Kjeldahl analyses were identical in control rats and optimally treated diabetic rats. In the poorly treated diabetic rats carcass-nitrogen and heart-nitrogen contents were reduced to 89% of the control value (p<0.01), whereas the kidney-nitrogen content was increased to 112% of the control value (p<0.01).Strict insulin therapy in experimental diabetes leads to a normalisation of nitrogen metabolism and hyperglucagonaemia, whereas less than optimally insulin treated rats show marked abnormalities in nitrogen metabolism as well as hyperglucagonaemia.     

Disappearance rate of insulin antibodies after discontinuing insulin treatment in 42 Type 2 (non-insulin-dependent) diabetic patients

Summary The disappearance rate of insulin antibodies was studied after cessation of insulin treatment which had been given for 3 months to 6 years in 42 Type 2 (non-insulin-dependent) diabetic patients. Insulin antibodies were measured before and 15 days after interruption of insulin treatment, and every 30 days until the disappearance of insulin antibodies. The mean ± SD value of insulin binding in the entire group before the interruption of insulin treatment was 32±14%. There was no relationship between the antibody level at that time and the duration of insulin treatment. However, the insulin antibody level was significantly higher in 17 diabetic patients on an insulin dose of >20 U/day (p<0.02) than in 25 on an insulin dose of <20 U/day (39±13% versus 28±12%). A positive correlation was found between intial insulin binding and the time required for it to fall below 10% (r = 0.74). Antibodies were absent 60 days after discontinuing insulin treatment in eight of ten subjects presenting with initial binding of <20%. In contrast, in only two of 12 patients with an initial binding of >40% were insulin antibodies detectable 150 days after discontinuation of insulin therapy. Disappearance of insulin antibodies sometimes took up to 1 year and occasionally even more than 2 years.     

Simple empirical assessment of Beta-cell function by a constant infusion of glucose test in normal and Type 2 (non-insulin-dependent) diabetic subjects

Summary The plasma insulin or C-peptide response to a 90-min constant glucose infusion 5 mg · kg ideal body weight^–1·min^–1 provides Beta-cell assessment comparable to more intensive methods. In 14 diet-treated Type 2 (non-insulin-dependent) diabetic subjects and 12 non-diabetic subjects, plasma insulin and C-peptide concentrations gave near linear plots against simultaneous glucose values. The glucose-insulin and glucose-C-peptide vectors (G-I and G-C vectors), could be extrapolated to predict insulin and C-peptide levels during a 12 mmol/l hyperglycaemic clamp. Predicted concentrations correlated with clamp concentrations, r = 0.94 and r = 0.98 respectively, p<0.001, validating the vectors as empirical glucose dose-response curves. The vector slopes correlated highly with % Beta, a mathematical model-derived measure of Beta-cell function using constant infusion of glucose model assessment, Spearman r = 0.95 and 0.93 for insulin and C-peptide, respectively. G-I vector slopes in 21 diet-treated Type 2 diabetic subjects with fasting glucose (mean +1 SD) 7.5±2,3 mmol/1, were lower than in 28 non-diabetic subjects, (geometric mean, 1 SD range, 8.4 pmol/mmol (3.3–21.0) and 25.1 pmol/mmol (14.3–44.1), p<0.001, respectively), indicating an impaired Beta-cell response. The G-I vector slopes correlated with obesity in both groups (r = 0.54 p<0.02 and 0.72, p<0.001 respectively), and, in 15 non-diabetic subjects, correlated inversely with insulin sensitivity as measured by a euglycaemic clamp (r = –0.66, p<0.01).Thus,Beta-cell function needs to be interpreted in relation to obesity/insulin resistance and, taking obesity into account, only 4 of 21 diabetic patients had Betacell function (G-I vector slope) in the non-diabetic range. The fasting plasma glucose in the diabetic subjects correlated inversely with the obesity-corrected G-I and G-C vector slopes (partial r = –0.57, p <0.01 and –0.86, p<0.001, respectively). The insulin or C-peptide response to the glucose infusion provides a direct empirical measure of the Beta-cell function, which can be interpreted in relation to obesity or to insulin resistance to assess underlying pancreatic responsiveness.