Effect of insulin on GLUT-4 mRNA and protein concentrations in skeletal muscle of patients with NIDDM and their first-degree relatives

Summary We examined whether insulin resistance, i. e. impaired insulin stimulated glucose uptake in NIDDM patients and their first-degree relatives is associated with alterations in the effect of insulin on the expression of the GLUT-4 gene in skeletal muscle in vivo. Levels of GLUT-4 mRNA and protein were measured in muscle biopsies taken before and after a euglycaemic insulin clamp from 14 NIDDM patients, 13 of their first-degree relatives and 17 control subjects. Insulin stimulated glucose uptake was decreased in the diabetic subjects (19.8±3.0 mol · kg LBM^–1 · min^–1, both p<0.001) compared with control subjects (44.1±2.5 mol · kg LBM^–1 · min^–1) and relatives (39.9±3.3 mol · kg LBM^–1 · min^–1). Basal GLUT-4 mRNA levels were significantly higher in diabetic subjects and relatives compared to control subjects (99±8 and 108±9 pg/g RNA vs 68±5 pg/g RNA; both p<0.01). Insulin increased GLUT-4 mRNA levels in all control subjects (from 68±5 to 92±6 pg/ug RNA; p<0.0001), but not in the diabetic patients (from 99±8 to 90±8 pg/g RNA, NS), or their relatives (from 94±9 to 101±11 pg/g RNA, NS). In the relatives, individual basal GLUT-4 mRNA concentrations varied between 55 and 137 pg/g RNA. Insulin-resistant (n=6, mean glucose uptake rate=30.6±3.4 mol · kg LBM^–1 · min^–1) but not insulin-sensitive relatives (n=7, mean glucose uptake rate=47.4±3.2 mol · kg LBM^–1 · min^–1) had higher basal GLUT-4 mRNA concentrations compared to control subjects (108±9 vs 68±5 pg/ug RNA, p<0.01). GLUT-4 protein content in muscle did not differ between the groups in the basal state and remained unchanged in all groups after insulin infusion. Neither insulin-stimulated GLUT-4 mRNA nor protein concentrations correlated with insulin-stimulated glucose uptake in any of the groups studied. We conclude, that impaired glucose uptake in NIDDM is not related to insulin-stimulated GLUT-4 mRNA or protein concentrations. Acute stimulation of GLUT-4 mRNA by insulin is altered in skeletal muscle of NIDDM patients and their first-degree relatives. This might be a consequence of chronic hyperinsulinaemia elevating basal GLUT-4 mRNA concentrations rather than the cause of insulin resistance.Abbreviations NIDDM Non-insulin dependent diabetes mellitus - GLUT-4 glucose transporter 4 - LBM lean body mass - IDDM insulin-dependent diabetes mellitus     

Islet B-cell dysfunction and the time course of recovery following chronic overinsulinisation of normal rats

Summary Appropriate insulin therapy may preserve or improve islet B-cell function whereas the effects of overinsulinisation are unclear. Pancreatic islet B-cell function was therefore studied after overinsulinisation of normal rats for 4 weeks (fed blood glucose 2.2–4.5 mmol/l, controls 4.1–7.0 mmol/l). Insulin secretion was assessed by a 3-h hyperglycaemic clamp (10.0 mmol/l) performed 1, 48, and 120 h after insulin withdrawal (n=6 in each group). When the clamp was performed 1 h after insulin withdrawal, clamp insulin concentration was 1.6±0.1 g/l, compared to 9.3±1.0 g/l in control rats. The integrated area under the plasma insulin concentration curve was also significantly decreased (4.8±0.4 vs 20.3±2.2 g·l^–1·h^–1, p<0.001), but recovered to 9.4±1.0 g·l^–1·h^–1 after 48 h, and to 17.5±1.4 g·l^–1·h^–1 after 120 h. Pancreatic insulin contents were decreased at 1 h (6±1 g/g wet wt) and 48 h (54±12 g/g wet wt) but not at 120 h (221±30 g/g wet wt) after withdrawal (controls, 303±29 /g wet wt) and there was a strong relationship with pancreatic preproinsulin mRNA and the clamp insulin response. Thus, overinsulinisation with prolonged periods of low blood glucose concentrations impairs islet B-cell function, but is reversible over 5 days.     

Non-esterified fatty acids do not contribute to insulin resistance in persons at increased risk of developing Type 2 (non-insulin-dependent) diabetes mellitus

Summary The mechanisms underlying insulin resistance in Type 2 (non-insulin-dependent) diabetes mellitus are not fully understood. An enhanced lipid/non-esterified fatty acid oxidation could provide an explanation. To test this hypothesis we examined the relationship between glucose and lipid metabolism in 44 first-degree relatives (28 glucose-tolerant and 16 glucose-intolerant) of Type 2 diabetic patients and in 18 healthy control subjects. Total body glucose disposal was impaired among both glucose-tolerant and glucose-intolerant relatives compared with control subjects (36.3±3.8 and 30.4±2.7 vs 47.7±3.4 mol · kgLBM^/s-1· min^–1; p < 0.05). The impairment in glucose disposal among the relatives was primarily accounted for by impaired non-oxidative glucose metabolism (14.8±3.0 and 12.5±1.8 vs 25.3±3.1 mol · kgLBM^–1 · min^–1; p <0.05). Plasma non-esterified fatty acid concentrations were similar in both glucose-tolerant and glucose-intolerant relatives and control subjects (646±36,649±43 and 615±41 mol/l) and showed the same degree of suppression by insulin (99±8, 86±7 and 84±9 mol/l). Basal lipid oxidation was similar in all groups (1.29±0.09, 1.52±0.13 and 1.49±0.21 mol · kgLBM^–1· min^–1). Furthermore, insulin suppressed lipid oxidation to the same degree in glucose-tolerant, glucose-intolerant relatives and control subjects (0.65±0.13, 0.88±0.15 and 0.59±0.09mol · kgLBM^–1 · min^–1). An inverse correlation between plasma non-esterified fatty acid concentration and total body glucose disposal was observed in the group of control subjects (r=–0.540; p<0.05), but not among the relatives (r=0.002; p=N.S.). In conclusion the present data challenge the view that the glucose-fatty acid cycle contributes to the insulin resistance seen in first-degree relatives of patients with Type 2 diabetes.     

Age-related Variations in the Neuroendocrine Control,More Than Impaired Receptor Sensitivity,Cause The Reduction in the GH-releasing Activity of GHRPs in Human Aging

The mechanisms underlying the reduction in the GH-releasing activity of GHRPs in aging are still unclear. Aim of our study was to verify in man whether age-related impairment of the neurohormonal control of GH secretion and/or receptor alterations are involved in the reduced GH response to GHRPs in aging. To this goal, in 16 normal elderly subjects (E, 66–81 yr) and 12 young controls (Y, 24–28 yr) we studied the effects of 1.0, 2.0 and 3.0 g/kg iv Hexarelin (HEX), a synthetic hexapeptide, or GHRH, as well as the interaction among HEX (2.0 g/kg), GHRH (2.0 g/kg) and arginine (ARG, 0.5 gr/kg) on GH secretion. In Y the GH response to increasing doses of HEX (1.0 vs. 2.0 vs. 3.0 g/kg; AUC0;v–120 ± SEM: 1728.4 ± 406.4 vs. 2265.9 ± 298.4 vs. 2934.3 ± 482.2 g//L/h, p < 0.05 for 1.0 vs. 2.0 g/kg) and GHRH (649.6 ± 111.4 vs. 792.2 ± 117.6 vs. 1402.6 ± 363.0 g/L/h) showed a progressive increase. Two g/kg HEX and 1 g/kg GHRH were the maximal effective doses. Similarly, in E the GH response to increasing doses of HEX (336.7 ± 50.0 vs. 742.8 ± 157.9 vs. 1205.1 ± 178.1 g/L/h, p < 0.05 for 1.0 vs. 2 g/kg, p < 0.001 for 1.0 vs. 3.0 g/kg and p < 0.03 for 2.0 vs. 3.0 g/kg) and GHRH (183.8 ± 27.3 vs. 260.9 ± 17.3 vs. 356.1 ± 46.3 g/L/h, p < 0.005 for 1.0 vs. 3.0 g/kg and p < 0.05 for 2.0 vs. 3.0 g/kg) showed a progressive increase. In E the GH response to 3 g/kg HEX or GHRH were clearly higher than those to 2 g/kg. However, at each dose the GH responses to HEX or GHRH in E were lower (p < 0.05) than those in Y. In Y the GH response to HEX + GHRH was synergistical (4259.2 ± 308.0 g/L/h, p < 0.05). ARG strikingly potentiated the GHRH-induced GH rise (2640.8 ± 273.6 g/L/h, p < 0.01) but not the HEX-induced one (2371.7 ± 387.2 g/L/h) as well as the synergistical effect of HEX and GHRH (4009.1 ± 360.8 g/L/h). In E the GH response to HEX and GHRH was still synergistical (1947.7 ± 306.0 g/L/h, p < 0.05) but these responses were lower than those in young (p < 0.01). On the other hand, in E ARG restored the GH response to GHRH (1858.9 ± 172.8 g/L/h, p < 0.01) and even those to HEX (2069.5 ± 528.7 g/L/h, p < 0.01) and HEX + GHRH (4406.0 ± 1079.2 g/L/h, p < 0.05). Our present results indicate that the impairment of GHRP and GHRH receptor activity may have a role in the reduction of the somatotrope responsiveness in aging. However, the age-related reduction in the GH-releasing activity of GHRPs seems mainly dependent on age-related variations in the neural control, i.e. concomitant GHRH hypoactivity and somatostatinergic hyperactivity.     

A study of zinc distribution in erythrocytes of normal humans

Summary The assignment of zinc bound to carbonic anhydrase isoenzymes (CA-I and CA-II) and Cu₂ Zn₂ superoxide dismutase (SOD₁) was investigated in the hemolysates from 21 normal male subjects.Sufficient care was taken to remove leukocytes and platelets. The following values of zinc distribution were obtained:total zinc, 1113.8±22.7 (mean±S.E.) g · 100 ml^–1; CA-I-derived zinc, 866.6±26.2 g · 100 ml^–1; CA-II-derived zinc, 99.9±3.9 g · 100 m^–1; SOD₁-derived zinc, 60.3±1.9 g · 100 ml^–1; the other zinc, 87.0±12.6 g · 100 ml^–1. Namely, 7.6% of the zinc in human erythrocytes is not bound to the carbonic anhydrases and Cu₂Zn₂ superoxide dismutase, but present in available form or attached to other enzymes.     

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.     

Antioxidant effects and anti-elastase activity of the calcium antagonist nicardipine on activated human and rabbit neutrophils--a potential antiatherosclerotic property of calcium antagonists?

Activated neutrophils which produce certain proteases, such as elastase and reactive oxygen species (ROS) are involved in oxidative stress and inflammation. In the present study, we have shown that nicardipine, a calcium channel blocker, affects the release of elastase and superoxide anion radicals (O₂ ^–) in vitro during human and rabbit neutrophil respiratory bursts. The drug inhibited the release of elastase and O₂ ^– by fMLP (N-formyl-methionylleucin-phenylalaninin), calcium ionophore (A23187) and PMA (phorbol-myristate-acetate)-stimulated human and rabbit neutrophils. Besides the release of elastase, strongly inhibited in the fMLP and A23187 stimulated systems, nicardipine affected elastase and O₂ ^– in a dose-dependent manner. The corresponding 50% inhibitory concentration (IC₅₀) of nicardipine for elastase, released in PMA-stimulated human and rabbit neutrophils, was 15.95 ± 0.17 M and 18.06 ± 0.08 M, respectively, whereas for O₂ ^–, the IC₅₀ of nicardipine in PMA, fMLP and A23187-stimulated human and rabbit neutrophils was 55.41 ± 0.09 M and 58.43 ± 0.03 M, 45.21 ± 0.13 M and 37.19 ± 0.53 M, 33.54 ± 0.09 M and 30.54 ± 0.29, respectively. The mechanisms underlying the inhibition of elastase and superoxide anion radicals by nicardipine appear related to an inhibiting effect on the mobilisation of cytosolic calcium and on activation of protein kinase C (PKC). These antioxidant and anti-elastasic activities contribute to the properties of nicardipine, as positive side effects of its antihypertensive activity and may be useful to prevent inflammatory disorders (tissue damage, oxidative injury) involved in the pathogenesis of hypertension.     

Modulation of hepatic glucose production by non-esterified fatty acids in Type 2 (non-insulin-dependent) diabetes mellitus

Summary To study the effect of changes in plasma non-esterified fatty acid concentration on suppression of hepatic glucose production by insulin eight Type 2 (non-insulin-dependent) diabetic patients participated in three euglycaemic, hyperinsulinaemic (108pmol · m^2–1 · min^–1) clamp studies combined with indirect calorimetry and infusion of [3-³H]-glucose and [1-¹⁴C]palmitate; (1) a control experiment with infusion of NaCl 154 mmol/l, (2) heparin was infused together with insulin, and (3) an antilipolytic agent, Acipimox, was administered at the beginning of the experiment. Six healthy volunteers participated in the control experiment. Plasma non-esterified fatty acid concentrations during the insulin clamp were in diabetic patients: (1) 151±36 mol/1, (2) 949±178 mol/l, and (3) 65±9 mol/l; in healthy control subjects 93±13 mol/l. Non-esterified fatty acid transport rate, oxidation and non-oxidative metabolism were significantly higher during the heparin than during the Acipimox experiment (p<0.001). Suppression of hepatic glucose production by insulin was impaired in the diabetic compared to control subjects (255±42 vs 51±29 mol/min, p<0.01). Infusion of heparin did not affect the suppression of hepatic glucose production by insulin (231±49 mol/min), whereas Acipimox significantly enhanced the suppression (21±53 mol/min, p<0.001 vs 154 mmol/l NaCl experiment). We conclude that insulin-mediated suppression of hepatic glucose production is not affected by increased non-esterified fatty acid availability. In contrast, decreased non-esterified fatty acid availability enhances the suppression of hepatic glucose production by insulin.     

Liver and muscle insulin sensitivity,glycogen concentration and glycogen synthase activity in a rat model of non-insulin-dependent diabetes

Summary Mild diabetes was induced in adult rats with streptozotocin (45 mg/kg body weight), and insulin sensitivity, glycogen deposition and glycogen synthase activity assessed in liver and muscle 5 weeks later. Diabetic rats had significantly elevated fasting blood glucose concentrations (5.6±0.1 versus 3.6±0.1 mmol/l, p<0.001), and blood glucose concentrations 2 h after a 1 g/kg glucose load (12.0±0.6 versus 3.7±0.2 mmol/l, p<0.001). After a 20-h fast hepatic glucose output was significantly elevated (58±3 versus 47±3 mol·min^–1·kg^–1, p<0.05), and failed to suppress at high insulin concentrations during a euglycaemic clamp (hepatic glucose output 21±4 versus 2±4 mol·min^–1·kg^–1, p<0.02). Liver glycogen was lower in the diabetic rats by the end of the clamp (16±3 versus 38±6 mol/g wet wt, p<0.05). At the end of the clamp total glucose turnover was lower in the diabetic rats (107±4 versus 161±17 mol·min^–1· kg^–1, p<0.01), as was skeletal muscle glycogen synthase activity (0.46±0.04 versus 0.67±0.05 U/g wet wt, p<0.01) and glycogen concentration (22±2 versus 33±3 mol/g wet wt, p<0.05). Blood lactate and pyruvate responses suggested that glycolytic pathways were similarly affected. Thus, insulin insensitivity develops in both liver and skeletal muscle after 5 weeks of mild streptozotocin-induced diabetes.     

Urinary albumin excretion in diabetic pregnancy

Summary We have analysed the results of urinary albumin excretion in timed overnight urine samples once every two weeks during pregnancy and post-natally in 25 non-diabetic women and 14 women with Type 1 (insulin-dependent) diabetes who were Albustix negative and had urinary albumin excretion <15 g· min^–1 at conception. Urinary albumin excretion did not vary significantly in the first two trimesters in either group and at 28 weeks was 2.73 g·min^–1 (0.32–251.68) (median and range) in the diabetic women and 2.53 g·min^–1 (0.90–13.37) in control patients (not significant). During the third trimester urinary albumin excretion increased, and levels were significantly higher in diabetic patients from 36 weeks (9.37 (0.9–31.78) vs 3.52 (0.19–33.74) g· min^–1, p<0.01) until delivery. In both groups, urinary albumin excretion reached a peak within the week following delivery — diabetic 17.42 g·min^–1 (2.03–46.64), control subjects 16.29 g· min^–1 (1.53–35.56), but six weeks after delivery, levels were similar to those in early pregnancy. The effect of pregnancy on urinary albumin excretion in these diabetic women would appear to be an exaggeration of the normal pattern, with levels returning to normal post-delivery. It is not possible to know if this has significance for future renal function, but it would be important to investigate this phenomenon in patients who already have raised urinary albumin excretion at conception.     

Granulocyte Elastase in Cirrhotic Patients with Spontaneous Bacterial Peritonitis

Granulocyte elastase (GE) is a powerfulproteolytic enzyme that is released by PMNs whendegranulated in infectious processes. The aim of thisstudy was to measure GE in ascites and plasma ofcirrhotic patients with spontaneous bacterial peritonitis(SBP). We studied 29 cirrhotic patients, 17 of themhaving SBP (group A). Twelve patients with noninfectedascites formed the control group (group B). At the time of diagnosis of SBP, GE levels inascites (183.17 ± 86.11 g/liter) and plasma(114.6 ± 35.99 g/liter) were higher in groupA than in group B (27.41 ± 11.54 g/liter, P< 0.00001 and 82.54 ± 20.52 g/liter, P = 0.01,respectively). Levels of GE in ascites had a high valuefor discriminating between patients with and withoutSBP. In the patients who responded to the initialantibiotic treatment, these values significantly decreasedin ascites (67.69 ± 54.22 g/liter, P = 0.003)and plasma (67 ± 22.39 g/liter, P = 0.01) 48hr after therapy was started, in parallel with thedecrease of PMN in ascites. In patients who did notrespond, the production of GE remained elevated.Patients who developed renal insufficiency following SBPhad more marked elevation of GE in plasma (144.8± 33.43 g/liter) than those with normal renalfunction (99.5 ± 27.53 g/liter, P = 0.02).These results suggest that the measurement of GE may behelpful for the diagnosis of SBP in patients withcirrhosis and for assessing the efficacy of therapy. Inaddition, the release of GE into plasma may contributeto the impairment of renal function that follows SBP insome patients.     

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.     

Determination of Biomechanical Properties in Guinea Pig Esophagus by Means of High Frequency Ultrasound andImpedance Planimetry

Impedance planimetry and high-frequency ultrasound were used to determine circumferential stress and strain from measurements of luminal cross-sectional area and wall thickness during balloon distension of the guinea pig esophagus in vitro (N = 30). The excised esophagus was mounted on two plastic tubes in an organ bath containing oxygenated calcium-free Krebs-Ringer solution with 10^–2 M MgCl₂ to abolish smooth muscle contractile activity. One of the plastic tubes was movable in order to stretch the esophagus longitudinally by 15% (elongated state). The impedance planimetry probe was placed with the balloon inside the lumen of the esophagus. A 20-MHz ultrasound transducer was mounted above the esophagus and provided scans in the transverse and longitudinal directions. The luminal cross-sectional area at the highest applied pressure of 2.9 kPa was 13.3 ± 0.3 mm² in the resting state. In the elongated state the luminal cross-sectional area at the highest pressure was 12.5 ± 0.1 mm² (P < 0.02). The wall thickness decreased from 990 ± 21 m at 0 kPa to 640 ± 9 m at 2.9 kPa at in vitro length. In the elongated state, the values were 940 ± 32 m to 480 ± 13 m (P < 0.01). The stress–strain relation was exponential ( = (e – 1), r ² > 0.98, P < 0.01). The circumferential elastic modulus calculated at a Green strain of 0.95 was 44.5 ± 10.5 kPa in the in vitro state and 81.7 ± 13.1 kPa in the elongated state. The elastic modulus differed between the resting and elongated states (P < 0.02).     

Determination of human ketone body kinetics using stable-isotope labelled tracers

Summary In order to avoid the use of radioactive tracers for the determination of human ketone body turnover, we have developed a method using a primed-continuous infusion of ¹³C-labelled acetoacetate or D--hydroxybutyrate. Determination of the mole percent enrichment of blood acetoacetate and D--hydroxybutyrate was performed by gas chromatography/mass spectrometry. In the post-absorptive state, the mean total ketone body appearance rate, determined in four subjects, was 3.74 mol · kg^–1 · min^–1 using [3,4-13 C2] acetoacetate and 2.76 mol · kg^–1 · min^–1 using [3-¹³C]D--hydroxybutyrate, values in agreement with those reported in studies with ¹⁴C-labelled tracers. In order to evaluate the usefulness of the method for determination of ketone body kinetics in non steady-state conditions, we infused four subjects with natural sodium acetoacetate and calculated the isotopically determined total ketone body appearance rate using a single compartment model (volume of distribution 0.201/kg; functional pool fraction: 1). During the tests with [3,4-¹³C2]-acetoacetate, the actual infusion rates of natural acetoacetate were 7.3±0.3, 14.6±0.8, 21.9±1.2 and 10.9 ± 0.6 mol · kg^–1 · min^–1 whereas the corresponding isotopically determined total ketone body appearance rates were respectively 9.2±1.0, 16.3±0.7, 23.1±1.1 and 10.7±0.8 mol· kg^–1 · min^–1. During the tests with [3-¹³C]D--hydroxybutyrate, the actual infusion rates were 8.4 ± 0.5, 16.8 ± 0.9, 25.2 ±1.4 and 12.6±0.8 mol · kg^–1 · min^–1, and the isotopically determined appearance rates respectively 11.1±0.7, 16.7±0.7, 25.0±1.1 and 11.1 ± 0.7 mol · kg^–1 · min^–1. Thus, using either tracer we found a good agreement between acetoacetate infusion rate and tracer-determined appearance rate of ketone bodies. In conclusion, the present method may be used to determine human ketone body kinetics under steady-state and non steady-state conditions.     

A Three-dimensional Analysis of Blood Vessels in Bronchioloalveolar Carcinoma

The three-dimensional architecture of blood vessels within lung adenocarcinomas has not been well studied. In 19 cases with bronchioloalveolar carcinoma with central fibrosis, we three-dimensionally examined blood vessel architecture in 150 m thick sections stained with elastin staining and anti-CD34 antibody. We examined four regions: normal alveoli and three regions within the tumor including an area adjacent to the normal alveoli (external area), an area in which tumor cells were replacing epithelial cells (replacement area), and a central fibrotic area (fibrotic area). Elastin staining showed that elastic fibers formed the framework of the alveoli, and the alveolar structure shrank more strongly to the center of the tumor due to folding of alveolar walls invaded by adenocarcinoma cells. We also measured three vessel parameters in these four regions. The vessel diameters were 4.08±1.10 m, 3.95±1.02 m, 5.04±1.56 m, and 6.11±2.23 m, the circumferences of those vessels seen as complete circles were 43.11±12.78 m, 43.71±12.87 m, 95.21±39.32 m, and 126.77±54.65 m; the lengths between vessel bifurcations were 13.28±3.08 m, 13.47±4.58 m, 24.91±9.66 m, and 41.82±28.08 m in the normal alveoli, and the external, replacement, and fibrotic areas, respectively. Blood vessel architecture changed such that the vessels became larger and coarser towards the center of the tumor. Our three-dimensional analysis suggests continuous remodeling of alveolar capillaries rather than angiogenesis within bronchioloalveolar carcinoma.     

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.     

Synovial fluid nitric oxide levels in patients with knee osteoarthritis

Nitric oxide (NO) has an important role in the inflammatory arthropathies. This study investigated NO levels in the synovial fluid and plasma of patients with primary osteoarthritis (OA) of the knee. Twenty-seven cases with primary knee OA and 13 controls were recruited for the study. Nitrate/nitrite levels of synovial fluid and plasma were measured by Griess reaction, and interleukin-1 (IL-1) levels were measured quantitatively by a sandwich immunoassay technique. We found a significant increase in the synovial fluid nitrate/nitrite levels in cases with primary OA of the knee compared to controls (50.26±23.63 g/l vs 32.49±10.05 g/l, p=0.002) as well as increased plasma nitrate/nitrite levels (57.06±23.32 g/l vs 39.98±16.36 g/l, p=0.012). There was no difference in plasma and synovial fluid IL-1 concentrations between the study and control groups. These results may be considered as supporting evidence that NO might be one of the factors responsible for cartilage destruction in primary osteoarthritis of the knee.Abbreviations NO Nitric oxide - OA Osteoarthritis - TMJ Temporomandibular joint     

Gastric diversion or pylorus ligation for gastric mucosal integrity and acid secretion studies in the rat?

The advantages of gastric diversion over pylorus ligation in rat gastric mucosal integrity and acid secretion studies over 6 hr were investigated. Mucosal injury developed in 80% of pylorus-ligation controls. Atropine (5 mg/kg) or cimetidine (40 mg/kg) had no effect on this injury (2.9 mm²±0.9 and 2.8 mm²±0.7, respectively, vs 3.1 mm²±1, mean±sem, N=10; however vagotomy increased it (13.7 mm²±Pylorus-ligation H⁺ output was higher than that of gastric diversion (390.5 mol±54.8 vs 61 mol±2.5, mean±sem, N=10, P<0.001). Cimetidine (40 mg/kg) depressed H⁺ output of gastric diversion (21.3 mol±1.2 vs 61 mol±2.5, mean±sem, N=10, P<0.001), but not of pylorus ligation (424 mol±74.2 vs 390.5 mol±54.8, mean±sem, N=10). Vagotomy or atropine depressed pylorus-ligation H⁺ output (P<0.001), but each allowed an output (36.6 mol±5.5 and 120 mol±29, respectively, mean±sem, N=10) significantly (P<0.001) higher than that associated with it in gastric diversion (16 mol±1.4 and 17.1 mol±1.6, respectively, mean±sem, N=10). This study demonstrates that in the rat pylorus ligation, in contrast to gastric diversion, injures the gastric mucosa, stimulates H⁺ secretion, and overshadows the efficacy of antisecretory agents.     

Surgery or chemoneurolysis for complete vagal denervation of rat stomach?

This study compared the completeness of vagal denervation of the rat stomach by transection vagotomy to that by chemoneurolysis (30% ethyl alcohol) alone or supplemented by truncal vagotomy. The H⁺ output over 6 hr with vagotomy by chemoneurolysis (10.5±0.7 mol, mean±sem,N=10) or truncal vagotomy and chemoneurolysis (10.9 ±1.1 mol, mean±sem,N=10) was similar to that with transection vagotomy, but significantly (P<0.01) lower than that with truncal vagotomy by chemoneurolysis (17.9 ±1.1 mol,N=10). The latter output was similar to that of truncal vagotomy performed surgically (18.2±1.3 mol,N=10). Reserpine (0.1 mg/kg intraperitoneal) stimulated gastric acid secretion relative to control values (207±3.1 mol vs 67±3.2 mol,N=10),P<0.001) and transection vagotomy, vagotomy by chemoneurolysis, or truncal vagotomy and chemoneurolysis were more effective (P<0.01) than truncal vagotomy performed surgically or by chemoneurolysis in preventing this stimulation. Insulin stimulated the H⁺ output (184±2.9 mol vs 62±3.1 mol,N=10,P<0.001) and transection vagotomy, vagotomy by chemoneurolysis, or truncal vagotomy and chemoneurolysis were more effective (P<0.01) than truncal vagotomy done surgically or by chemoneurolysis in preventing this action. These results were reproducible in experiments done after three months. This investigation shows that transection vagotomy, vagotomy by chemoneurolysis, and truncal vagotomy plus chemoneurolysis are equally effective in achieving vagal denervation of the rat stomach and are superior in this respect to truncal vagotomy done surgically or by chemoneurolysis.     

Pharmacological evidence for beta3 adrenoceptors in the control of rat gastric acid secretion

The effect of the ₃-adrenoceptor agonist BRL37344 on gastric acid secretion evoked by different secretory stimuli was investigated in anaesthetized rats with lumen-perfused stomachs in comparison with the ₂-adrenoceptor agonist clenbuterol. Intravenous injections of BRL37344 (1–10 mol/kg) and clenbuterol (0.01–1 mol/kg) dose-dependently reduced 2-deoxy-D-glucose-induced acid secretion, with BRL37344 about forty times less potent than clenbuterol. BRL37344 (0.1–3 mol/kg) inhibited pentagastrin-induced acid output, whereas clenbuterol was effective only at high doses (10–100 mol/kg). The inhibitory effect of BRL37344 on pentagastrin-induced acid secretion was not modified by the nonselective –adrenoceptor antagonist propranolol, but it was prevented by bupranolol, a ₃-adrenoceptor antagonist. Furthermore, neither BRL37344 (10 mol/kg) nor clenbuterol (100 mol/kg) modified the acid secretion induced by histamine. These data suggest that ₃ adrenoceptors have an inhibitory role in the control of rat gastric acid secretion induced by indirect stimuli.