Effect of glucagon on carbohydrate-mediated secretion of glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (7-36 amide) (GLP-1).

BACKGROUND: The insulinotropic hormones, glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (7-36 amide) (GLP-1), regulate insulin secretion to nutrient intake and constitute the endocrine arm of the entero-insular axis. Glucagon has been implicated in the pathophysiology of conditions characterised by abnormal glucose tolerance such as obesity and diabetes mellitus although its effect on the entero-insular axis is not fully understood. Materials and methods We investigated the effect of exogenous glucagon on the entero-insular axis and its relation to gastric emptying in six healthy men aged [mean (+/-S.E.M. )] 23.6 (0.9) years with a body mass index of 24.0 (1.5) kg/m(2). Plasma glucose, GIP, GLP-1, insulin and paracetamol concentrations were measured before and after a 100 g oral carhohydrate load containing 1.5 g of paracetamol for 6 h during intravenous infusion of either glucagon or saline. RESULTS: When compared to the saline infusion, peak and integrated insulin and glucose concentrations were higher (p<0.05) following glucagon infusion. After 60 min paracetamol concentrations were lower (p<0.05) following glucagon infusion. Integrated responses for GIP and GLP-1 were markedly reduced following glucagon infusion. CONCLUSIONS: Exogenous glucagon in addition to its well-documented action of increasing glucose and insulin concentrations and delaying gastric emptying also markedly reduces GIP and GLP-1 secretion. The inhibition of GLP-1 soon after commencement of glucagon infusion supports a direct effect of glucagon on intestinal L-cells. We speculate that the marked inhibition of postprandial GLP-1 secretion by glucagon may be of importance in the pathogenesis of relative insulinopenia in Type 2 diabetes and in the development of reduced satiety in obesity and diabetes.

p53 protein expression in malignant, pre-malignant and non-malignant lesions of the lip.

AIM: To elucidate the role of the p53 tumour suppressor gene in the pathogenesis of lip cancer. METHODS: Expression of p53 was evaluated immunocytochemically in a retrospective study of formalin fixed, paraffin wax embedded tissue. Five cases each of four types of lip lesions were studied; these comprised squamous cell carcinoma (SCC), solar keratosis (SK), chronic hyperplastic candidosis (CHC), and lichen planus (LP). Five cases each of normal lip mucosa, SCC, and SK from sun exposed facial skin as well as LP, CHC, and SCC from buccal mucosa were also analysed. Immunolocalisation of p53 was scored semiquantitatively. The degree of apoptosis was also assessed in selected lesions by determining cell nuclear fragmentation. RESULTS: All SCCs from lip lesions were immunopositive for p53. All cases of SK and two of five CHC lip lesions were also p53 positive. Normal lip mucosa samples were p53 negative. Sun exposed skin lesions of SCC and SK were all positive for p53, but only three of five cases of SCC from the buccal mucosa had detectable levels of p53. p53 expression was not detected in CHC and LP lesions of the buccal mucosa. CONCLUSIONS: The aberrant expression of p53 is likely to occur early in the pathogenesis of lip cancer and may be related to exposure to the sun. The immunopositive p53 cells identified in the benign LP lesions do not necessarily correlate with commitment of cells within the lesion to programmed cell death. In light of the prior reports which indicate that p53 positive cells may progress to form malignant tumours, it is suggested that patients with p53 positive but otherwise benign lesions should be followed more closely.

L-canavanine restores blood pressure in a rat model of endotoxic shock.

Administration of lipopolysaccharide to anaesthetised rats produced a reduction in mean arterial pressure, an increase in heart rate, and death at 4-6 h. Intravenous infusion of NG-nitro-L-arginine methyl ester (50 mg/kg), an inhibitor of constitutive and inducible nitric oxide (NO) synthase, 60 min after challenge with lipopolysaccharide, caused an immediate increase in blood pressure followed by a precipitous fall in pressure, and death. In contrast, intravenous infusion of L-canavanine (100 mg/kg), reported to be a selective inhibitor of inducible NO synthase in vitro, 60 min and 180 min after lipopolysaccharide challenge, produced an increase in mean arterial pressure and reversed the lipopolysaccharide induced hypotension. However, in lipopolysaccharide challenged animals protected from hypotension by administration of L-canavanine (60 min post challenge), intravenous infusion of NG-nitro-L-arginine methyl ester at 180 min post challenge caused an immediate rise in mean arterial pressure, followed by a rapid fall in blood pressure and heart rate, and sudden death. In contrast, a second dose of L-canavanine at 180 min post challenge maintained blood pressure for the duration of the experiment. These findings indicate that inhibition of both constitutive and inducible NO synthase during endotoxaemia is lethal. However, the use of a selective inhibitor of inducible NO synthase restores mean arterial pressure to baseline, and offers a therapeutic approach to managing hypotension in shock.

Clinical studies of IGFBP-2 by radioimmunoassay.

A specific radioimmunoassay for human IGFBP-2 was developed using a polyclonal antiserum directed against a partial sequence (hIGFBP-2(176-190)). The tracer was prepared by radioiodination of a [Tyr]o-hIGFBP-2(176-190) derivative. The assay was used to study IGFBP-2 levels in numerous clinical and experimental situations. There was little circadian fluctuations of serum level which showed a marked age-dependence with high levels at birth and senescence and low levels during puberty. Decreased IGFBP-2 levels were present in untreated insulin-dependent diabetes mellitus (IDDM), in acromegaly and during dexamethasone suppression test. GH deficiency, fasting, IGF-I administration to patients with GH receptor deficiency, hepatic failure and insulinomas caused a moderate increase of serum IGFBP-2. Markedly elevated levels were found in chronic renal failure, non-islet cell tumour induced hypoglycemia and leukaemias. The fact that all possible relationships between insulin secretion and IGFBP-2 levels could be identified suggests that insulin is not a major regulator. In general, there was an inverse relationship with serum IGFBP-3 (except IDDM) and IGFBP-2 levels were high in situations where free IGF-II should be expected to be high. The tentative conclusion would therefore be that free IGF-II is a major regulator of circulating IGFBP-2.

Inhibition of nitric oxide synthesis improves survival in a murine peritonitis model of sepsis that is not cured by antibiotics alone.

Inhibition of nitric oxide synthesis was investigated in a murine model of advanced sepsis in which antibiotic therapy alone did not improve survival. Seven hours after receiving a lethal intraperitoneal challenge with live Escherichia coli, mice were given either NG-monomethyl-L-arginine (L-NMMA) intravenously, imipenem-cilastatin subcutaneously or a combination of both. L-NMMA (3-300 mg/kg) or imipenem-cilastatin (10 or 50 mg/kg) given alone did not improve survival; co-administration of L-NMMA and either 10 or 50 mg imipenem-cilastatin/kg improved survival significantly. These findings suggest that nitric oxide contributes to the morbidity associated with advanced sepsis and that nitric oxide synthase inhibition may improve the efficacy of conventional antimicrobial treatment of severe infections.

Twin very low birth weight infants: a study of nutritional and hormonal status.

Nonidentical twin male infants (twin 1,950 g birth weight, twin 2,970 g) had their nutritional and hormone status studied for up to 59 days. Both infants received parenteral nutrition up to 32 days postnatally; enteral feeding was then established in twin 1; in twin 2 parenteral feeding was recommenced on day 35, for the remainder of the study. Serial 72-hr metabolic balances were performed in both infants at 4, 32, 45, and 56 days postnatally. Insulin-like growth factor I (IGF-I) and growth hormone were assayed on day 2 of each balance. During the course of the study growth was similar in each infant. Overall mean daily energy intakes were 90 kcal/kg/day and 84 kcal/kg/day and percentage nitrogen retention was 62% and 55% in twin 1 and twin 2, respectively. No differences were observed between the two infants in IGF-I or growth hormone. Despite low energy intakes incremental weights were within an acceptable range for both infants.

Hyperinsulinaemic hypoglycaemia in small for dates babies.

Blood glucose concentrations were measured prospectively in 27 small for dates infants in the first 48 hours after birth: 10 infants became hypoglycaemic. Of these, five had inappropriately raised plasma insulin concentrations. Plasma free fatty acids were lower and carbohydrate intake higher in these five infants, further supporting the diagnosis of hyperinsulinism. The hypoglycaemia recurred in four of the five hyperinsulinaemic infants, but in none of those who were not hyperinsulinaemic. Hyperinsulinism is common in small for dates babies. It is important to recognise this because hypoglycaemia is likely to recur and appropriate treatment is needed to prevent long term sequelae.

Development of an enzyme-linked immunosorbent assay for caffeine.

A competitive enzyme-linked immunosorbent assay suitable for the measurement of caffeine in plasma and serum has been developed. Sheep immunised with an immunogen prepared by coupling 7-(5-carboxypentyl)1,3-dimethylxanthine to egg albumin produced antibodies with little crossreactivity with the metabolites of caffeine. The enzyme label was prepared by coupling 7-(5-carboxypentyl)-1,3-dimethylxanthine to peroxidase using the mixed anhydride method. The assay, which has a sensitivity of 0.01 mumol/l, permits direct measurement of caffeine in plasma and serum samples. 50 plasma samples measured by ELISA and by an established radioimmunoassay showed a correlation of r = 0.97 (P less than 0.001).

Insulin-like growth factor 1 in patients with hypoglycaemia.

Plasma insulin-like growth factor 1 (IGF-1) concentrations were measured in a group of patients with hypoglycaemia due to endogenous hyperinsulinism, and compared with those in a group of patients matched for age and sex with hypoglycaemia and appropriately suppressed insulin levels. Insulin-like growth factor 1 concentrations were significantly higher in the hyperinsulinaemic hypoglycaemic group than in either the hypoinsulinaemic hypoglycaemia group or a group of euglycaemic control subjects. These data provide further evidence that insulin promotes IGF-1 production and release from the liver.

Bone disease in chronic alcoholism: the value of plasma osteocalcin measurement.

Osteoporosis is more common in chronic alcoholics than in age-matched controls. Possible aetiological factors are: malabsorption of calcium and vitamin D; liver disease and abnormal parathyroid function. The possibility that alcohol may directly affect osteoblastic function has, however, received little attention. We measured plasma osteocalcin, a protein synthesised specifically by osteoblasts, in chronic alcoholics. Our data show that these have low plasma osteocalcin but normal calcium, magnesium and parathormone, which suggest that alcohol may be directly toxic to osteoblasts.

A direct assay for proinsulin in plasma and its applications in hypoglycaemia.

A direct radioimmunoassay in unextracted plasma is described. The assay has a sensitivity of 4 pmol/l (2 standard deviation from zero). The proinsulin antiserum was immuno-adsorbed against human C-peptide and insulin coupled to glass beads. Cross-reactivity of the antiserum was assessed and shown to be less than 0.01% with both peptides. In normal healthy fasting subjects the plasma proinsulin level was 6.7 +/- 1.7 pmol/l (n = 17) (mean +/- SD). Fasting proinsulin levels in non-insulin dependent diabetics were significantly elevated compared with non diabetics (14.2 +/- 2 pmol/l (n = 11) vs 6.7 +/- 1.7 (n = 17) P less than 0.005). The insulin/proinsulin ratio was 3.4:1 in the non-insulin dependent diabetic compared with 6:1 in non-diabetics. Samples from 21 insulinoma patients were assayed and mean fasting plasma proinsulin level was 255 pmol/l +/- 479 when the patients were hypoglycaemic. The range in pro-insulin levels was large (30-2300 pmol/l). Mean fasting proinsulin level in three hypoglycaemic subjects due to sulphonylurea overdose was 15.7 +/- 2.3 pmol/l. The molar ratio of proinsulin to insulin was 1:6 in healthy subjects, 1:1 in insulinoma patients and 10:1 in sulphonylurea induced hypoglycaemic patients.

Modulation of type IV collagenase and plasminogen activator in a hamster fibrosarcoma by basement membrane components and lung fibroblasts.

The effect of basement membrane components (laminin, fibronectin and type IV collagen) and lung fibroblasts on type IV collagenase and plasminogen activator activity was investigated in a primary HSV-2-induced hamster fibrosarcoma, and its in vivo derived sublines and in vitro derived clones of varying metastatic potential. Fibronectin and type IV collagen were ineffective at influencing the expression of either type IV collagenase or plasminogen activator activity. Laminin, however, at concentrations of 1-10 micrograms ml-1 added to the serum-free culture supernatants, increased the release of type IV collagenase by up to 100% for the parental cell line. Three highly metastatic sublines (two from in vivo origin and one from in vitro cloning) showed increases of up to 300%. Non-metastatic sublines (two from in vivo origin and one from in vitro cloning), however, showed no increase in type IV collagenase activity. Plasminogen activator release from either the parental line cell or its metastatic sublines and clones, was unaffected by the addition of laminin. Addition of tumour cells to lung fibroblast monolayers resulted in an increased expression of PA activity in the supernatant, whilst type IV collagenase activity was reduced.

Sex hormones, insulin, lipids, and prevalent ischemic heart disease.

The relations between estradiol, testosterone, insulin, lipids, and prevalent ischemic heart disease were examined using the cross-sectional data from the Caerphilly Heart Disease Study, a cohort of 2,512 men (aged 45-59 years) surveyed between 1978 and 1982. Endogenous levels of estradiol were associated directly with high density lipoprotein (HDL) cholesterol (r = 0.106, p less than 0.001), but this relation was removed after adjustment for testosterone and insulin levels. Estradiol was not associated with prevalent ischemic heart disease. Endogenous levels of testosterone were associated directly with HDL cholesterol (r = 0.148, p less than 0.001) and inversely with triglyceride (r = -0.217, p less than 0.001). Persons with prevalent ischemic heart disease had significantly lower testosterone levels than persons without ischemic heart disease (mean levels 20.9 vs. 22.0 nmol/liter, p less than 0.01). These relations were confounded by associations with insulin. The associations between testosterone and the lipids persist after adjusting for body mass index, age, and insulin. The association between testosterone and prevalent ischemic heart disease was reduced after adjusting for insulin and/or triglyceride levels. The results suggest that insulin and testosterone may have an interdependent regulatory effect on lipid metabolism. The effect of testosterone on ischemic heart disease appears to be primarily mediated through its association with insulin. Future work on sex hormones and ischemic heart disease will need to account for the effects of insulin.

Type IV collagenase activity of a primary HSV-2-induced hamster fibrosarcoma and its in vivo metastases and in vitro clones.

The expression of a basement membrane (BM) collagen-degrading metalloprotease (Type IV collagenase) was studied in a herpes simplex virus (HSV)-2 transformed hamster fibrosarcoma and its in vivo derived sublines and in vitro derived clones of varying metastatic potential. The primary parent tumor was shown to release more or less Type IV collagenolytic activity compared with its sublines (derived from lung nodules that developed after resection of the primary tumor). Normal baby hamster kidney and hamster embryo fibroblasts did not secrete detectable amounts of BM collagenase, whereas normal hamster lung fibroblast secreted intermediate levels of Type IV collagenase activity. The collagenase IV activity of the parent tumor and its in vivo and in vitro derived sublines was assayed in vitro and compared with the ability of the cells lines to spontaneously metastasize in vivo. No correlation between the ability to secrete type IV collagenase and metastatic propensity was detected. Although all cell lines secreted type IV collagenase, the highest activity was recorded for a nonmetastatic variant.

Therapy of spontaneously metastatic HSV-2 induced hamster tumours with cortisone acetate administered with or without heparin.

Subcutaneous injection of cortisone acetate administered with or without oral heparin retarded the growth of two HSV-2 induced hamster fibrosarcomas. Histological sections showed no obvious difference between the vasculature of treated and untreated tumours although there were fewer infiltrating lymphocytes in treated tumours. Treatment was, however, found to be ineffective against metastatic development following resection of primary tumours. Natural killer cell activity was found to be greatly reduced in animals receiving heparin and/or cortisone acetate treatment and this may influence the effectiveness of treatment on the metastatic process. We conclude that treatment of tumours with cortisone plus heparin is no different from the response to cortisone used alone.

Metastatic heterogeneity in a spontaneously metastatic HSV-2 induced hamster fibrosarcoma: association of phenotypic and genotypic properties with metastatic potential.

A spontaneously metastatic tumour of hamsters and cell lines derived from its in vivo metastatic deposits, or from in vitro cloning were compared for immunobiological and genotypic variation and correlation sought with regard to metastatic potential. Cell lines were established from seven individual lung metastases following primary tumour resection of the parent (HSV-2-333-2-26) cell line. When inoculated subcutaneously, and upon resection of the subsequent tumour mass, four cell lines (MetA, MetB, MetE and MetF) demonstrated greater, one similar (MetG) and two (MetC and MetD) less metastatic capacity compared with the parent tumour line. In further studies cell lines were established in vitro by single cell cloning of the parent tumour either by limiting dilution or soft agar cloning. In vivo tumour resection experiments showed eight cell lines (S4, S7, S4A, S7A, S7B, C1.1, C1.4 and C1.5) to have an increased metastatic potential and five cell lines (S8, S9, S8B, S9D and S9E) a decreased metastatic potential compared to the parental line. Karyotypic analysis of the cells revealed that all highly metastatic cell lines were of a near diploid genotype, whilst non- and weakly metastatic cell lines, including the parental line, were aneuploid or near tetraploid. The immunobiological characteristics of these cell lines was studied. Assessment of in vivo immunogenicity showed that eight clones (MetA, MetB, MetE, MetF, S7A, C1.1, C1.4 and C1.5) were non-immunogenic whilst the parental tumour line and three clones (MetC, MetD and MetG) exhibited a strong transplantation rejection antigen; immunogenicity showed an inverse correlation with metastatic potential. Susceptibility to NK cytolysis was demonstrated for cell lines exhibiting a weak or non-metastatic/immunogenic phenotype. The origin of metastatic variants and their association with genotype and immunobiological properties is discussed.

Production of plasminogen activator by a primary HSV-2-induced hamster fibrosarcoma and its in vivo derived sublines.

The plasminogen activator (PA) content of a primary HSV-2-induced hamster fibrosarcoma and sublines derived from its in vivo metastases was investigated using the indirect 125I-labeled fibrin plate method. Fresh tissue culture lines established from primary HSV-2-333-2-26 tumors were shown to produce levels of PA similar to sublines derived from lung or kidney foci that developed after resection of primary tumors. In comparison, normal hamster embryo fibroblast (NHEF) and baby hamster kidney cells produced little or no PA, although baby hamster lung fibroblasts produced intermediate levels of PA. In addition, no correlation was found between PA levels of the sublines and their ability to metastasize from subcutaneous tumors, although tumor cell lines were shown to produce significantly more PA than normal cells.