Effect on insulin sensitivity of angiotensin converting enzyme inhibitors with or without a sulphydryl group: Bradykinin may improve insulin resistance in dogs and humans

Summary The present study compared the effect on insulin sensitivity of ACE inhibitors with a sulphydryl group (captopril) or those without a sulphydryl group (delapril and enalapril) during the hyperinsulinaemic euglycaemic clamp test in both animal and clinical experiments. A possible contribution of bradykinin to the improvement of insulin sensitivity by ACE-inhibition was also studied. In healthy control and depancreatized dog experiments, administration of captopril either intravenously (3.0 mmol · kg^–1) or orally (5.0 mmol · kg^–1) increased insulin sensitivity indices and plasma bradykinin concentrations. In comparison, intravenous administration of an active metabolite of delapril (3.0 mmol · kg^–1) and oral administration of either delapril or enalapril (5.0 mmol · kg^–1) showed slight, but not significant increases in insulin sensitivity indices and plasma bradykinin concentrations. Infusion of a bradykinin antagonist (N--adamantaneacetyl-d-Arg-[Hyp³, Thi^5,8,d-Phe⁷]-bradykinin) (0.5 nmol · kg^–1 · min^–1) abolished the effect of captopril on insulin sensitivity. Furthermore, intravenous administration of bradykinin (0.1 nmol · kg^–1 · min^–1) increased insulin sensitivity indices. In clinical experiments, insulin sensitivity indices decreased in the following order: normotensive healthy subjects, hypertensive non-diabetic patients, normotensive NIDDM patients and hypertensive NIDDM patients. In these four groups, oral administration of captopril (2.0 mmol · kg^–1) significantly increased insulin sensitivity indices, and a concomitant increase in plasma bradykinin concentrations was observed. By contrast, oral administration of enalapril or delapril showed slight, but not significant effects on insulin sensitivity indices and plasma bradykinin concentrations. From these studies, it is concluded that ACE inhibitors with a sulphydryl group have more potent action on the improvement in insulin sensitivity than those without a sulphydryl group. Bradykinin may also possibly be involved in the mechanism underlying the improvement in insulin sensitivity associated with ACE-inhibition.Abbreviations ACE Angiotensin converting enzyme - NIDDM non-insulin-dependent diabetes mellitus - IDDM insulin-dependent diabetes mellitus - AEP artificial endocrine pancreas     

Interactions between essential fatty acid,prostanoid, polyol pathway and nitric oxide mechanisms in the neurovascular deficit of diabetic rats

Summary Impaired -6 essential fatty acid metabolism and exaggerated polyol pathway flux contribute to the neurovascular abnormalities in streptozotocin-diabetic rats. The potential interactions between these mechanisms were examined by comparing the effects of threshold doses of aldose reductase inhibitors and evening primrose oil, alone and in combination, on neurovascular deficits. In addition, highdose aldose reductase inhibitor and evening primrose oil treatment effects were challenged by co-treatment with the cyclo-oxygenase inhibitor, flurbiprofen, or the nitric oxide synthase inhibitor, N^G-nitro-l-arginine. Eight weeks of diabetes caused an 18.9% reduction in sciatic motor conduction velocity (p<0.001). This was only modestly ameliorated by a 0.1% dietary supplement of evening primrose oil or the aldose reductase inhibitors ZD5522 (0.25 mg · kg^–1 · day^–1) and WAY121509 (0.2 mg · kg^–1· day^–1) for the final 2 weeks. However, joint treatment with primrose oil and ZD5522 or WAY121509 caused marked 71.5 and 82.4% corrections, respectively, of the conduction deficit. Sciatic nutritive blood flow was 43.1% reduced by diabetes (p<0.001) and this was corrected by 67.8% with joint ZD5522 and primrose oil treatment (p<0.001). High-dose WAY121509 (10 mg · kg^–1 · day^–1) and primrose oil (10% dietary supplement) prevented sciatic conduction velocity and nutritive blood flow deficits in 1-month diabetic rats (p<0.001). However, these effects were abolished by flurbiprofen (5 mg · kg^–1 · day^–1) and N^G-nitro-l-arginine (10 mg · kg^–1 · day^–1) co-treatment (p<0.001). Thus, the data provide evidence for synergistic interactions between polyol pathway/nitric oxide and essential fatty acid/cyclo-oxygenase systems in the control of neurovascular function in diabetic rats, from which a potential therapeutic advantage could be derived.Abbreviations ARI Aldose reductase inhibitor - EPO evening primrose oil - NCV nerve conduction velocity - NO nitric oxide - NOLA N^G-nitro-l-arginine     

Comparison of the effects of human recombinant insulin-like growth factor I and insulin on plasma amino acid concentrations and leucine kinetics in humans

Summary We examined the effects of recombinant human insulin-like growth factor I (IGF-I) and insulin on the plasma amino acid (AA) profile and leucine kinetics in eight normal subjects. IGF-I was infused at 52 pmol·kg^–1·min^–1, in combination with prime-continuous [1-¹⁴C] leucine infusion, to obtain steady-state plasma concentrations of total (54±3 nmol/l) and free (7.3±1 nmol/l) IGF-I (study 1). In response to IGF-I, plasma AA levels declined by 37±3% (1975±198 to 1368±120 mol/l) and total branched chain amino acids (BCAA) declined by 34±3% (390±21 to 256±13 mol/l). This hypoaminoacidaemic effect was associated with a decline in endogenous leucine flux of 17±2% (1.88±0.05 to 1.57±0.04 mol·kg^–1·min^–1) and leucine oxidation of 17±1% (0.31±0.02 vs 0.26±0.02 mol·kg^–1·min^–1) (both p<0.01 vs basal). The same subjects underwent a second study (study 2) in which insulin was infused at 6.22 pmol·kg^–1·min^–1 to obtain a steady-state plasma insulin concentration of 530±25 pmol/l while maintaining euglycaemia. The infusion rate was designed to match the declines in plasma BCAA levels and leucine turnover observed during IGF-I infusion. The rates of glucose infusion necessary to maintain euglycaemia during IGF-I and insulin infusion were 4.9±1.0 and 7.8±0.6 mg·kg^–1 ·min^–1, respectively. During insulin infusion total BCAA declined by 39% from 369±23 to 226±20 mol/l, leucine flux declined by 16±2% from 1.90±0.05 to 1.61±0.03 mol·kg^–1·min^–1, and leucine oxidation declined by 19±2% from 0.32±0.02 to 0.26±0.02 mol·kg^–1·min^–1. On a molar basis IGF-I was 7.3% as potent as insulin in inhibiting proteolysis. These results demonstrate that in humans: (i) the hypoaminoacidaemic response to IGF-I can be entirely ascribed to the inhibition of proteolysis; (ii) qualitatively, the effects of IGF-I and insulin on plasma AA profile and protein metabolism are similar; (iii) quantitatively, IGF-I is 14-fold less potent than insulin in suppressing protein degradation.Abbreviations IGF-I Insulin-like growth factor I - AA aminoacid - BCAA branched chain amino acids - KIC alphaketoisocaproate - ELF endogenous leucine flux - NOLD non-oxidative leucine disposal     

Effects of nitric oxide synthase inhibitor NG-nitro-l-arginine methyl ester on duodenal alkaline secretory and ulcerogenic responses induced by mepirizole in rats

The inhibition of nitric oxide (NO) production by NO synthase inhibitors stimulates HCO ₃ ^– secretion in the rat duodenal mucosa. Therefore, we examined the effects of N^G-nitro-l-arginine methyl ester (l-NAME, the NO synthase inhibitor) and nitroprusside (the exogenous NO donor) on the duodenal HCO ₃ ^– and ulcerogenic responses in anesthetized rats. Animals were administered mepirizole (200 mg/kg, subcutaneously) for induction of duodenal ulcers, and gastric acid and duodenal HCO ₃ ^– secretions were measured with or without pretreatment withl-NAME (5 mg/kg, intravenously) or nitroprusside (4 mg/kg, intravenously). Mepirizole increased acid secretion, decreased the acid-induced duodenal HCO ₃ ^– secretion, and induced hemorrhagic lesions in the proximal duodenum. The inhibition of NO production byl-NAME potentiated the acid secretory response, increased the duodenal HCO ₃ ^– secretion, and prevented the duodenal lesions, and these changes were all antagonized by simultaneous administration ofl-arginine (200 mg/kg, intravenously) but notd-arginine. On the other hand, nitroprusside slightly reduced the acid response but further decreased the HCO ₃ ^– output, resulting in aggravation of duodenal lesions induced by mepirizole. These data suggest that the inhibition of endogenous NO production by the NO synthase inhibitorl-NAME increases duodenal HCO ₃ ^– secretion and protects the duodenal mucosa against acid injury.     

Increased glucose turnover and glucose cycling in acromegalic patients with normal glucose tolerance

Summary To characterize the diabetogenic effects of growth hormone, we simultaneously measured glucose turnover with 2-³H- and 6-³H-glucose in six acromegalic patients with normal fasting blood glucose and oral glucose tolerance tests. Eight healthy volunteers served as controls. All subjects were studied under both basal conditions and during glucose infusion (2 mg · kg^–1 · min^–1). We determined true glucose production and irreversible glucose uptake using 6-³H-glucose and glucose cycling (difference between 2-³H- and 6-³H-glucose). After an overnight fast, glucose production was higher than normal in the acromegalic patients (2.18±0.15 vs 1.85±0.03 mg · kg^–1 · min^–1,p < 0.05) despite hyperinsulinaemia. The metabolic clearance rate was normal. During the glucose infusion, glucose production was not suppressed as effectively in the acromegalic patients as in controls nor was glucose uptake augmented, while metabolic clearance rate was decreased. In acromegaly, basal glucose cycling was increased (0.44 ± 0.08 vs 0.25 ± 0.07 mg · kg^–1 · min^–1, p < 0.05). Furthermore cycling of endogenous glucose measured during glucose infusion was also augmented (0.41 ± 0.05 vs 0.24 ± 0.05 mg · kg^–1 · min^–1, p < 0.05). Hence the increase of glucose cycling (70%) was much more pronounced than that of glucose production (17%). In conclusion, small defects in glucose metabolism in acromegaly can be detected with sensitive tracer methods. These derangements are confined to the liver under fasting conditions, but are of both hepatic and extrahepatic origin during glucose loading.     

An evaluation of protein requirements in methylmalonic acidaemia

A 3-month-old girl and a 13-month-old boy with vitamin B₁₂-unresponsive methylmalonic acidaemia were studied to determine responses to varying levels of protein intake of growth, nitrogen balance and organic acid metabolism. A linear increase in the excretion of methylmalonic acid was observed in both patients above a critical level of protein intake. The inflection point was judged to reflect a ceiling above which amino acid intake exceeded requirements and catabolism was initiated. Below this point in each infant there was a plateau of minimal excretion of methylmalonic acid. Within this plateau level a reasonable rate of growth and metabolic stability were achieved at intakes between 0.70 and 0.75 and between 0.75 and 1.17 g protein kg^–1, respectively, indicating that there is a range of protein tolerance and the importance of an individual approach to the provision of protein in patients with methylmalonic acidaemia. In the 3-month-old infant, nitrogen equilibrium was achieved at protein intakes above 0.6 g kg^–1 and modest nitrogen retention was attained at a protein intake of 0.75 g kg^–1, a level at which the excretion of methylmalonic acid was minimal and weight gain satisfactory. A protein intake of 1.25 g kg^–1 was required to achieve a level of nitrogen retention often considered optimal for normal growth; however, this infant demonstrated an elevated excretion of methylmalonic acid and was close to clinical illness at this level of protein intake. The 13-month-old infant demonstrated a normal level of nitrogen retention, minimal excretion of methylmalonic acid, and a satisfactory rate of growth at protein intakes of 1.0–1.17 g kg^–1. The values should prove useful guidelines for the management of infants requiring minimal intakes of protein. In studies carried out at 18–20 months of age, supplementation of the basic diet containing 0.75 g kg protein^–1 with a mixture of amino acids not containing the precursors of methylmalonic acid was associated with increase of retention of nitrogen and increased concentrations of some essential amino acids in plasma, but effects on growth and the excretion of methylmalonic acid were not significant.     

Der Einfluß des selektiven Pharmakonverbrauches der Krebszellen bei regionaler Perfusion von Tumoren mit Glycerinaldehyd

Zusammenfassung Brock und Niekamp konnten an Sarkomen der Ratte nach lokaler Perfusion mit d-Glycerinaldehyd bei einer maximalen intravasalen Wirkdosis von 5 mol · l^–1 min keine mit den Kontrollen (N-Oxyd-Lost-Behandlung) vergleichbare Heilungstendenz beobachten. Von Ardenne u. Mitarb. zeigen, daß hierbei die auf die Krebszellen wirkende Dosis d _G nur etwa ein 50stel der im Kreislauf wirkenden Dosis d _K beträgt (max. 0,1 mol · l^–1 min) und somit keine Heilung zu erwarten ist. Durch eine mittels Extremhyperthermie (44°C) im Tumorgewebe erzielbare Pharmakonverbrauchsminderung kann die effektive Wirkdosis bedeutend erhöht werden (max. 0.55 mol · l^–1 min), was bei Krebszellen der Resistenz von Ehrlich-Mäuse-Ascites-Zellen eine hohe Abtötungsquote in den Herden bzw. sogar eine Heilung ermöglichen müßte (Abtötung in vitro bei 0,2 mol · l^–1 min). Über den cytostatischen Charakter von Glycerinaldehyd unter den genannten in-vivo-Bedingungen kann zur Zeit noch kein endgültiges Urteil abgegeben werden.

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Summary After perfusing sarcomas of the rat locally with d-glycerolaldehyde (with a maximal intravascular dose of 5 mol · l^–1 min) Brock and Niekamp were unable to observe a healing tendency as compared with the controls (given N-oxide-mustard therapy). Von Ardenne and coworkers showed, that with this the dose acting on the cancer cells (d _G ) was only about 1/50 of the dose (d _K ) acting in the circulation (max. 0.1 mol · l^–1 min). Thus, no curative effect was to be expected. By means of a reduction in the consumption of drug obtained by an extreme hyperthermia (44°C) in the tumor tissue the effective acting dose can be appreciably increased (max. 0.55 mol · l^–1 min), which should make a cure possible (destruction in vitro at 0.2 mol · l^–1 min). At present no definite statement can be made regarding the cytostatic character of glycerolaldehyde.

     

Galactose metabolism in transferase-deficient galactosaemic and normal long-term lymphoid cell lines

Summary The activity (mean±SD) of galactose-1-phosphate uridyl transferase in two long-term lymphoid cell lines from Caucasian patients with transferase deficiency galactosaemia, a heterozygote, and eight normal subjects was 0, 78 and 168±55 nmol UDPG consumed (mg protein)^–1h^–1, respectively. Also, no activity was found in erythrocytes and cultured fibroblasts from the patients. A small number of cells of the galactosaemic lines cultured in medium, in which galactose was substituted for glucose, survived for 37 days. Normal and galactosaemic lines incubated withD-galactose-[1-¹⁴C] liberated 218.2±65.6 and 18.1 pmol¹⁴CO₂ (mg cellular protein)^–1 (6h)^–1, respectively. The evolution of¹⁴CO₂ fromD-glucose-[1-¹⁴C] was similar in normal and galactosaemic lines. In the presence of [³H] galactose the radioactivity incorporated into TCA-precipitated material of the galactosaemic lines was 6.8% of the normal lines. Approximately 26% and 1.3% of the total radioactivity was incorporated into molecular species with a molecular weight greater than 400000 daltons in normal and galactosaemic cells, respectively. Similar molecules were identified in the cell-free medium of both normal and deficient cells except for an 18000 daltons molecule identified only in the medium of the normal cells. These findings indicate that a small amount of galactose is metabolized in galactosaemic lines with no transferase activity.     

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.     

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.     

Transient hyperphenylalaninaemia with a high neopterin to biopterin ratio in urine

A case of transient hyperphenylalaninaemia with a maturational delay of dihydropteridine synthesis is described. With the Guthrie test, the patient showed a blood phenylalanine level of 38 mg dl^–1, which had fallen to a normal value without a phenylalanine restricted diet by 3 months of age. The neopterin level and the neopterin to biopterin ratio in the patient's urine were very high at 19 days of age. The blood phenylalanine level did not decrease when tetrahydrobiopterin (2.5 mg kg^–1) was administered at 19 days of age, while administration of tetrahydrobiopterin (7.5 mg kg^–1) at 20 days of age had decreased the blood phenylalanine level to 50% of the preloading level after 24 h. The oral phenylalanine loading test showed the pattern of classic phenylketonuria (PKU) at 15 days of age, but it showed the normal pattern at 1 year 8 months of age.     

Pancreatic islet function in omega3 fatty acid-depleted rats: Glucose metabolism and nutrient-stimulated insulin release

In order to gain information on the determinism of the perturbation of fuel homeostasis in situations characterized by a depletion in long-chain polyunsaturated ω3 fatty acids (ω3), the metabolic and hormonal status of ω3-depleted rats (second generation) was examined. When required, these rats were injected intravenously 120 min before sacrifice with a novel medium-chain triglyceride-fish oil emulsion able to provoke a rapid and sustained increase of the ω3 content in cell phospholipids. The measurement of plasma glucose, insulin, phospholipid, triglyceride, and unesterified fatty acid concentration indicated modest insulin resistance in the ω3-depleted rats. The plasma triglyceride and phospholipid concentrations were decreased in the ω3-depleted rats with abnormally low contribution of ω3 in both circulating and pancreatic islet lipids. The protein, insulin, and lipid content of the islets, as well as their intracellular and extracellular spaces, were little affected in the ω3-depleted rats. The metabolism of d-glucose in the islets of ω3-depleted rats was characterized by a lesser increase in d-[5-³H]glucose utilization and d-[U−¹⁴C]glucose oxidation in response to a given rise in hexose concentration and an abnormally low ratio between d-glucose oxidation and utilization. These abnormalities could be linked to an increased metabolism of endogenous fatty acids with resulting alteration of glucokinase kinetics. The release of insulin evoked by d-glucose, at a close-to-physiological concentration (8.3mM), was increased in the ω3-depleted rats, this being considered as consistent with their insulin resistance. Relative to such a release, that evoked by a further rise in d-glucose concentration or by non-glucidic nutrients was abnormally high in ω3-depleted rats, and restored to a normal level after of the intravenous injection of the ω3-rich medium-chain triglyceride-fish oil emulsion. Because the latter procedure failed to correct the perturbation of d-glucose metabolism in the islets of ω3-depleted rats, it is proposed that the anomalies in the secretory behaviour of islets in terms of their response to an increase in hexose concentration or non-nutrient secretagogues is mainly attributable to alteration in K⁺ and Ca²⁺ handling, as indeed recently documented in separate experiments.     

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     

Increases in plasma lysosomal enzymes in Type 1 (insulin-dependent) diabetes mellitus: relationship to diabetic complications and glycaemic control

Summary Lysosomal enzymes degrade membrane glycoconjugates, and increased circulating enzyme activity may be an important mechanism in the pathogenesis of diabetic microangiopathy. We have assayed a profile of seven lysosomal enzyme activities (nmol·h^–1·ml^–1) in platelet-free plasma from 54 Type 1 (insulin-dependent) diabetic subjects (median age 31 years) and 42 matched normal control subjects. A significant increase in median (interquartile range) enzyme activity was measured in diabetic compared to control subjects for -d-glucuronidase, 121 (97.7–171) vs 88.8 (62.8–113), p <0.001; -d-Nacetylglucosaminidase, 693 (568–799) vs 568 (462–686), p <0.001; -d-mannosidase, 23.8 (16.7–28.9) vs 14.5 (10.1–20.0), p <0.001; and -d-galactosidase, 6.94(6.11–9.99) vs 6.66(4.78–8.33), p <0.04. In contrast, -l-fucosidase, -d-galactosidase and -d-mannosidase activities were similar in diabetic and control subjects. None of the enzyme activities differed significantly (p <0.05) between 24 diabetic patients with clinical complications and 30 complication-free diabetic patients with similar glycaemic control which does not support the hypothesis that enzyme increases in diabetes arise simply by leakage from damaged tissues. In the diabetic subjects HbA₁, median (interquartile range) 9.10 (7.40–10.60), was significantly related to -d-glucuronidase (r _s = 0.56, p <0.001) and -d-Nacetylglucosaminidase (r _s = 0.55, p <0.001). We have therefore demonstrated in diabetic subjects an increase in certain lysosomal glycosidases, that correlates with glycaemic control. Such increased enzyme activities acting on endothelial and basement membrane substrates may reduce glycoconjugate content, modify charge density, and thereby influence membrane permeability, a hallmark of microangiopathy.     

5-aminoimidazole-4-carboxy-amide-1-β-D-ribofuranoside treatment ameliorates hyperglycaemia and hyperinsulinaemia but not dyslipidaemia in KKAy-CETP mice

Aim/hypothesis: 5-aminoimidazole-4-carboxy-amide-1-β-d-ribofuranoside increases 5'-AMP-activated kinase activity in insulin-sensitive tissues known to control glucose homeostasis. We hypothesised that 5-aminoimidazole-4-carboxy-amide-1-β-d-ribofuranoside treatment could have a beneficial effect on glucose homeostasis in KKA^y-CETP mice, a model of Type II (non-insulin-dependent) diabetes mellitus. Our aim was to examine potential effects of acute and chronic (7-day) 5-aminoimidazole-4-carboxy-amide-1-β-d-ribofuranoside treatment on glucose homeostasis in KKA^y-CETP diabetic mice. Methods: Female KKA^y-CETP mice were treated with 5-aminoimidazole-4-carboxy-amide-1-β-d-ribofuranoside by a single daily injection for 7 days (100, 300, or 500 mg · kg^–1· day^–1). Results: After 7 days of treatment with 500 mg · kg^–1· day^–1 5-aminoimidazole-4-carboxy-amide-1-β-d-ribofuranoside, blood glucose and plasma insulin concentrations were reduced (p < 0.01). Body weight and food intake were also reduced after treatment (p < 0.01 and p < 0.05, respectively). Glucose and insulin tolerance were improved (p < 0.05), whereas endogenous glucose production was suppressed (p < 0.05). The beneficial effect of 5-aminoimidazole-4-carboxy-amide-1-β-d-ribofuranoside on hyperglycaemia and hyperinsulinaemia was due to an inhibition of endogenous glucose production, since in vivo and in vitro basal and insulin-stimulated glucose uptake in skeletal muscle was not affected by 5-aminoimidazole-4-carboxy-amide-1-β-d-ribofuranoside. Other features of the treatment included increased plasma of free fatty acid concentration (1.9-fold, p < 0.01) and triglycerides (1.3-fold, p < 0.05). Conclusion/interpretation: 5-aminoimidazole-4-carboxy-amide-1-β-d-ribofuranoside treatment attenuated hyperglycaemia and hyperinsulinaemia but not dyslipidaemia in KKA^y-CETP mice, a model of Type II diabetes. The blood glucose lowering effects of 5-aminoimidazole-4-carboxy-amide-1-β-d-ribofuranoside occurs mainly as a consequence of reduced endogenous glucose production because insulin-stimulated skeletal muscle glucose uptake has not been altered. [Diabetologia (2001) 44: 2180–2186] Received: 21 June 2001 and in revised form: 6 August 2001     

Anomalous leucine metabolism in total lipoatrophic diabetes: a possible mechanism of muscle mass hypertrophy

Total acquired lipoatrophic diabetes (LD) is characterized by muscle mass hypertrophy, non-ketotic hyperglycaemia and insulin resistance with respect to glucose and lipid metabolism. To assess whether the defect in insulin action extends to leucine/protein metabolism, a female subject (age=33 years; body weight=44 kg; HbA_lc=9.5%) with LD was studied twice: in study I we used a three-step euglycaemic hyperinsulinaemic clamp (40, 80 and 200 mU · m^–2 · min^–1) combined with [3-³H]glucose and [1-¹⁴C]leucine infusions along with indirect calorimetry. In study II we used a 40 mU · m^–2 · min^–1 euglycaemic hyperaminoacidaemic (plasma leucine 160 mol/l) hyperinsulinaemic clamp. Five controls were also studieD. In the basal state the patient with LD had plasma leucine (130 mol/l), isoleucine (63), valine (169) and phenylalanine levels (48) comparable to those of the controls. Basal hepatic glucose production (3.2 vs 2.0±0.2 mg · kg^–1 · min^–1), endogenous leucine flux (ELF=45.4 vs 40±1 mol · m^–2 · min^–1) and non-oxidative leucine disposal (NOLD=37.2 vs 34±1 mol · m^–2 · min^–1) were increased in the patient with LD, while basal leucine oxidation (LO=8.2 vs 6.0±2 mol · m^–2 · min^–1) was similar in LD and controls. Following the three-step insulin infusion, insulin-stimulated glucose metabolism was defective in the subject with LD (glucose oxidation=60%, 50%, and 52% of controls; non-oxidative glucose disposal =39%, 34% and 30% of controls at 40, 80 and 200 mU · m^–2 · min^–1 respectively). The decrease of leucine, isoleucine, valine and phenylalanine, as well as the suppression of ELF, LO and NOLD was defective in the subject with LD at each insulin step. In vitro studies demonstrated a defect in receptor insulin binding on erythrocytes, the absence of anti-insulin receptor antibodies and the presence of insulin antibodies in the serum of the patient with LD. Following combined hyperaminoacidaemia/hyperinsulinaemia a similar stimulation of protein synthetic rate (20 vs 30%) was demonstrated in the patient with LD and controls respectively. In conclusion the patient with LD shows a reduced insulin sensitivity and a reduced maximal response to insulin in both glucose and protein metabolism. The present data support the hypothesis that in LD the defect in insulin action is both at the receptor and post-receptorial sites. The patient with LD showed a normal stimulation of protein synthesis under combined hyperinsulinaemic/hyperaminoacidaemic conditions. Our results may explain the muscle mass hypertrophy in LD.     

Hypoglycaemic brain damage: effect of a dihydropyridine calcium channel antagonist in rats

Summary Hypoglycaemic brain damage consists of selective necrosis of cerebral neurons related to the extracellular release of excitatory amino acids. Neuronal excitatory amino acid receptors are activated and calcium channels are opened. The present investigation was designed to test the effectiveness of dihydropyridine blockade of voltage-sensitive calcium channels in hypoglycaemic brain damage. Sixty-four rats were given either high-dose nimodipine, consisting of an initial bolus of 300 g/kg nimodipine administered at the stage of EEG slowing (blood glucose levels of 1.0–1.5 mmol/l), followed by continuous intravenous nimodipine infusion at 1.5 g · kg^–1 · min^–1, low-dose nimodipine, consisting of an initial bolus of 30 g/kg at the time of EEG slowing, followed by 0.15 g · kg^–1 · min^–1, an equal volume of vehicle solution, or 154 mmol/l NaCl. Animals receiving either low- or high-dose nimodipine had higher mortality, and increased brain damage compared with controls. Examination of the perfusion-fixed brains 1 week after recovery with glucose revealed that quantitated neuronal necrosis was worsened by nimodipine in the hippocampus, caudate nucleus and cerebral cortex. The present results in profound hypoglycaemia (accompanied by a flat EEG) contrast with the beneficial effect of nimodipine in brain ischaemia.Abbreviations EAA Excitatory amino acid - NMDA N-methyl-d-aspartate - VSCC voltage-sensitive calcium channels - ANOVA analysis of variance     

Comparison of the effects on glucose and lipid metabolism of equipotent doses of insulin detemir and NPH insulin with a 16-h euglycaemic clamp

Aims/hypothesis The association of insulin detemir with non-esterified fatty acid binding sites on albumin may limit its transfer from the circulation into the extravascular extracellular space in adipose tissue and muscle, due to the capillary endothelial cell barrier. In the liver, the open sinusoids may expose hepatocytes to insulin detemir, enabling it to have a greater effect in the liver than in peripheral tissues.Methods We investigated the effects of equipotent doses of insulin detemir and NPH insulin on hepatic glucose rate of appearance (R_a), peripheral glucose rate of disposal (R_d) and glycerol R_a (a measure of lipolysis) using stable isotope techniques. We also investigated the effects of these insulins on NEFA concentrations in seven healthy volunteers during a 16-h euglycaemic clamp. A higher dose of insulin detemir was also studied.Results There was no difference in the glucose infusion profile between insulin detemir and NPH. Insulin detemir had a greater effect on mean suppression of glucose R_a (mean difference 0.24 mg kg^–1 min^–1; CI 0.09–0.39; p<0.01), and minimum glucose R_a, with minimum low dose detemir –0.10±0.15 mg·kg^–1·min^–1 and minimum NPH 0.17±0.10 mg·kg^–1·min^–1 (p<0.02). However, it had a lesser effect on mean suppression of NEFA concentrations (mean difference –0.10 mmol/l; CI –0.03 to –0.17; ANOVA, p<0.02) than NPH. The effect of insulin detemir on glucose R_d and glycerol R_a was not different from NPH. Following high-dose detemir, total glucose infused and maximum glucose R_d were higher (p<0.02, p<0.03) and plasma NEFA concentrations lower (p<0.01) than with low-dose determir.Conclusions/interpretation This study suggests that insulin detemir, when compared to NPH insulin, has a greater effect on the liver than on peripheral tissues and thus has the potential to restore the physiological insulin gradient.     

d-Lysine reduces the non-enzymatic glycation of proteins in experimental diabetes mellitus in rats

Summary d-Lysine, the non-physiological isomer of l-lysine, can competitively reduce protein non-enzymatic glycation in vitro. To study the effect of d-lysine in vivo, 6–8-week old Sprague-Dawley rats with streptozotocin-induced diabetes mellitus were treated from diagnosis for 45 days with two daily subcutaneous injections of d-lysine (0.5 g·ml^–1·day^–1). Another group of diabetic rats was only injected with equal volumes of physiological saline (0.9% NaCl). Glycated haemoglobin was measured by ion exchange chromatography, and glycated serum and lens proteins by boronate affinity gel chromatography. Serum and urinary creatinine concentrations were evaluated by the alkaline-picrate reaction. Urinary lysine concentrations at mid- and end-study were evaluated by cation exchange chromatography. Blood glucose concentrations, serum creatinine levels and creatinine clearances, measured at the end of the study, were similar in both diabetic groups (> 22.0 mmol/l, 106 mol/l and 0.02 ml/s, respectively). Urinary lysine concentration in d-lysine-treated diabetic animals was more than 50-fold higher than in placebo-treated diabetic rats. In d-lysine-treated vs placebo-treated diabetic animals, a statistically significant reduction was found in the levels of glycated haemoglobin (stable HbA₁; mean ± SD=3.00±0.74% vs 4.02±0.46%, p<0.05; labile HbA₁=3.92±0.89% vs 5.84±0.61%, p<0.005), glycated serum proteins (1.40±0.47% vs 2.52±1.15%, p<0.05) and glycated lens proteins (4.90±0.96% vs 5.98±0.65 %,p<0.05). Thus, d-lysine (i) is not nephrotoxic and (ii) causes a significant reduction of the early glycation products at the protein level. Therefore, the d-amino acid could be useful in attempting to control damaging phenomena associated with or due to an enhanced protein non-enzymatic glycation.