Diabetologia

Shum, L.; Sadler, T.

doi: 10.1007/BF00404998pmid: 1864482

125 34 34 5 5 L. Shum T. W. Sadler Department of Cell Biology and Anatomy, Laboratories for Developmental Biology, The School of Medicine The University of North Carolina at Chapel Hill North Carolina USA Summary Previous studies have shown that the ketone body D,L,-beta-hydroxybutyrate was teratogenic to mouse embryos exposed in culture during the period of neurulation. Inhibition of closure of the cranial and caudal neuropores was the most frequently occurring defect and these abnormalities were thought to be the forerunner of anencephaly and spina bifida, respectively. However, additional studies demonstrated that embryos could recover morphologically from these effects if the ketone body was removed from the culture medium and if the recovery period was of sufficient duration. In an attempt to define further the phenomenon responsible for this recovery and to determine the extent of the recovery process, the present study examining the cross-sectional area, cell number, and mitotic index of cranial neuroepithelial cells was conducted in mouse embryos cultured from the early somite stage under one of the following conditions: 1) control medium for 60 h; 2) medium containing 32 mmol/1 D,L,-beta-hydroxybutyrate for 24 h followed by culture in control medium for an additional 36 h (recovery group); 3) medium containing 32 mmol/1 D,L,-beta-hydroxybutyrate for 60 h (continuously exposed group). The results indicate that although neural tube closure occurred in the recovery group, complete recovery was limited to the ventral regions of the forebrain and that the remainder of the prosencephalon as well as the rhombencephalon failed to undergo complete catch-up growth. Thus, cell numbers in these areas were approximately 70% of control values. Therefore, while the gross anatomical disturbances produced by the ketone body may be compensated for, histological alterations in the affected tissues remain. Ultimately, these data suggest that neurological deficits may be an outcome of ketone body exposure during the early stages of embryogenesis.

Gottlieb, P.; Handler, E.; Appel, M.; Greiner, D.; Mordes, J.; Rossini, A.

doi: 10.1007/BF00404999pmid: 1864483

125 34 34 5 5 P. A. Gottlieb E. S. Handler M. C. Appel D. L. Greiner J. P. Mordes A. A. Rossini Departments of Medicine University of Massachusetts Medical School Worcester Massachusetts Departments of Pathology University of Massachusetts Medical School Worcester Massachusetts Department of Pathology University of Connecticut Health Center Farmington Connecticut USA Summary Prophylactic insulin administration is known to prevent hyperglycaemia in diabetes prone BB rats and non-obese diabetic mice. This study investigated the effect of insulin treatment on the development of overt diabetes, clinically inapparent anti-islet autoreactivity, and thyroiditis in RT6-depleted diabetes resistant BB rats. Fewer than 1% of these animals develop spontaneous diabetes, but if depleted of RT6 + T cells >50% become hyperglycaemic. We treated 30-day-old diabetes resistant rats with anti-RT6.1 monoclonal antibody, exogenous insulin, or both. Up to 60 days of age, 16 of 20 rats given antibody alone became diabetic, compared with 1 of 20 also treated with antibody plus insulin. Up to 110 days of age, only 1 of 10 rats treated with both insulin and antibody between 30 and 60 days became diabetic. Histologic study of non-diabetic insulin plus anti-RT6 antibody treated rats revealed insulitis in 3 of 9 at 60 days old, and insulitis in 3 of 8 and thyroiditis in 6 of 7 at 110 days of age. Non-diabetic animals were also found to harbour autoreactive spleen cells that adoptively transferred diabetes. Splenocytes from 60 or 110-day-old non-diabetic donors that had been treated with insulin and antibody between 30 and 60 days of age induced diabetes in 7 of 13 and 6 of 8 adoptive recipients respectively. We conclude that insulin treatment prevents clinical diabetes in the RT6-depleted diabetes resistant BB rat, but this treatment does not prevent the development of autoreactive cell populations that cause thyroiditis and adoptively transfer diabetes.

Chakrabarti, S.; Ma, N.; Sima, A.

doi: 10.1007/BF00405000pmid: 1864484

125 34 34 5 5 S. Chakrabarti N. Ma Prof. A. A. F. Sima Neuropathology Research Laboratories, Department of Pathology University of Manitoba Winnipeg Manitoba Canada Michigan Diabetes Research and Training Center The University of Michigan 1331 East Ann Street Bldg. 48109-0580 Ann Arbor MI USA Summary Basement membrane anionic sites, thought to be responsible for charge selective permeability barriers, were investigated in retinal, endoneurial, and muscle capillary basement membranes and in Bruch's membrane of diabetic, and age- and sex-matched non-diabetic BB-rats using an ultrastructural quantitative histochemical technique. Six months of diabetes was associated with significant basement membrane thickening which was linearly related to a decrease in anionic site density suggesting a relative loss of proteoglycans. Calculation of anionic sites per unit length of basement membrane, reflecting their absolute number, revealed a significant loss in basement membrane, constituting part of normal blood-tissue barrier systems such as retinal and endoneurial capillary basement membranes, and the basement membrane of the retinal pigment epithelium. The absolute number of anionic sites in normally permeable microvessels, such as those of muscle and choriocapillaries, was unaltered by diabetes. We conclude that this specific loss of anionic sites in basement membranes of tissues affected by chronic diabetic complications may in part be responsible for permeability abnormalities seen in these tissues.

Bornfeldt, K.; Gidlöf, R.; Wasteson, A.; Lake, M.; Skottner, A.; Arnqvist, H.

doi: 10.1007/BF00405001pmid: 1713869

125 34 34 5 5 K. E. Bornfeldt R. A. Gidlöf A. Wasteson M. Lake A. Skottner H. J. Arnqvist Department of Pharmacology, Faculty of Health Sciences Linköping University Linköping Department of Cell Biology, Faculty of Health Sciences Linköping University Linköping Department of Internal Medicine, Faculty of Health Sciences Linköping University Linköping Department of KabiGen Stockholm Sweden Department of Kabi Peptide Hormones Stockholm Sweden Summary Binding and growth promoting effects of insulin, insulin analogues modified in the B chain, proinsulin, insulin-like growth factor-I and -II were studied in cultured rat aortic smooth muscle cells. Specific binding of 125 I-insulin was 0.9±0.2% of total 125 I-insulin added, and the IC 50 -value was estimated to 8.9 pmol/1. The insulin analogue B10 Asp tended to be more potent than insulin in displacing 125 I-insulin, B28 Asp was equipotent, B9 Asp/B27 Glu was approximately 100 times less potent and insulin-like growth factor-I more than 1000 times less potent than insulin. Specific binding of 125 I-insulin-like growth factor-I after 4 h incubation at 10 °C was five times higher than the specific binding of insulin (4.4±0.4% of total 125 I-insulin-like growth factor-I added), and the IC 50 -value was 0.3 nmol/l. Insulin was approximately 500 times less potent than insulin-like growth factor-I in displacing 125 I-insulin-like growth factor-I. The insulin analogue B10 Asp was slightly more potent and analogue B28 Asp was equipotent with insulin. Analogue B9 Asp/B27 Glu was ten times less potent and proinsulin was more than ten times less potent than insulin. The order of potency was similar for 3 H-thymidine incorporation into DNA: insulin-like growth factor-I > B10 Asp > insulin-like growth factor-II > insulin > B28 Asp > B9 Asp/B27 Glu > proinsulin. The maximal effect of insulin-like growth factor-I on 3 H-thymidine incorporation was 71±16% higher than the maximal effect of insulin. The maximal effect of insulin-like growth factor-II was at least as high as the effect of insulin-like growth factor-I. Furthermore, the maximal effect of B10 Asp was 62±10% higher than the maximal effect of insulin. Insulin-like growth factor-I and B10 Asp tended to increase cell number more than insulin. In conclusion, this study shows that insulin analogues interact with different potencies with receptors for insulin and insulin-like growth factor-I in vascular smooth muscle cells and that insulin-like growth factors and the insulin analogue B10 Asp have more pronounced growth effects than insulin. Substitution of the amino acid Asp for His at position B10 in insulin makes the molecule more similar to insulin-like growth factor-I, chemically and probably also biologically.

Thivolet, C.; Goillot, E.; Bedossa, P.; Durand, A.; Bonnard, M.; Orgiazzi, J.

doi: 10.1007/BF00405002pmid: 1864485

125 34 34 5 5 C. H. Thivolet E. Goillot P. Bedossa A. Durand M. Bonnard J. Orgiazzi INSERMU197 France INSERM U80 Lyon France Hopital Antoine Beclere Paris France Centre de Transfusion sanguine Lyon France Summary Early intensive insulin treatment is thought to improve subsequent Beta-cell function in Type 1 (insulin-dependent) diabetic patients. Prophylactic insulin administration also reduced diabetes incidence in diabetes-prone animals. To study the mechanisms by which these effects occur, we tested the ability of insulin therapy in the model of non-obese-diabetic mice, to prevent the penetration of committed T cells into the islets and subsequent Beta-cell destruction. Sublethally irradiated non-obese-diabetic males of 8 weeks of age were adoptively transferred with splenocytes from diabetic donors and treated with the maximum tolerable dosage of fast-acting insulin (0.5 U, twice daily) until 30 days after cell transfer. Diabetes incidence was compared to control animals injected with the same concentration of insulin diluent. After one month of treatment, the cumulative diabetes frequency was significantly less within the insulin-treated group (4 of 15, 26.6%) than in the control group (15 of 18, 83.3%; p <0.01). Pancreatic histological analysis of insulin-treated animals revealed a lower severity of insulitis and Beta-cell necrosis and a higher percentage of normal islets (46.6±10% vs 2.3±2%, p < 0.01), including five (33%) mice with no lesions. Immunoperoxydase staining of pancreatic sections indicated similar insulin and ganglioside staining of Beta cells from insulin-treated mice and control animals. Insulin-treated mice had comparable pancreatic insulin content to normal mice. Flow cytometry analysis of spleen cell populations indicated that insulin increased the number of Thy1,2 + and Lyt-2 + T cells. Although an effect at the Beta cell level cannot be definitely excluded, several lines of evidence suggest that insulin may influence the capacity of effector T cells to invade the islets and cause Beta-cell destruction. These effects may have potential interest for future immunointervention trials in Type 1 diabetic patients of recent onset.

Annapurna, V.; Senciall, I.; Davis, A.; Kutty, K.

doi: 10.1007/BF00405003pmid: 1864486

125 34 34 5 5 V. Annapurna I. Senciall A. J. Davis K. M. Kutty Dr. Charles A. Janeway Child Health Centre St. John's Newfoundland Canada Faculty of Medicine Memorial University of Newfoundland St. John's Newfoundland Canada Summary This study was designed to understand the reasons for the increase in serum pseudocholinesterase activity in diabetes mellitus. Streptozotocin-induced diabetic rats were used for the study. Serum pseudocholinesterase activity increased with the induction of diabetes (381.5 units/l ± 11.8) compared to the non-diabetic rats (243.1 units/l ± 7.2). Serum triglycerides, total low density lipoprotein and glycerol also increased concurrently with the development of diabetes. Insulin treatment of the diabetic rats normalized serum glucose concomitant with the reduction of pseudocholinesterase activity, triglycerides, total low density lipoprotein and glycerol. Heparin injection appeared to activate lipoprotein lipase in the diabetic rats by showing a marked fall in serum triglyceride and total low density lipoprotein levels but not in pseudocholinesterase activity. Administration of tetraisopropylpyrophosphoramide a specific pseudocholinesterase inhibitor, inhibited serum and adipose tissue pseudocholinesterase activity by > 80% and liver > 50%. Concurrent with the inhibition of pseudocholinesterase activity serum triglyceride, low density lipoprotein and glycerol decreased significantly. In normal rats treatment with tetraisopropylpyrophosphoramide also reduced serum lipoproteins markedly, while glycerol only showed a marginal decrease. Glycerol was used as a marker of adipose tissue lipolysis and total low density lipoprotein which is defined as lipoproteins of density < 1.063 (LDL + VLDL). Our findings suggest (a) that the increase in serum pseudocholinesterase activity in diabetic rats is due to the overproduction of very low density lipoprotein triglyceride in the liver, and (b) decreased lipolysis in the adipose tissue of the diabetic rats treated with tetraisopropylpyrophosphoramide is accompanied by an inhibition of pseudocholinesterase activity.

Eriksson, U.; Borg, L.

doi: 10.1007/BF00405004pmid: 1864487

125 34 34 5 5 U. J. Eriksson L. A. H. Borg Department of Medical Cell Biology University of Uppsala Uppsala Sweden Summary This study addresses the possibility that the teratogenic effects of a diabetic pregnancy are associated with increased embryonic activities of free oxygen radicals. Rat embryos were cultured in 50 mmol/l glucose for 48 h and subsequently showed pronounced growth retardation and severe malformations. The enzyme inducer citiolone and the free oxygen radical scavenging enzymes Superoxide dismutase, catalase and glutathione peroxidase protected against the disturbed growth and development of the embryos at 50 mmol/l glucose when added to the culture media. Enzymatic measurements indicated that citiolone induced an increased activity of superoxide dismutase in the embryonic tissues and that the added enzymes were taken up by both the yolk sac and the embryo proper. The protection against embryonic maldevelopment was thus conferred by agents that increased the free oxygen radical scavenging capacity of the embryonic tissues. The results suggest that a high glucose concentration in vitro causes embryonic dysmorphogenesis by generation of free oxygen radicals. An enhanced production of such radicals in embryonic tissues may be directly related to the increased risk of congenital malformations in diabetic pregnancy.

Ishii, H.; Umeda, F.; Hashimoto, T.; Nawata, H.

doi: 10.1007/BF00405005pmid: 1907585

125 34 34 5 5 H. Ishii F. Umeda T. Hashimoto H. Nawata 3rd Department of Internal Medicine, Faculty of Medicine Kyushu University Fukuoka Japan Summary Enhanced platelet functions have been reported in patients with diabetes mellitus. Our recent study demonstrated that phosphoinositide turnover is increased in platelets from diabetic patients. In the present study, we evaluated the abnormality in platelet intracellular calcium mobilization in patients with Type 2 (non-insulin-dependent) diabetes mellitus using fura-2, a fluorescent calcium indicator. Washed platelets were prepared from six diabetic patients with increased platelet aggregation rates (DM-A group), seven diabetic patients with normal platelet aggregation rates (DM-B group), and eight age-matched healthy control subjects. The basal intracellular free calcium concentrations in platelets were similar among the three groups. Thrombin (0.025–0.1 U/ml) induced a dose-dependent increase in intracellular calcium in both the presence and the absence of extracellular calcium. This increase in the presence of extracellular calcium, which depends on calcium influx and release, was significantly higher in the DM-A group than in the DM-B and control groups. However, there was no significant difference between the control group and the DM-B group. In the absence of extracellular calcium, thrombin-induced calcium increase, which depends only on calcium release, was also significantly enhanced in the DM-A group. Furthermore, the calcium increase stimulated by platelet-activating factor (10 nmol/l) with and without extracellular calcium was significantly higher in the DM-A group than in the other groups. Additionally, calcium ionophore A23187 (100 nmol/l) caused a significantly higher calcium increase in the DM-A group with extracellular calcium, while the calcium increase without extracellular calcium showed no significant difference among the three groups. These observations suggest that enhanced intracellular calcium mobilization due to increased calcium influx and release may be closely related to platelet hyperfunctions in diabetes mellitus.

Langan, S.; Deary, I.; Hepburn, D.; Frier, B.

doi: 10.1007/BF00405006pmid: 1864488

125 34 34 5 5 S. J. Langan I. J. Deary D. A. Hepburn B. M. Frier Department of Psychology University of Edinburgh Edinburgh UK Diabetic Department Royal Infirmary Edinburgh UK Summary To examine the hypothesis that episodes of severe hypoglycaemia may cause cumulative cognitive impairment, 100 Type 1 (insulin-dependent) diabetic patients were examined. Their age range was 25–52 years, and the onset of diabetes had occurred after the age of 19 years. Patients with evidence of organic brain disease, including cerebrovascular disease, were excluded. A questionnaire was used to assess the number, frequency and severity of hypoglycaemic episodes experienced during treatment with insulin and the accuracy of this retrospective information was verified from general practice and hospital case-notes. A detailed neuropsychological assessment was undertaken, including tests of pre-morbid and present IQ (Wechsler-Revised), memory and information-processing speed. Significant correlations were observed between the frequency of severe hypoglycaemia and the magnitude of intellectual decline, Performance IQ, inspection time and reaction time (patients with the more frequent hypoglycaemia had poorer performance). Two sub-groups of patients were identified on the basis of their experience of severe hypoglycaemia, and were balanced for pre-morbid IQ, age and duration of diabetes. One sub-group ( n =23) had never experienced severe hypoglycaemia (Group A), whilst the other sub-group ( n =24) had suffered at least five episodes of severe hypoglycaemia (Group B). Group B had greater intellectual impairment than Group A, and Group B also had a significantly slower mean reaction time and higher reaction time variance when compared with Group A. It is concluded that recurrent severe hypoglycaemia is associated with cumulative cognitive impairment in adult diabetic patients treated with insulin.

Wilczek, H.; Gunnarsson, R.; Felig, P.; Östman, J.; Groth, C.; Wahren, J.

doi: 10.1007/BF00405007pmid: 1864489

125 34 34 5 5 H. Wilczek R. Gunnarsson P. Felig J. Östman C. -G. Groth J. Wahren Department of Transplantation Surgery, Huddinge Hospital Karolinska Institute Stockholm Sweden Department of Medicine, Huddinge Hospital Karolinska Institute Stockholm Sweden Department of Clinical Physiology, Huddinge Hospital Karolinska Institute Stockholm Sweden Summary With current surgical techniques for pancreatic transplantation, the graft is anastomosed to the iliac vessels, resulting in delivery of insulin to the systemic circulation rather than to the portal vein as in healthy man. The possible influence of the altered route of insulin delivery on the regulation of splanchnic glucose metabolism was studied in four patients with Type 1 (insulin-dependent) diabetes mellitus at 6–19 months after combined pancreatic and kidney transplantation. Four non-diabetic, age-matched renal transplant recipients and two groups of age-matched healthy subjects served as controls. The studies were carried out in the basal state and during two rates of intravenous glucose infusion (2 and 4 mg · kg −1 · min−1). Fasting arterial glucose and splanchnic glucose output was similar in all groups. Basal hyperinsulinaemia was present in pancreatic graft recipients compared to healthy subjects. During low rate intravenous glucose infusion splanchnic glucose output decreased to a similar extent in all groups. With the higher glucose infusion rate (4 mg · kg −1 · min −1 ) a net glucose uptake was observed which was similar in all three groups. Peripheral glucose uptake was unchanged at the lower glucose infusion rate but increased by 45–55% at the higher rate. It is concluded that despite systemic insulin delivery from a heterotopic pancreatic graft, hepatic glucose metabolism appears normal both in the post-absorptive state and in response to glucose-stimulated endogenous insulin secretion. Portal insulin delivery is thus not necessary for normal hepatic glucose metabolism in the Type 1 diabetic patient.