Diabetologia

Reimer, R. A.; Field, C. J.; McBurney, M. I.

doi: 10.1007/s001250050762pmid: 9267980

Weaning onto chow diets causes the highest incidence of diabetes in the BB rat. Changes in gut development and absorption of nutrients in the diabetes prone rat and the subsequent effect on pancreatic function may play a role in the ultimate development of the disease. BB diabetes prone (dp) and BB normal (n) dams were fed chow diets. Pups were killed at various ages ranging from 7 to 30 days. BBdp rats had higher small intestine and colon weights expressed per body weight at all ages ( p < 0.0001). RNA content (mg/g) in the jejunum, ileum and colon was higher in the BBdp rats beginning at the critical period at 21 days and maintained at 24 days and 30 days ( p < 0.0001). Proglucagon message decreased with age in both BBdp and BBn animals ( p < 0.0001). Levels of proglucagon mRNA were higher in BBdp compared to BBn animals only in the ileum at 10 days ( p < 0.01). Adjusting for total ileal and colonic RNA content resulted in BBdp animals having higher total colonic proglucagon mRNA at 21, 24 and 30 days ( p < 0.0001). Plasma GLP-1(7–36) amide was more than doubled in BBdp compared to BBn animals ( p < 0.0005) at 30 days. Expressing sodium-dependent D-glucose co-transporter (SGLT-1), GLUT2 and GLUT5 mRNA per total jejunal RNA shows increased transporter mRNA in BBdp compared to BBn rats at weaning (21 days) ( p < 0.05). Radical differences exist between BBdp and BBn animals at ’critical periods' in both proglucagon and glucose transporter gene expression. These differences may help explain altered growth and diseases incidence between these two strains (Diabetologia (1997) 40: 871–878).

Podestá, F.; Roth, T.; Ferrara, F.; Cagliero, E.; Lorenzi, M.

doi: 10.1007/s001250050763pmid: 9267981

Thickening of basement membranes is an early and characteristic feature of diabetic vessels, but its consequences on the properties of vascular cells remain undefined. We investigated whether and how excess extracellular matrix (ECM) alters the replication of vascular endothelial cells in vitro. To test the effects of endogenous excess matrix, human umbilical vein endothelial cells (HUVEC) were plated on ECM produced under culture conditions (high ambient glucose) that increase ECM synthesis. Four of six HUVEC isolates plated on such ECM yielded a lower cell number (68 ± 18 %) than cells plated on control ECM. Growth inhibition was observed in HUVEC cultured on elevated concentrations (10 and 50 μg/ml) of exogenous fibronectin, when compared with HUVEC plated on tissue culture plastic or 0.25, 1.0, and 5.0 μg/ml fibronectin; the decreased replication was attributable to delayed transit through the G 1 phase of the cell cycle. HUVEC grown on both 1 and 10 μg/ml fibronectin exhibited a modest upregulation of the fibronectin-specific integrin receptor α5 β 1, and increased attachment to fibronectin substratum. However, unique to the HUVEC plated on growth-inhibitory concentrations of fibronectin was a redistribution in situ of integrins and vinculin to form more numerous focal adhesions, and an increased polymerization of cytoskeletal actin to form stress fibers. Concentrations (0.01 μg/ml) of cytochalasin D intended to prevent excess actin polymerization prevented the growth inhibition. Thus, excess ECM hampers endothelial cell replication in vitro through increased cell-ECM adhesion and attendant cytoskeletal rearrangements. These phenotypic changes provide probes to test whether cell-ECM interactions are altered in diabetic vessels in a direction that may compromise orderly endothelial cell renewal and its antithrombogenic function. (Diabetologia (1997) 40: 879–886)

Wang, R. N.; Rehfeld, J. F.; Nielsen, F. C.; Klöppel, G.

doi: 10.1007/s001250050764pmid: 9267982

In adult rats islet cell neogenesis can be stimulated by partial duct ligation. Duct to islet cell differentiation is thought to be regulated by growth factors such as gastrin and transforming growth factor-α (TGFα). To test this hypothesis, we examined the expression of gastrin and TGFα at the mRNA and protein level in pancreatic tissue following partial duct ligation. Pancreatic specimens were investigated on days 3, 5, 7 and 14 after duct ligation by means of non-isotopic in situ hybridization, immunocytochemistry and Western blotting. Gastrin mRNA was strongly expressed in newly developed duct-like cell structures in the ligated tail portion of the pancreas before the period of pronounced islet cell neogenesis (days 5 and 7), and immunostaining for gastrin peptides was positive at days 5–7. In the non-ligated head portion and in control pancreases, gastrin was not expressed. Expression of TGFα was found to be increased in the ligated tail portion of the pancreas on day 3 and particularly on day 5, while there was no enhanced signal in the non-ligated part. Western blotting revealed two different TGFα isoforms (18 kDa and 42 kDa) in the ligated tail part and three isoforms (18 kDa, 24 kDa and 42 kDa) in the non-ligated head part and in untreated pancreases. The induction of gastrin and TGFα expression preceded the peak in the bromodeoxyuridine pulse labelling index of beta cells, known from a previous study to occur on day 7. We conclude that pancreas duct ligation induces the overexpression of gastrin and TGFα in the first days following ligation. Since ductal cells are known to give rise to endocrine cells after duct ligation, gastrin and TGFα may play a role as growth factors in islet neogenesis. (Diabetologia (1997) 40: 887–893)

Usui, I.; Takata, Y.; Imamura, T.; Morioka, H.; Sasaoka, T.; Sawa, T.; Ishihara, H.; Ishiki, M.; Kobayashi, M.

doi: 10.1007/s001250050765pmid: 9267983

Saturated fatty acids cause insulin resistance but the underlying molecular mechanism is still unknown. We examined the effect of saturated non-esterified fatty acids on insulin binding and action in transfected Rat-1 fibroblasts, which over-expressed human insulin receptors. Incubation with 1.0 mmol/l palmitate for 1–4 h did not affect insulin binding, insulin receptor autophosphorylation, insulin-stimulated tyrosine kinase activity toward poly(Glu 4 :Tyr 1 ), pp185 and Shc phosphorylation and PI3-kinase activity in these cells. However, the dose response curve of insulin-stimulated glucose transport was right-shifted. Palmitate inhibited the maximally insulin-stimulated mitogen activated protein (MAP) kinase activity toward synthetic peptide to 7 % that of control. The palmitate treatment influenced neither cytosolic protein kinase A activity nor cAMP levels. These results suggested that 1) palmitate did not inhibit the early steps of insulin action from insulin binding to pp185 or Shc phosphorylation but inhibited insulin-stimulated MAP kinase, and that 2) palmitate decreased insulin sensitivity as manifested by inhibited insulin-stimulated glucose uptake. In conclusion, the mechanism of saturated non-esterified fatty acid induced insulin resistance in glucose uptake may reside at post PI3-kinase or Shc steps, including the level of MAP kinase activation. (Diabetologia (1997) 40: 894–901)

Hartmann, B.; Bellmann, K.; Ghiea, I.; Kleemann, R.; Kolb, H.

doi: 10.1007/s001250050766pmid: 9267984

Oral administration of insulin suppresses the development of diabetes in nonobese diabetic (NOD) mice and deviates the cytokine balance in the islets of Langerhans from a Th1 to a Th2 type cytokine pattern. However, the effect of oral insulin is limited and disease suppression is limited to a narrow dose range. Therefore we tried to improve the outcome of suboptimal insulin dosing by bacterial adjuvant. Mice treated with a suboptimal dose of oral insulin showed no change in diabetes incidence although a shift from Th1 towards Th2 cytokine expression occurred in inflamed islets. Significant suppression of diabetes development was only seen in NOD mice receiving both, insulin and the bacterial preparation OM-89 as adjuvant. OM-89 is a protein extract of Escherichia coli , with nonspecific immunostimulatory properties. Potentiation of the effect of oral insulin by the adjuvant was associated with upregulation of interleukin (IL)-4 Th2 cells in infiltrated islets and sustained local IL-2 gene expression. RT PCR analyses of cytokine expression in the gut showed a clear deviation to Th2 type reactivity and downregulation of inducible nitric oxide (NO) synthase (iNOS) expression by the bacterial adjuvant but not by oral insulin alone. Since macrophages are the primary target cells of adjuvant action we tested its effect on mouse macrophages in vitro. Treatment with OM-89 induced transient release of tumour necrosis factor alpha and nitrite but rendered macrophages refractory to restimulation by the potent macrophage activator lipopolysaccharide. In conclusion, the protective effect of oral insulin can be potentiated by pretreatment with the bacterial adjuvant OM-89. This effect correlates with enhanced Th2 cytokine and decreased iNOS gene expression in the gut, probably due to the downregulation of proinflammatory mediators by exposure to the adjuvant. (Diabetologia (1997) 40: 902–909)

Pieper, G. M.; Siebeneich, W.; Moore-Hilton, G.; Roza, A. M.

doi: 10.1007/s001250050767pmid: 9267985

We examined the effects of acute supplementation with arginine in vitro on endothelium-dependent relaxation in aortic rings taken from female genetic, diabetes-prone BB rats. Sensitivity to norepinephrine-induced contraction was unaltered in rings of diabetic BB rats compared to rings from non-diabetic littermates. In precontracted rings, acetylcholine produced a concentration-dependent relaxation which was impaired by diabetes. This relaxation was blocked by l -nitroarginine in both control and diabetic rings. Addition of 3 mmol/l l -arginine (but not d -arginine) enhanced relaxation in diabetic rings similar to that seen in control rings without arginine. l -arginine had no effect on acetylcholine-induced relaxation in control rings. In contrast, relaxation-induced by nitroglycerin in diabetic rings without endothelium was not altered by l -arginine treatment. Thus, a defect in the utilization of arginine by nitric oxide synthase exists in the endothelium of the diabetic BB rat. (Diabetologia (1997) 40: 910–915)

Movassat, J.; Saulnier, C.; Serradas, P.; Portha, B.

doi: 10.1007/s001250050768pmid: 9267986

In the endocrine pancreas of the GK rat, a genetic model of non-insulin-dependent diabetes mellitus (NIDDM), it is not clear whether the histopathological changes reported up to now are related to the pathogenesis of hyperglycaemia or whether they occur secondarily to metabolic alterations. Using GK rats from the Paris colony, our study chronicles for the first time the pathophysiologic changes that occur in the GK pancreas from the late fetal period (day 21.5) until adult age (18 weeks). As compared to Wistar controls, GK fetuses exhibited higher plasma glucose level, lower plasma insulin level and normal plasma glucagon level. Their pancreatic insulin content and the relative volume and the total mass of their beta cells were sharply decreased, representing only 23, 38 and 23 % of control values, respectively. During the period from 4 days to 14 days after birth, GK neonates exhibited normal basal plasma glucose and glucagon levels despite decreased plasma insulin level. Their pancreatic insulin content represented only 31–40 % of values found in the age-related control pancreases and their total beta-cell mass was only 35 % on day 4, 30 % on day 7 and 37 % on day 14. The adult diabetic GK rats exhibited higher basal plasma glucose and insulin levels while their basal plasma glucagon level remained normal. Their pancreatic insulin content and the total beta-cell mass remained decreased, representing only 32 % and 47 % of control values, respectively. Moreover, the adult GK pancreases exhibited noticeable alteration in the architecture of the large islet sub-population which displayed considerable fibrosis with clusters of beta cells widely separated from each other by strands of connective tissue. Concerning the development of alpha cells in the GK rats, their relative volume was found to be normal during fetal and early neonatal periods. It was found to be moderately decreased (representing 64–67 % of corresponding control values) in 14-day-old neonates and adult GK rats. Our findings demonstrate that in the GK rat, the deficit of total beta-cell mass as observed in the adult animal is related to impaired beta-cell development. The restriction of the beta-cell mass must be considered as a primary and crucial event in the sequence leading to overt diabetes in this NIDDM model. (Diabetologia (1997) 40: 916–925)

Bott, S.; Bott, U.; Berger, M.; Mühlhauser, I.

doi: 10.1007/s001250050769pmid: 9267987

The objectives of the present analyses were to assess the association between HbA 1 c levels and severe hypoglycaemia (SH, treatment with glucose i. v. or glucagon injection) and to identify predictors of SH in a prospective multicentre trial. The study population consisted of 636 insulin-dependent diabetic patients who had participated in a structured 5-day in-patient group treatment and teaching programme for intensification of insulin therapy (ITTP) in one of 10 hospitals and who were re-examined after 1, 2, 3, and 6 years including assessment of demographic, disease and treatment related parameters, diabetes-related knowledge, behaviour, and emotional coping. At baseline, age (mean ± SD) was 27 ± 7 years, diabetes duration 9 ± 7 years and HbA 1 c 8.3 ± 1.9 %. During the 6-year follow-up, the mean HbA 1 c value improved to 7.6 %, and in patients with a diabetes duration of more than 1 year at entry into the study ( n = 538) the incidence of SH decreased from 0.28 cases/patient/year during the year preceding the ITTP to 0.17 cases/patient/year. The patient group was divided into decile groups according to mean follow-up HbA 1 c values. In each group more than 230 patient years could be analysed. Groups with mean HbA 1 c values of 5.7, 7.0, 7.4, 7.7 and 8.9 % had comparable risks of SH (0.15–0.19 cases/patient/year). In a logistic regression analysis, mean HbA 1 c during follow-up, a history of SH during the year preceding the ITTP, C-peptide level, emotional coping, carrying emergency carbohydrates (as assessed at the 1-year follow-up), and age at onset of diabetes were significant independent predictors of SH. The incidence of SH between centres varied between 0.05 and 0.27 cases/patient/year. In conclusion, in the present analyses no linear or exponential relationship between HbA 1 c and severe hypoglycaemia could be identified by using simple group comparisons. Applying complex regression analyses, various patient-related predictors of severe hypoglycaemia were identified. (Diabetologia (1997) 40: 926–932)

McKinney, P. A.; Parslow, R.; Gurney, K.; Law, G.; Bodansky, H. J.; Williams, D. R. R.

doi: 10.1007/s001250050770pmid: 9267988

Environmental risk factors for childhood insulin-dependent diabetes mellitus (IDDM) have been investigated using data abstracted from the obstetric records of mothers participating in a population-based case-control study of children (0–15 years) diagnosed with IDDM during 1993–1994. A univariate analysis of 196 age and sex matched sets (129 triplets, 67 pairs) gave significantly raised odds ratios (OR) for mothers over 35 years (OR 2.13, 95 %CI 1.04–4.36) and the following exposures in pregnancy: amniocentesis (3.85, 1.34–11.04), oedema, proteinuria and/or hypertensive disorders (1.62, 1.03–2.54), excessive weight gain (7.12, 1.50–33.79) and complications in labour (1.49, 1.00–2.21). The risk previously associated with caesarean deliveries was confirmed and the trend of increasing risk with age was significant. Adjusting separately for mothers with IDDM (4 cases, 0 control subjects), parity and small for gestational age failed to have any influence on the pregnancy risk factors or caesarean delivery. Case mothers undergoing amniocentesis were significantly younger compared to their control counterparts ( p = 0.02) and the majority were given the test to determine fetal maturity, late in pregnancy, rather than to identify chromosomal abnormalities. Oedema, proteinuria and/or hypertension conferred an increased risk throughout pregnancy, particularly in the first two trimesters. No specific pattern of risk was present for any age group. Multivariate modelling of the significant OR, using conditional logistic regression, retained excessive weight gain as significant. The overall results present a risk profile of older mothers whose babies may be exposed to adverse intrauterine conditions and delivery by caesarean section. (Diabetologia (1997) 40: 933–939)

Hansen, L.; Arden, K. C.; Rasmussen, S. B.; Viars, C. S.; Vestergaard, H.; Hansen, T.; Møller, A. M.; Woodgett, J. R.; Pedersen, O.

doi: 10.1007/s001250050771pmid: 9267989

Activation of glycogen synthesis in skeletal muscle in response to insulin results from the combined inactivation of glycogen synthase kinase-3 (GSK-3) and activation of the protein phosphatase-1, changing the ratio between the inactive phosphorylated state of the glycogen synthase to the active dephosphorylated state. In a search for genetic defects responsible for the decreased insulin stimulated glycogen synthesis seen in patients with non-insulin-dependent diabetes mellitus (NIDDM) and their glucose-tolerant first-degree relatives we have performed mutational analysis of the coding region of the 2 isoforms of GSK-3α and GSK-3 β in 72 NIDDM patients and 12 control subjects. No structural changes were detected apart from a few silent mutations. Mapping of the GSK-3α to chromosome 19q13.1–13.2 and the GSK-3 β to chromosome 3q13.3-q21 outside known genetic loci linked to NIDDM further makes it unlikely that these genes are involved in the pathogenesis of common forms of NIDDM. (Diabetologia (1997) 40: 940–946)