Importance of glucagon in the control of futile cycling as studied in alloxan-diabetic dogs

Summary In order to determine the role of glucagon in futile or substrate cycling in diabetes, we measured tracer determined glucose kinetics during a combined infusion of 2-³H-glucose (total glucose production) and 6-³H-glucose (glucose production) in six alloxan-diabetic dogs. The animals received either a 420 min infusion of (1) somatostatin alone (0.3 g·kg^–1· min^–1), (2) somatostatin with insulin replacement (100 U·kg^–1min^–1) or (3) glucagon (6 ng·kg^–1· min^–1) together with somatostatin and transient insulin replacement. When somatostatin was given alone, plasma glucagon (p<0.004) and insulin (p<0.0001) were suppressed. Glucose production and disappearance and plasma glucose concentrations fell (p<0.0001), but the metabolic clearance of glucose did not change significantly. In the basal state, futile cycling comprised 29±4%, 33±4% and 33±3% of total glucose production in the three goups of studies, which is high compared to normal dogs. The absolute rate of futile cycling fell slightly but significantly from 10.0±1.7 to 8.3±1.7 mol·kg·^–1min^–1 (p<0.0008). When insulin replacement was given during somatostatin infusion to correct for the small somatostatin-induced insulin suppression, there were similar changes in plasma glucagon, glucose concentrations and glucose kinetics as seen during the infusion of somatostatin alone. Futile cycling decreased to a slightly greater extent from 12.8±2.8 to 9.5±1.7mol·kg^–1·min.^–1 (p<0.02). When glucagon was infused together with somatostatin and insulin replacement, plasma glucagon (p<0.0002) increased and plasma glucose levels rose (p<0.001) due to a transient increase in glucose production. Metabolic clearance of glucose did not change significantly. There was a marked increase in futile cycling from 12.2±1.7 to 21.7±1.7mol· kg^–1·min^–1 (p<0.0001) in response to exogenous glucagon excess. There was a slight (p<0.01) drop in free fatty acid levels with somatostatin. Free fatty acid levels nearly doubled (p<0.025) with the infusion of glucagon together with somatostatin. In conclusion, (a) futile cycling was increased in alloxan-diabetic dogs; (b) glucagon suppression can suppress futile cycling only if total insulin deficiency is prevented; and (3) hyperglucagonaemia increases futile cycling, and this effect is more pronounced during insulin deficiency.     

Contribution of glucose/glucose 6-phosphate cycle activity to insulin resistance in Type 2 (non-insulin-dependent) diabetes mellitus

Summary It has been suggested that increased glucose/glucose 6-phosphate substrate cycling impairs net hepatic glucose uptake in Type 2 (non-insulin-dependent) diabetes mellitus and contributes to hyperglycaemia. To investigate glucose/glucose 6-phosphate cycle activity and insulin action in Type 2 diabetes we studied eight patients and eight healthy control subjects, using the euglycaemic glucose clamp and isotope dilution techniques with purified [2-³H]- and [6-³H] glucose tracers, in the post-absorptive state and eight patients and five healthy control subjects during consecutive insulin infusions at rates of 0.4 and 2.0 mU·kg^–1·min^–1. [2-³H]glucose and [6-³H]glucose radioactivity in plasma samples were determined using selective enzymatic detritiation, allowing calculation of glucose turnover rates for each isotope, the difference being glucose/glucose 6-phosphate cycling. Endogenous glucose production ([6-³H]glucose) was greater in diabetic than control subjects in the post-absorptive state (15.6±1.5 vs 11.3±0.4 mol·kg^–1·min^–1, p<0.05) and during the 0.4 mU insulin infusion (10.1±1.3 vs 5.2±0.3 mol·kg^–1·min^–1, p<0.01) indicating hepatic insulin resistance. Glucose/glucose 6-phosphate cycling was significantly greater in diabetic than in control subjects in the post-absorptive state (2.6±0.4 vs 1.6±0.2 mol·kg^–1·min^–1, p<0.05) but not during the 0.4 mU insulin infusion (2.0±0.4 vs 2.0±0.3 mol·kg^–1·min^–1). During the 2.0 mU insulin infusion endogenous glucose production was suppressed to a similar degree in both groups (2.6±0.5 vs 3.4±0.7 mol · kg^–1·min^–1) but glucose disappearance was lower in the diabetic subjects (30.8±2.0 vs 52.4±4.6 mol·kg^–1·min^–1, p<0.01). During the 2.0 mU insulin infusion glucose/glucose 6-phosphate cycling was greater in the diabetic subjects (3.8±0.7 vs 0.8±0.6 mol·kg^–1·min^–1, p<0.05). In conclusion, both hepatic and peripheral insulin action are impaired in Type 2 diabetes. Increased glucose/glucose 6-phosphate cycling is seen in the post-absorptive state and also during marked hyperinsulinaemia, when insulin resistance is predominantly due to reduced peripheral tissue glucose uptake.     

Decreased hepatic glucagon responses in Type 1 (insulin-dependent) diabetes mellitus

Summary The effect of glucagon infusion on hepatic glucose production during euglycaemia was evaluated in seven Type 1 (insulin-dependent) diabetic patients and in ten control subjects. In the diabetic subjects normoglycaemia was maintained during the night preceding the study by a variable intravenous insulin and glucose infusion. During the study endogenous insulin secretion was suppressed by somatostatin (450 g/h) and replaced by insulin infusion (0.15 mU·kg^–1·min^–1). ³H-glucose was infused for isotopic determination of glucose turnover. Plasma glucose was clamped at 5 mmol/1 for 2 h 30 min and glucagon (1.5 ng· kg^–1·min^–1) was then infused for the following 3 h. Hepatic glucose production and glucose utilisation were measured during the first, second and third hour of the glucagon infusion. Basal hepatic glucose production (just prior to glucagon infusion) was similar in diabetic (1.2±0.3 mg·kg^–1·min^–1) and control (1.6±0.1 mg·kg^–1·min^–1) subjects. In diabetic patients hepatic glucose production rose slowly to 2.1±0.5 mg·kg^–1·min^–1 during the first hours of glucagon infusion and stabilized at this level (2.4±0.5 mg·kg^–1·min^–1) in the third hour. In control subjects hepatic glucose production increased sharply to higher levels than in the diabetic subjects (3.4±0.3 mg·kg^–1·min^–1) during the first and second hour of glucagon infusion (p<0.05) and then gradually fell (2.9±0.4 mg·kg^–1·min^–1) during the third hour. In conclusion, when stimulated with glucagon at a physiologic plasma concentration diabetic patients had 1) an overall reduced hepatic glucose production response and 2) an abnormal sluggish response pattern. These abnormalities may imply inappropriate counter-regulation following a hypoglycaemic episode.     

Increased glucose carbon recycling in severely insulin deficient Type 1 (insulin-dependent) diabetic subjects

Summary Six Type 1 (insulin-dependent) diabetic subjects were studied in order to determine the contribution of recycling of glucose carbon to the overproduction of glucose which is characteristic of the fasting hyperglycaemia produced by insulin withdrawal. The subjects were studied on two occasions, once after an overnight insulin infusion and once following 24 h of insulin withdrawal. The difference in turnover rates of 1-¹⁴C-glucose and 3-³H-glucose was used as a measure of glucose recycling. Insulin withdrawal caused a marked metabolic derangement with a rise in non-esterified fatty acids from 0.69±0.23 to 1.11±0.21 mmol/l (mean±SEM, p<0.05), total ketones from 0.27±0.06 to 2.06±0.51 mmol/l (p<0.01), cortisol from 341±43 to 479±31 nmol/l (p<0.05) and growth hormone from 1.1±0.3 to 19+5-mu/l (p<0.05). Glucose turnover rose from 13.8±2.3 mol·kg^–1·min^–1 at a glucose of 6.9±0.7 mmol/l in the insulin infused study to 25.8±4.4 mol·kg^–1·min^–1 (p<0.05) at a glucose of 16.4±0.7 mmol/l in the insulin withdrawn study. Recycling also rose from 3.0±0.4 mol· kg^–1·min^–1 to 9.4±2.2 mol·kg^–1·min^–1 (p<0.05) when insulin withdrawn, accounting for 23±3% and 36±3% of glucose turnover, respectively. We conclude that in the severely insulin deficient Type 1 diabetic subject recycling of glucose carbon is a major contributor to the excess glucose production.     

Insulin regulation of glucose and lipid metabolism in massive obesity

Summary Eight obese patients and 12 normal individuals underwent a euglycaemic insulin clamp (20 and 40 mU · m^2–1 · min^–1) along with continuous infusion of 3-³H-glucose and 1-¹⁴C-palmitate and indirect calorimetry. Basal plasma glucose concentration (4.7±0.3 vs 4.4±0.2 mmol/l) was similar in the two groups, whereas hepatic glucose production was slightly higher in obese individuals (1.11±0.06 vs 0.84±0.05 mmol/min) in spite of higher plasma insulin levels (17±2 vs 6±1 mU/l; p<0.01). Insulin inhibition of hepatic glucose production was impaired in obese subjects. Glucose disposal by lean body mass was markedly reduced both at baseline (11.7±1.1 vs 15.6±0.6 mol · kg^–1 · min^–1; p<0.05) and during clamp (15.0±1.1 vs 34.4±2.8 and 26.7±3.9 vs 62.2±2.8 mol · kg^–1 · min^–1; p<0.01) Oxidative (12.2±1.1 vs 17.8±1 and 16.1±1.1 vs 51.1±1.7 mol · kg^–1 · min^–1; p<0.05–0.002) and non-oxidative glucose metabolism (3.9±1.1 vs 15.0±2.8 and 12.8±3.3 vs 38.3±2.2 mol · kg^–1 · min^–1; p<0.01–0.001) were impaired. Basal plasma concentrations of non-esterified fatty acids (635±75 vs 510±71 mol/l) and blood glycerol (129±17 vs 56±5 mol/l; p<0.01) were increased in obese patients. Following hyperinsulinaemia, plasma non-esterified fatty acids (244±79 vs 69±16 and 140±2 vs 36±10 mol/l; p<0.01) and blood glycerol levels (79±20 vs 34±6 and 73±22 vs 29±5 mol/l; p<0.01) remained higher in obese subjects. Baseline non-esterified fatty acid production rate per kg of fat body mass was significantly larger in normal weight subjects (37.7±6.7 vs 14.0±1.8 mol/l; p<0.01) and insulin inhibition was reduced in obese patients (–41±9 vs –74±3 and –53±11 vs –82±3%; p<0.05). Basal plasma non-esterified fatty acid utilization by lean body mass was similar in the two groups (9.8±0.9 vs 8.8±2.0 mol · kg^–1 · min^–1), whereas during clamp it remained higher in obese patients (6.0±1.2 vs 2.8±2.5 and 4.9±1.3 vs 1.5±0.6 mol · kg^–1 · min^–1; p<0.1–0.05). Lipid oxidation was higher in obese individuals in spite of hyperinsulinaemia (3.7±0.3 vs 2.4±0.4 and 2.3±0.4 vs 0.9±0.3 mol · kg^–1 · min^–1; p<0.05– 0.02). An inverse correlation was found between lipid oxidation and glucose oxidation (r=0.82 and 0.93; p<0.001) and glucose utilization (r=0.54 and 0.83; p<0.05–0.001) both in obese and control subjects. A correlation between lipid oxidation and non-oxidative glucose metabolism was present only in normal weight individuals (r=0.75; p<0.01). We conclude that in obesity all tissues (muscles, liver, and adipose tissue) are resistant to insulin action. Insulin resistance involves glucose as well as lipid metabolism.     

Artificial induction of intravascular lipolysis by lipid-heparin infusion leads to insulin resistance in man

Summary Although extensive evidence indicates that free fatty acids can decrease glucose utilization in vitro, it is still controversial how an increase in lipolysis affects glucose metabolism in man. To test the hypothesis that an increase in lipolysis is related to insulin resistance, we examined the effect of lipid-heparin infusion on glucose metabolism in ten normal subjects by the euglycaemic glucose clamp technique and isotopic determination of glucose turnover. In the control euglycaemic clamp studies with insulin infusion at 0.2 and 1.0 mU·kg^–1·min^–1, endogenous glucose production was suppressed from the basal rate of 2.0±0.3 mg· kg^–1min^–1 to 1.1±0.7 mg·kg^–1·min^–1 and -0.4±0.7mg· kg^–1min^–1 respectively. Glucose utilization increased from the basal rate of 2.0±0.3 mg·kg^–1min^–1 to 2.3±0.5mg· kg^–1min^–1 and 5.9±1.8 mg·kg^–1min^–1 respectively. When the euglycaemic clamp studies were coupled with lipid-heparin infusion at comparable low and high rates of insulin infusion, endogenous glucose production increased (1.8± 0.7 mg·kg^–1·min^–1, p<0.001, and 0.3±0.6 mg·kg^–1· min^–1, p<0.05, respectively), and glucose utilization decreased (2.1±0.3 mg·kg^–1·min^–1, not significant, and 3.2±0.7 mg·kg^–1·min^–1, p<0.001 respectively). These data suggest that the artificial induction of intravascular lipolysis by lipid-heparin infusion leads to a state of insulin resistance in man.     

Enhanced glucose cycling and suppressed de novo synthesis of glucose-6-phosphate result in a net unchanged hepatic glucose output in ob/ob mice

Aims/hypothesis Leptin-deficient ob/ob mice are hyperinsulinaemic and hyperglycaemic; however, the cause of hyperglycaemia remains largely unknown.Methods Glucose metabolism in vivo in 9-h fasted ob/ob mice and lean littermates was studied by infusing [U-¹³C]-glucose, [2-¹³C]-glycerol, [1-²H]-galactose and paracetamol for 6 h, applying mass isotopomer distribution analysis on blood glucose and urinary paracetamol-glucuronide.Results When expressed on the basis of body weight, endogenous glucose production (109±23 vs 152±27 µmol·kg^–1·min^–1, obese versus lean mice, p<0.01) and de novo synthesis of glucose-6-phosphate (122±13 vs 160±6 µmol·kg^–1·min^–1, obese versus lean mice, p<0.001) were lower in ob/ob mice than in lean littermates. In contrast, glucose cycling was greatly increased in obese mice (56±13 vs 26±4 µmol·kg^–1·min^–1, obese versus lean mice, p<0.001). As a result, total hepatic glucose output remained unaffected (165±31 vs 178±28 µmol·kg^–1·min^–1, obese vs lean mice, NS). The metabolic clearance rate of glucose was significantly lower in obese mice (8±2 vs 18±2 ml·kg^–1·min^–1, obese versus lean mice, p<0.001). Hepatic mRNA levels of genes encoding for glucokinase and pyruvate kinase were markedly increased in ob/ob mice.Conclusions/interpretation Unaffected total hepatic glucose output in the presence of hyperinsulinaemia reflects hepatic insulin resistance in ob/ob mice, which is associated with markedly increased rates of glucose cycling. Hyperglycaemia in ob/ob mice primarily results from a decreased metabolic clearance rate of glucose.     

Inhibition of muscle glycogen synthase activity and non-oxidative glucose disposal during hypoglycaemia in normal man

Summary The purpose of the present study was to evaluate the role of muscle glycogen synthase activity in the reduction of glucose uptake during hypoglycaemia. Six healthy young men were examined twice; during 120 min of hyperinsulinaemic (1.5 mU · kg^–1 · min^–1) euglycaemia followed by: 1) 240 min of graded hypoglycaemia (plasma glucose nadir 2.8 mmol/l) or 2) 240 min of euglycaemia. At 350–360 min a muscle biopsy was taken and indirect calorimetry was performed at 210–240 and 330–350 min. Hypoglycaemia was associated with markedly increased levels of adrenaline, growth hormone and glucagon and also with less hyperinsulinaemia. During hypoglycaemia the fractional velocity for glycogen synthase was markedly reduced; from 29.8±2.3 to 6.4±0.9%, p<0.05. Total glucose disposal was decreased during hypoglycaemia (5.58±0.55 vs 11.01±0.75 mg · kg^–1 · min^–1 (euglycaemia); p<0.05); this was primarily due to a reduction of non-oxidative glucose disposal (2.43±0.41 vs 7.15±0.7 mg · kg^–1 · min^–1 (euglycaemia); p<0.05), whereas oxidative glucose disposal was only suppressed to a minor degree. In conclusion hypoglycaemia virtually abolishes the effect of insulin on muscle glycogen synthase activity. This is in keeping with the finding of a marked reduction of non-oxidative glucose metabolism.Abbreviations HGP Hepatic glucose production - Rd glucose disposal - GH growth hormone - 3-OHB 3-hydroxybutyrate - G 6-P glucose 6-phosphate - NEFA non-esterified fatty acids - PP-1 phosphatase-1 - Ra rate of appearance - Rd-nonox non-oxidative glucose disposal - Rd-ox oxidative glucose disposal - A0.5 half-maximal activity     

ACE-inhibition increases hepatic and extrahepatic sensitivity to insulin in patients with Type 2 (non-insulin-dependent) diabetes mellitus and arterial hypertension

Summary To assess the effects of ACE-inhibition on insulin action in Type 2 (non-insulin-dependent) diabetes mellitus associated with essential hypertension, 12 patients with Type 2 diabetes (on diet and oral hypoglycaemic agents) and arterial hypertension were examined on two occasions, in a single blind, cross-over study, after two days of treatment with either captopril or a placebo. The study consisted of a euglycaemic-hyperinsulinaemic clamp (two sequential steps of insulin infusion at the rates of 0.25 mU·kg^–1·min^–1 and 1 mU·kg^–1·min^–1, 2 h each step), combined with an infusion of 3-³H-glucose to measure the rate of hepatic glucose production and that of peripheral glucose utilization. The results show that blood pressure was lower after captopril (sitting, systolic 148±5 mmHg, diastolic 89±2 mm Hg) compared to placebo (155±6 and 94±2 mm Hg) (p<0.05). Captopril treatment resulted in a more suppressed hepatic glucose production (2.7±0.4 vs 4.94±0.55 mol·kg^–1·min^–1), and a lower plasma non-esterified fatty acid concentration (0.143±0.05 vs 0.200±0.05 mmol/l) (captopril vs placebo, p<0.05) at the end of the first step of insulin infusion (estimated portal plasma insulin concentration 305±28 pmol/l); and in a greater glucose utilization (36.5±5.1 vs 28±3.6mol·kg^–1·min^–1, p<0.001) at the end of the second step of insulin infusion (arterial plasma insulin concentration of 604±33 pmol/l). We conclude that captopril improved insulin sensitivity in Type 2 diabetes associated with hypertension at the level of the liver and extrahepatic tissues, primarily muscle and adipose tissue. Thus, in contrast to other antihypertensive drugs such as diuretics and beta-blockers which may have a detrimental effect on insulin action, ACE-inhibitors appear to improve insulin action in Type 2 diabetes and essential hypertension, at least on a short-term basis.     

Multiple defects of both hepatic and peripheral intracellular glucose processing contribute to the hyperglycaemia of NIDDM

Summary Non-insulin-dependent diabetic (NIDDM) patients were studied during a modified euglycaemic state when fasting hyperglycaemia was normalized by a prior (–210 to –150 min) — and later withdrawn (–150–0 min) — intravenous insulin infusion. Glucose metabolism was assessed in NIDDM patients (n=10) and matched control subjects (n=10) using tritiated glucose turnover rates, indirect calorimetry and skeletal muscle glycogen synthase activity determinations. Total and non-oxidative exogenous glycolytic flux rates were measured using appearance rates of tritiated water. A+180 min euglycaemic hyperinsulinaemic (40 mU·m^–2·min^–1) clamp was performed to determine the insulin responsiveness of the various metabolic pathways. Plasma glucose concentration increased spontaneously during baseline measurements in the NIDDM patients (–120 to 0 min: 4.8±0.3 to 7.0±0.3 mmol/l; p<0.01), and was primarily due to an elevated rate of hepatic glucose production (3.16±0.13 vs 2.51±0.16 mg·kg FFM^–1·min^–1; p<0.01). In the NIDDM subjects baseline glucose oxidation was decreased (0.92±0.17 vs 1.33±0.14 mg·kg FFM^–1·min^–1; p<0.01) in the presence of a normal rate of total exogenous glycolytic flux and skeletal muscle glycogen synthase activity. The simultaneous finding of an increased lipid oxidation rate (1.95±0.13 vs 1.61±0.07 mg·kg FFM^–1·min^–1; p=0.05) and increased plasma lactate concentrations (0.86±0.05 vs 0.66±0.03 mmol/l; p=0.01) are consistent with a role for both the glucose-fatty acid cycle and the Cori cycle in the maintenance and development of fasting hyperglycaemia in NIDDM during decompensation. Insulin resistance was demonstrated during the hyperinsulinaemic clamp in the NIDDM patients with a decrease in the major peripheral pathways of intracellular glucose metabolism (oxidation, storage and muscle glycogen synthase activity), but not in the pathway of non-oxidative glycolytic flux which was not completely suppressed during insulin infusion in the NIDDM patients (0.55±0.15 mg·kg FFM^–1·min^–1; p<0.05 vs 0; control subjects: 0.17±0.29; NS vs 0). Thus, these data also indicate that the defect(s) of peripheral (skeletal muscle) glucose processing in NIDDM goes beyond the site of glucose transport across the cell membrane.Abbreviations NIDDM Non-insulin-dependent diabetes mellitus - FFM fat free mass - HGP hepatic glucose production - R_d peripheral glucose disposal (uptake) rate - G6P glucose 6-phosphate - UDPG uridine diphosphate glucose - FV fractional velocity     

Underestimation of glucose turnover determined using [6-3H]glucose tracer in non-steady state

Summary The use of tritiated glucose tracers may result in underestimation of glucose turnover during hyperinsulinaemic clamps giving paradoxical negative endogenous glucose production rates. While mathematical modelling errors in the analysis of tracer data are major determinants of this underestimate in the non-steady state, the relative importance of tracer contamination under these conditions remains in doubt. We have used high performance liquid chromatography to assess the possible contribution to this problem of a labelled tracer impurity found in [6-³H]glucose. In conventional 4 h hyperinsulinaemic clamps performed in six normal subjects, labelled impurity increased as a percentage of the neutral plasma radioactivity fraction from 5.3±0.9% after a 2 h equilibration period (0 min) to 13.5±2.2% at 120 min and 15.4±2.4% at 240 min, as plasma glucose specific activities fell following the infusion of insulin. Negative endogenous glucose production rates were observed both at 90–120 min (–8.8±1.6mol·kg^–1min^–1) and at 210–240 min (–8.5±1.4 mol·kg^–1min^–1) implying a persistent underestimate in isotopically determined glucose appearance rate. Using chromatography data to correct for impurity increased glucose appearance rates by 7.9±2.1% at 120 min and 11.0±2.5% at 240 min. Purified tracer was then used for a further six clamps. When the conventional protocol was used with unlabelled glucose infusion an obvious negative error persisted only at 90–120 min. In contrast, labelled infusions gave exclusively positive values for endogenous glucose production. We conclude that a labelled impurity of [6-³H]glucose may be an important source of error in measurement of glucose turnover and endogenous glucose production in the non-steady state. Use of chromatographically pure tritiated glucose tracers is recommended.     

Immunology in Diabetes '84

Summary To investigate the influence of opiates on insulin action in vivo, we induced mild physiological hyperinsulinaemia (15–20 mU/l) in five trained conscious dogs in the absence or presence of ongoing infusion with the opiate agonist D-met²-pro⁵-enkephalinamide (DMPE, 0.5 g·kg^-1· min^-1), or the opiate antagonist naloxone (1.25 mg followed by 1 g· kg^-1·min^-1). The effects on glucose production and glucose utilization were measured by isotope dilution using 3-³H-glucose. Glucose fell similarly over 30 min in response to insulin in controls (0.021±0.003 mmol·1^-1· min^-1), and both the DMPE and naloxone studies (0.016±0.002 mmol · 1^-1 · min^-1 and 0.017±0.003 mmol·1^-1 ·min^-1, respectively). In control dogs, insulin lowered glucose by transiently suppressing production by 0.028 ±0.006 mmol·kg^-1·min^-1 at 20–30 min without changing utilization. In contrast, in both the DMPE and naloxone studies insulin lowered glucose by markedly raising utilization at 20 min by 0.094 ±0.017 and 0.139±0.022 mmol·kg^-1·min^-1, respectively. Furthermore, insulin failed to suppress production in both DMPE and naloxone studies and, as plasma glucose fell, production rose in both treatment groups at 20 min by 0.045 ±0.012 and 0.089 ±0.022 mmol · kg^-1 · min^-1 respectively. The counter-regulatory hormone glucagon was transiently suppressed by insulin at 20 min in controls, but not in the treatment groups; cortisol and adrenaline rose at 30 and 45 min respectively in the naloxone group only. No other changes were noted in counterregulatory hormones. Thus hormonal changes do not appear to account for the early pronounced rise in glucose utilization leading to the fall in glucose in the DMPE and naloxone studies. We conclude that the morphine-like agent DMPE and high doses of the opiate antagonist naloxone modulate insulin-induced glucose fluxes in vivo, promoting both glucose utilization and production. These effects may be direct or indirect, and may serve a function in the redistribution of glucose during stress responses.     

Differential effects of insulin therapy on hepatic and peripheral insulin sensitivity in Type 2 (non-insulin-dependent) diabetes

Summary Hepatic glucose production and metabolic clearance rate of glucose were measured using (3-³H) glucose at steady state, basally and during two sequential 2 h insulin (25 and 40mU · kg^–1 · h^–1)/glucose(2 and 3mg · kg^–1 · min^–1) infusion periods. Eight diabetic subjects were studied before and after 1 week of twice daily insulin therapy; six control subjects matched for age, weight and degree of obesity were also studied. In the diabetic patients, pre-treatment hepatic glucose production was 20.0 ± 2.2, 9.9 ± 2.9, and 1.4 ± 0.8 mol · kg^–1 · min^–1 respectively (± SEM) for each of the three periods, and fell significantly with treatment to 12.8 ± 1.7,4.0 ± 1.5 and 1.9 ± 1.0 mol · kg^–1 · min^–1. Hepatic glucose production in normal subjects was 13.2 ± 0.6, 2.2 ± 0.8 and < 1 mol · kg^–1 · min^–1. The pre-treatment metabolic clearance rate in all diabetic studies with insulin levels 30 mU/l was 1.10 ± 0.14 ml · kg^–1 · min^–1 and remained virtually unchanged following insulin therapy; this was significantly lower than in the control subjects (6.83 ± 1.02, p < 0.001). Basal non-esterified fatty acid levels were higher (p < 0.02) in the pre-treated diabetic patients compared to post-treated diabetic patients and control subjects. Non-esterified fatty acids in each group fell to similar levels during the insulin infusions, but the rate of fall was slower in the pre-treated diabetic patients. Insulin receptor binding to erythrocytes was normal in the diabetic subjects and unchanged by treatment. Therefore, following insulin treatment of uncontrolled Type 2 (non-insulin-dependent) diabetes, the initially increased basal hepatic glucose production, and decreased hepatic sensitivity, return towards normal. However, the glucose clearance remains low, despite good diabetic control, and appears to be a major factor in the continuing glucose intolerance. As insulin receptor binding is normal, the defect of glucose clearance in Type 2 diabetes appears compatible with a post-receptor defect of glucose metabolism.     

No evidence for isotope discrimination of tritiated glucose tracers in measurements of glucose turnover rates in man

Summary Under non-steady-state conditions, glucose turnover rates determined with tritiated glucose tracers are often underestimated. To examine whether isotope discrimination or a tracer contaminant can contribute to this, we compared the turnover rates of unlabelled and tritiated glucose under isotopic steady-state conditions. The turnover rates were measured in 20 healthy subjects at two insulin concentrations (79±3 mU·l^–1 and 704±62 mU·l^–1). Euglycaemia was maintained by infusing unlabelled glucose mixed with (3³H)-or (6³H)-glucose. In both studies, the isotopically determined glucose disposal rate was virtually identical to the exogenous glucose infusion rate (low insulin 7.66±0.48 vs 7.58±0.44 mg·kg^–1·min^–1, high insulin 13.36±0.74 vs 13.55±0.98 mg·kg^–1·min^–1). The individual values were correlated in both the low (r = 0.85, p<0.001) and high dose insulin (r=0.81, p<0.001) studies. Tritiated glucose specific activities were also compared in arterialized and deep venous blood across forearm tissues during the high-dose insulin infusion. Glucose specific activities were similar in arterilized and deep venous blood when analysed with HPLC and conventional methods. In summary: (1) Under isotopic steady-state conditions the turnover rates of unlabelled and labelled glucoses are similar. (2) Unlabelled and labelled glucose are handled identically across forearm tissues. (3) We found no tracer impurity in our tritiated glucose preparations. We conclude that (3³H)- and (6³H)-glucose tracers can be used to reliably measure glucose turnover rates in man.     

Acetyl-salicylic acid impairs insulin-mediated glucose utilization and reduces insulin clearance in healthy and non-insulin-dependent diabetic man

Summary The effect of acetyl-salicylic acid (ASA, 3 g per day for 3 days) on glucose utilization and insulin secretion was studied in healthy volunteers and Type 2 diabetic patients using the hyperglycaemic and euglycaemic insulin clamp technique. When in healthy subjects arterial plasma glucose was acutely raised and maintained at +7 mmol/l above fasting level, the plasma insulin response was enhanced by ASA (70±7 vs. 52±7mU/l), whereas the plasma C-peptide response was identical. Despite higher insulin concentrations, glucose utilization was not significantly altered (control, 61±7; ASA, 65±6mol·kg^–1·min^–1) indicating impairment of tissue sensitivity to insulin by ASA. Inhibition of prostaglandin synthesis was not likely to be involved in the effect of ASA, since insulin response and glucose utilization were unchanged following treatment with indomethacin. In the euglycaemic insulin (1 mU·kg^–1·min^–1) clamp studies, glucose utilization was unaltered by ASA despite higher insulin concentrations achieved during constant insulin infusion (103±4vs. 89±4mU/l). In Type 2 diabetic patients, fasting hyperglycaemia (10.6 ±1.1 mmol/l) and hepatic glucose production (15±2 mol·kg^–1·min^–1) fell upon ASA treatment (8.6±0.7 mmol/l; 13±1 mol·kg^–1· min^–1). During the hyperglycaemic clamp study, the plasma response of insulin, but not of C-peptide, was enhanced by ASA, whereas tissue sensitivity to insulin was reduced by 30 percent. It is concluded that in healthy and Type 2 diabetic man, ASA impairs tissue sensitivity to the action of insulin. This effect is counterbalanced by an augmented plasma insulin response to glucose, which results from a reduced insulin clearance rate. In Type 2 diabetic patients, the reduction in hepatic glucose production may be responsible for the amelioration of hyperglycaemia following ASA treatment.     

Impaired insulin action in newly diagnosed type 1 (insulin-dependent) diabetes mellitus

Summary Hepatic and peripheral insulin sensitivity were investigated in five newly diagnosed Type 1 (insulin-dependent) diabetic subjects before and after 1 week of twice daily insulin therapy. Eight weight-matched control subjects were also studied. Hepatic glucose production and glucose utilization were measured basally and during two sequential 2-h insulin (25 and 40 mU· kg^–1· h^–1)/glucose infusion periods. In the untreated hyperglycaemic diabetic patients hepatic glucose production was 16.3±2.6, 8.1±1.1 and 3.6±2.8|mol· kg^–1· min^–1 respectively for each of the three periods (mean±SEM), and fell with treatment to 12.5±1.4, 0.5±0.5 and 0.5±0.5 mol· kg^–1· min^–1. Hepatic glucose production for normal subjects was 13.4±0.7, 2.3±0.8 and <0.1 mol-kg^–1· min^–1. Glucose utilization was 12.7±1.4,18.2±0.7 and 22.1±3.4mol· kg^–1· min^–1 before treatment in the diabetic subjects, and 11.8±1.7, 20.9±3.3 and 30.1±3.6 after treatment. These values compare with those in the euglycaemic control subjects (13.4±0.7, 18.7±1.6 and 36.3±2.7 mol · kg^–1· min^–1). The pre-treatment metabolic clearance rate of glucose in all diabetic studies with insulin levels >30mU/l was 2.6 ±0.4 and rose to 3.9 ±0.5 ml· kg^–1· min^–1 following insulin therapy. This was significantly lower than in the control subjects (6.7±0.8 ml· kg^–1 · min^–1; p<0.005). Basal nonesterified fatty acid levels were high in the untreated, but normal in the treated diabetic subjects, and fell in response to insulin infusion. Basal -hydroxybutyrate levels were high in both diabetic groups, but also fell in response to insulin infusion. Erythrocyte insulin receptor binding was normal in the untreated diabetic subjects, and was not changed by treatment. Therefore, treatment of newly diagnosed Type 1 diabetic subjects with insulin reverses the hepatic insensitivity to insulin. In contrast, treatment only partially improves peripheral glucose disposal. Since erythrocyte insulin receptor binding is normal, it is likely that a post-receptor defect in peripheral glucose metabolism exists in Type 1 diabetic patients despite insulin therapy and good diabetic control for a period of 1 week.     

Peripheral and hepatic insulin sensitivity in subjects with impaired glucose tolerance

Summary Recent evidence suggests that the post-prandial hyperglycaemia in impaired glucose tolerance is primarily due to impaired suppression of basal hepatic glucose output. This in turn appears to be secondary to decreased first phase insulin secretion, although decreased hepatic insulin sensitivity, which is a feature of non-insulin-dependent diabetes mellitus, might also play a role. Eight mildly overweight subjects with impaired glucose tolerance and eight closely matched control subjects with normal glucose tolerance underwent an intravenous glucose tolerance test to assess first phase insulin secretion. Insulin sensitivity was examined by a 150-min hyperinsulinaemic-euglycaemic clamp. Somatostatin was infused from 150 min to suppress endogenous insulin secretion, and glucagon and insulin were replaced by constant infusion. Glucose with added dideuterated glucose (labelled infusion technique) was infused to maintain euglycaemia. First phase insulin secretion ( 0–10 min insulin area ÷ 0–10 min glucose area) was significantly decreased in the subjects with impaired glucose tolerance (median [range]: 1.2 [0.2–19.4] vs 9.1 [2.6–14.5] mU·mmol^–1; p<0.01). During the clamp, circulating insulin (93±8 [mean±SEM] and 81±10 mU·l^–1) and glucagon (54±4 and 44±6 ng·l^–1) levels were comparable. Total glucose disposal was decreased in subjects with impaired glucose tolerance (2.78±0.27 vs 4.47±0.53 mg·kg^–1·min^–1; p<0.02), and was primarily due to decreased non-oxidative glucose disposal. However, hepatic glucose output rates were comparable during the clamp (0.38±0.10 and 0.30±0.18 mg·kg^–1·min^–1). Therefore, the main defects in subjects with impaired glucose tolerance are decreased first phase insulin secretion and peripheral non-oxidative glucose disposal, but hepatic glucose output shows normal responsiveness to insulin.Abbreviations FPIS First phase insulin secretion - PG plasma glucose - NIDDM non-insulin-dependent diabetes mellitus - IGT impaired glucose tolerance - HGO hepatic glucose output - IVGTT intravenous glucose tolerance test - OGTT oral glucose tolerance test     

The contribution of hyperglycaemia and hypoinsulinaemia to the insulin resistance of streptozotocin-diabetic rats

Summary The relative contribution of hyperglycaemia and hypoinsulinaemia was evaluated in rats made diabetic by streptozotocin administration. Four groups of rats were studied: untreated normal rats; streptozotocin-diabetic; streptozotocin-diabetic treated with phlorizin (0.4 mg/kg body weight per day); streptozotocin-diabetic mildly treated with insulin (0.7 IU/day). In all groups, insulin action (responsiveness) was assessed with the euglycaemic (5.3 mmol/l) hyperinsulinaemic (524 mU/l) clamp technique combined with ³H-2-deoxy-D-glucose method, enabling determination of the glucose utilization index in various tissues. Responsiveness of the overall glucose utilization process to insulin was reduced by 28% in streptozotocin-diabetic rats (12.0±1.2 vs 16.5±0.6 mg·kg^–1·min^–1, p<0.001). This was associated with a significant reduction (p<0.05) in the glucose utilization index in all muscles studied (average=17.0 vs 32.1 ng·mg of tissue^–1·min^–1), in the heart (19.6 vs 39.5 ng·mg^–1·min^–1), brown adipose tissue (98.9 vs 178.0 ng·mg^–1·min^–1), skin (6.4 vs 13.1 ng·mg^–1·min^–1). Phlorizin treatment normalized plasma glucose levels without affecting those of insulin, and restored overall glucose utilization to normal (16.6±1.0mg·kg^–1·min^–1). This normalization was accompanied by a normalization of the glucose utilization index in all muscle types studied (29.2 ng·mg^–1·min^–1), in the heart (50.0ng·mg^–1·min^–1), brown adipose tissue (157.2 ng·mg^–1·min^–1), and skin (10.0 ng·mg^–1·min^–1). White adipose tissue, brain and gut were not affected. Mild insulin treatment with persistent hyperglycaemia was not able to significantly ameliorate glucose disposal (14.5±0.9 mg·kg^–1·min^–1) or the glucose utilization index of most individual tissues (muscle=18.4; heart=36.2; brown adipose tissue=148.0; skin=7.7 ng· mg^–1· min^–1). These data show that correction of hyperglycaemia in streptozotocin-diabetic rats normalizes insulin action, while partial correction of the hypoinsulinaemia fails to do so.     

Normal insulin sensitivity during the phase of glucose intolerance but insulin resistance at the onset of diabetes in the spontaneously diabetic BB rat

Summary In diabetes-prone BB rats, 30 to 50% of animals undergo autoimmune destruction of the pancreatic B-cells leading to a short period of glucose intolerance, followed by an abrupt onset of diabetes. We have examined whether the glucose intolerance period and the onset of diabetes are associated with changes in insulin sensitivity, using the euglycaemic hyperinsulinaemic clamp coupled with [3-³H] glucose infusion. Glucose intolerant rats were detected by a transient glycosuria one hour after an oral glucose load performed every four days. Insulin sensitivity studied in these rats the day following their detection was normal. Other diabetes-prone BB rats were tested daily and studied on the first day of glycosuria. In the basal state, glucose production was increased in diabetic rats (11.3±1.1 vs 7.1±0.8mg·min^–1·kg^–1, p<0.05). Tissue glucose utilization was similar in diabetic and control rats (8.3±0.5 vs 7.1±0.8mg·min^–1·kg^–1) despite a three fold higher glycaemia in the diabetic rats. During the hyperinsulinaemic clamps, glycaemia was clamped at 6.1–6.6 mmol/l in diabetic and control rats. A decreased insulin sensitivity was observed in diabetic rats at submaximal (200 U/ml) and maximal (1500 U/ml) insulin concentrations for both inhibition of hepatic glucose production and stimulation of glucose utilization. No autoantibodies against insulin could be detected in the plasma of diabetic rats. Plasma concentrations of glucagon, catecholamines, ketone bodies and fatty acids were similar in control and diabetic rats during the clamp studies. Our results suggest that the decrease of basal insulin concentration is responsible for the insulin resistance in the diabetic BB rat at onset of diabetes, either directly or through the increased glycaemia.     

Determination and kinetic analysis of non-insulin mediated glucose uptake in Type 1 (insulin-dependent) diabetes mellitus

Summary In man, total glucose uptake is the sum of insulin mediated glucose uptake and non-insulin mediated glucose uptake. The latter pathway has not been examined in Type 1 (insulin-dependent) diabetes mellitus. In order to assess non-insulin mediated glucose uptake in Type 1 diabetes, we measured steady-state rates of glucose uptake during glucose clamps at 5.27, 9.71 and 12.5 mmol/l using low (0.25 mU· kg^–1·min^–1), intermediate (0.75 mU·kg^–1·min^–1) and high (1.50 mU·kg^–1·min^–1) insulin infusion rates in 10 subjects with Type 1 diabetes. For insulin infusion rates of 0.25, 0.75 and 1.50 mU·kg^–1·min^–1 as plasma glucose rose from 5.27 to 9.71 mmol/l, total glucose uptake increased by 35, 43 and 52 percent respectively (p<0.05 for each insulin infusion rate). For all three insulin infusion rates, there was no significant increase in total glucose uptake as plasma glucose increased from 9.71 to 12.5 mmol/l. At each glycaemic level, glucose uptake correlated significantly with plasma free insulin (r=0.81, p<0.01 at 5.71 mmol/l; r=0.84, p<0.01 at 9.71 mmol/l; r=0.73, p<0.02 at 12.5 mmol/l). Linear regression analysis to a point corresponding to plasma free insulin equalling zero, yielded values for non-insulin mediated glucose uptake (mmol·kg^–1·min^–1) of 0.11,0.14,0.18 at plasma glucose of 5.27, 9.7 and 12.5 mmol/l respectively. Thus, increasing plasma glucose concentrations were associated with increasing rates of non-insulin mediated glucose uptake. For each insulin infusion rate used, the percent of total glucose uptake accounted for by non-insulin mediated glucose uptake remained independent of plasma glucose concentration, but decreased as insulin infusion rate increased. During the insulin infusion at 0.25 mU·kg^–1·min^–1, this percentage ranged from 83.7 to 91.4%. Analysis of glucose uptake data derived for theoretical plasma insulin levels of 0, 40, 80 and 160 U/ml yielded linear Eadie-Hofstee plots (r=– 0.83 to – 0.99), suggesting that insulin increased Vmax but did not alter Km. Hence, in these subjects with Type 1 diabetes, glucose uptake, both insulin mediated and non-insulin mediated can be described by Michaelis-Menten kinetics. Comparison of values obtained for Vmax and Km in the present studies of Type 1 diabetes with those obtained from non-diabetic subjects indicates that non-insulin dependent glucose uptake in Type 1 diabetes is quantitatively similar to that of non-diabetic subjects.