The impact of ethnicity on glucose regulation and the metabolic syndrome following gestational diabetes.

AIMS/HYPOTHESIS: We assessed the impact of ethnic origin on metabolism in women following gestational diabetes mellitus (GDM). MATERIALS AND METHODS: Glucose regulation and other features of the metabolic syndrome were studied at 20.0 (18.2-22.1) months (geometric mean [95% CI]) post-partum in women with previous GDM (185 European, 103 Asian-Indian, 80 African-Caribbean). They were compared with the same features in 482 normal control subjects who had normal glucose regulation during and following pregnancy. RESULTS: Impaired glucose regulation or diabetes by WHO criteria were present in 37% of women with previous GDM (diabetes in 17%), especially in those of African-Caribbean and Asian-Indian origin (50 and 44%, respectively vs 28% in European, p=0.009). BMI, waist circumference, diastolic blood pressure, fasting triglyceride and insulin levels, and insulin resistance by homeostatic model assessment (HOMA), were increased following GDM (p<0.001 for all, vs control subjects). Where glucose regulation was normal following GDM, basal insulin secretion (by HOMA) was high (p<0.001 vs control subjects). Irrespective of glucose regulation in pregnancy, Asian-Indian origin was associated with high triglyceride and low HDL cholesterol levels, and African-Caribbean with increased waist circumference, blood pressure, and insulin levels, together with insulin resistance and low triglyceride concentrations. Nonetheless, the GDM-associated features were consistent within each ethnic group. The metabolic syndrome by International Diabetes Federation criteria was present in 37% of women with previous GDM, especially in non-Europeans (Asian-Indian 49%, African-Caribbean 43%, European 28%, p=0.001), and in 10% of controls. CONCLUSIONS/INTERPRETATION: Following GDM, abnormal glucose regulation and the metabolic syndrome are common, especially in non-European women, indicating a need for diabetes and cardiovascular disease prevention strategies.

Women with a history of gestational diabetes of European and South Asian origin are shorter than women with normal glucose tolerance in pregnancy.

AIMS: It has been reported that short individuals are more likely to have abnormalities of glucose homeostasis. The aim of this study was to examine the relationship between adult height and gestational diabetes mellitus (GDM), taking into account possible artefactual or confounding explanations. METHODS: Three hundred and forty-six women with previous GDM (169 European, 102 South Asian, 75 Afro-Caribbean) and 470 control women with no previous history of GDM (282 European, 94 South Asian and 94 Afro-Caribbean) were studied. Post-partum glucose status and height were measured. RESULTS: European and South Asian women with previous GDM were shorter than control women from the same ethnic groups (European: (mean +/- SD) 162.9 +/- 6.1 vs. 165.3 +/- 6.8 cm, P < 0.0001; South Asian: 155.2 +/- 5.4 vs. 158.2 +/- 6.3 cm, P = 0.003, adjusted for age). A similar, but non-significant trend was observed among Afro-Caribbean women (162.2 +/- 6.2 vs. 163.7 +/- 6.1 cm, P = 0.1). Similar, significant height differences were observed in Europeans and South Asians when analysis was restricted to those GDM women who had received insulin during pregnancy. There was no association between height and glucose tolerance postpartum within the GDM group. CONCLUSIONS: European and South Asian women with previous GDM are shorter than control women from the same ethnic groups. The data demonstrate that this is unlikely to be an artefact resulting from the use of an fixed 75 g load in women of differing sizes, and suggest that there are likely to be common pathophysiological mechanisms underlying GDM and the determination of final adult height.

The prevalence of polycystic ovaries in women with a history of gestational diabetes.

OBJECTIVE: Women with a history of gestational diabetes mellitus (GDM) and women with polycystic ovary syndrome (PCOS) both demonstrate abnormalities in insulin action and secretion, and both are at increased risk of developing type 2 diabetes. To determine whether these similarities reflect a common pathophysiological basis, we examined the prevalence of ultrasound-based polycystic ovarian morphology in a large multiethnic group of women with a history of GDM and a group of women who had normal glucose tolerance during pregnancy. PATIENTS AND DESIGN: We studied 91 women with previous GDM (48 European, 20 South Asian, 10 Afro-Caribbean and 13 of other or mixed ethnicity) and 73 normoglycaemic control women (56 European, one South Asian, 14 Afro-Caribbean and two of other or mixed ethnicity), a median (interquartile range) of 20 (11-36) and 29 (17-49) months postpartum, respectively. A detailed history was taken, and the prevalence of PCO morphology on ultrasound scan was assessed. Fasting lipids, insulin, glucose status, gonadotrophins and testosterone were measured. Estimates of beta-cell function (%B) and insulin sensitivity (%S) were derived using the HOMA algorithm. RESULTS: Women with previous GDM had higher fasting glucose (5.4 (4. 8-6.0) vs. 4.7 (4.4-5.0) mmol/l, P<0.0001) and features reminiscent of syndrome X: higher BMI (26.4 (22.8-31.4) vs. 23.8 (21. 0-27.5) kg/m2, P = 0.002), waist/hip ratio (0.82 (0.79-0.88) vs. 0. 77 (0.73-0.81), P<0.0001), fasting insulin (165 (68-299) vs. 54 (24-156) pmol/l, P<0.0001), triglycerides (1.1 (0.8-1.6) vs. 0.8 (0.6-1.1) mmol/l, P<0.0001) and lower insulin sensitivity (%S) (27 (16-62) vs. 86 (34-139)%, P<0.0001) compared to control women. The prevalence of PCO was higher in the previous GDM group than in the control subjects (47/91 (52%) vs. 20/73 (27%), chi2 = 9.86, P = 0. 002 overall, odds ratio 2.7, P = 0.007 by logistic regression allowing for ethnicity). There was no difference in any metabolic parameter between the post-GDM PCO group and the post-GDM normal ovaries group, but irregular cycles were more prevalent in the PCO group (22/47 (47%) vs. 9/44 (21%), chi2 = 7.03, P = 0.008). CONCLUSIONS: We found a higher prevalence of polycystic ovarian morphology in women with a history of gestational diabetes. Among the women with previous gestational diabetes, irregular cycles were more prevalent in the PCO group than in the women with normal ovarian morphology, but no other differences in endocrine or metabolic parameters were detected. These findings confirm an association between PCO and gestational diabetes and suggest that women with gestational diabetes display metabolic abnormalities irrespective of ovarian morphology.

Parent-offspring trios: a resource to facilitate the identification of type 2 diabetes genes.

The transmission disequilibrium test with use of trios (an affected proband with both parents) is a robust method for assessing the role of gene variants in disease that avoids the problem of population stratification that may confound conventional case/control studies and allows the detection of parent-of-origin effects. Trios have played a major role in defining genes in a number of polygenic conditions, including type 1 diabetes. We assessed the prevalence, clinical characteristics, and suitability for defining type 2 susceptibility genes of European type 2 diabetes trios. In a Caucasian population in the U.K., only 2.5% of type 2 patients had both parents alive. Using a nationwide strategy, we collected 182 trios defined by strict clinical criteria. Immunological and genetic testing resulted in the exclusion of 25 trios as a result of latent autoimmune diabetes (n = 13), inconsistent family relationships (n = 7), and maternally inherited diabetes and deafness (n = 5). The 157 remaining probands had similar treatment requirements to familial type 2 diabetic subjects but presented at a younger age, were more obese, and more frequently had affected parents. Using this resource, we have not found any evidence for linkage disequilibrium between type 2 diabetes and the glucokinase gene markers GCK1 and GCK2 and the chromosome 20 marker D20S197. We conclude that European type 2 diabetes trios are difficult to collect but provide an important additional approach to dissecting the genetics of type 2 diabetes.

Implications of new diagnostic criteria for abnormal glucose homeostasis in women with previous gestational diabetes.

OBJECTIVE: To determine the consequences of applying revised American Diabetes Association (ADA) (1997) and World Health Organization (WHO) (1998) recommendations for the classification of glucose intolerance in women with previous gestational diabetes mellitus (GDM). RESEARCH DESIGN AND METHODS: There were 192 women with previous GDM who took an oral glucose tolerance test (OGTT) 1-86 months after delivery and were classified by WHO (1985), ADA (1997, fasting glucose), and revised WHO (1998) guidelines. RESULTS: Among the 165 women without a preexisting diagnosis of diabetes, WHO-1985 and ADA-1997 provided similar estimates of diabetes prevalence (13.3% vs. 11.5%) but widely differing estimates of impaired glucose homeostasis (31.5% impaired glucose tolerance [IGT] by WHO-1985 vs. 10.9% impaired fasting glucose by ADA-1997 criteria). Overall, 56 women (34%) showed a classification discrepancy between WHO-1985 and ADA-1997 criteria, including 44 with normal fasting glucose by ADA-1997 criteria, but abnormal 2-h glucose by WHO-1985 criteria (40 IGT, 4 diabetes). The cardiovascular risk profile of these women was more favorable than that of 18 women with impaired fasting glucose. WHO-1998 recommendations reproduced ADA-1997 findings when used as a fasting screen, but behaved similarly to WHO-1985 criteria when 2-h glucose values were also analyzed. CONCLUSIONS: All criteria produced similar estimates of diabetes prevalence. However, analyses based on a single fasting glucose screen (and a threshold of 6.1 mmol/l) failed to identify 60% of women with abnormal 2-h glucose levels. Screening women with previous GDM (and by analogy, other groups at high risk of diabetes) with a single fasting glucose has low sensitivity for the detection of abnormal glucose tolerance. Recent guidelines recommending this approach require reevaluation.

Dominant-negative inhibition of Flk-1 suppresses the growth of many tumor types in vivo.

Angiogenesis, the sprouting of new blood vessels from existing vessels, occurs in many physiological and pathological processes, including embryonic development, wound healing, and tumor growth. It is required for tumor growth because new blood vessel formation is necessary for tumors to expand beyond a minimum volume. Several growth factor receptor tyrosine kinases have been implicated in angiogenesis, including receptors for epidermal, fibroblast, and platelet-derived growth factors, as well as the receptors Flk-1/KDR, Flt-1 Tek/Tie-2, and Tie-1. Endothelial cells in the vessels of tumors express Flk-1/KDR, a receptor for vascular endothelial growth factor. Flk-1 was previously shown to play a role in angiogenesis and tumor formation of s.c. xenografts of C6 glioma cells using dominant-negative methodology. We now demonstrate that Flk-1 seems to be generally involved in the growth of a wide range of solid tumors, including mammary, ovarian, and lung carcinoma, as well as glioblastoma. Furthermore, survival times in rats bearing intracerebral tumors were prolonged using the same dominant-negative methodology. The involvement of Flk-1 in a variety of tumor types suggests an important role for Flk-1 in tumor angiogenesis.

Distinct structural characteristics of discoidin I subfamily receptor tyrosine kinases and complementary expression in human cancer.

Mammary carcinoma kinase 10 (MCK-10) and colon carcinoma kinase 2 (CCK-2) constitute a subclass of receptor tyrosine kinases characterized by a discoidin I motif in the extracellular domain and a large cytoplasmic juxtamembrane (JM) region. While the ectodomain structure suggests a common role in cell aggregation, the JM domains of MCK-10 and CCK-2 are structurally most divergent and display features that suggest an involvement in signal generation and definition. MCK-10 occurs in at least three isoforms, which contain alternatively spliced consensus sequences for internalization and SH3 domain interaction. The presence of the 37 amino acid insert affects receptor autophosphorylation and changes ectodomain glycosylation. Proteolytic cleavage within the extracellular domain of MCK-10 generates a membrane-anchored kinase domain and releases a soluble ectodomain fragment including the discoidin I homology domain. CCK-2 and MCK-10 expression was found in connective and epithelial tissues, respectively, which in cancers of epithelial origin results in mutually exclusive expression in stroma and tumor cells, indicating a possible involvement of this class of RTKs in tumor invasion.

Identification of alternatively spliced mRNAs encoding variants of MDK1, a novel receptor tyrosine kinase expressed in the murine nervous system.

A novel member of the eck/eph family of receptor tyrosine kinases (RTKs), termed mouse developmental kinase 1 (MDK1), was identified and shown to be closely related to the Eek, Ehk1/Cek7, Ehk2, Cek4/Mek4/hek and Sek/Cek8 subfamily. Northern blot analysis revealed MDK1 mRNA transcripts of 6.8, 5.7, 4.0, 3.2 and 2.6 kb that encode apparent splice variants. Sequence analyses of MDK1 cDNA clones from adult mouse brain predict the existence of at least five isoforms, including two truncated receptor variants lacking the kinase domain. Northern blot and in situ hybridization analysis indicate that in the adult mouse MDK1 RNA expression is restricted to brain, testes and spleen. The distinct patterns of MDK1 gene expression during mouse development suggest an important role in the formation of neuronal structures and possibly other morphogenic processes.

Glioblastoma growth inhibited in vivo by a dominant-negative Flk-1 mutant.

Angiogenesis, the sprouting of capillaries from pre-existing blood vessels, is a fundamental process in the formation of the vascular system during embryonic development. In adulthood, angiogenesis takes place during corpus luteum formation and in pathological conditions such as wound healing, diabetic retinopathy, and tumor-igenesis. Vascularization is essential for solid tumour growth and is thought to be regulated by tumour cell-produced factors, which have a chemotactic and mitogenic effect on endothelial cells. Vascular endothelial growth factor (VEGF), a homodimeric glycoprotein of relative molecular mass 45,000, is the only mitogen, however, that specifically acts on endothelial cells, and it may be a major regulator of tumour angiogenesis in vivo. Its expression has been shown to be upregulated by hypoxia, and its cell-surface receptor, Flk-1, is exclusively expressed in endothelial cells. Here we investigate the biological relevance of the VEGF/Flk-1 receptor/ligand system for angiogenesis using a retrovirus encoding a dominant-negative mutant of the Flk-1/VEGF receptor to infect endothelial target cells in vivo, and find that tumour growth is prevented in nude mice. Our results emphasize the central role of the Flk-1/VEGF system in angiogenesis in general and in the development of solid tumours in particular.

Up-regulation of vascular endothelial growth factor and its cognate receptors in a rat glioma model of tumor angiogenesis.

We have recently shown that vascular endothelial growth factor (VEGF) is produced by human malignant glioma cells and acts on tumor endothelial cells, which express VEGF receptors, suggesting that VEGF is a regulator of tumor angiogenesis. To investigate the feasibility of antiangiogenic brain tumor therapy, we developed an intracerebral (i.c.) rat glioma model. We used two transplantable rat glioma cells lines, C6 and GS-9L, to analyze VEGF regulation in vitro and expression of VEGF and its high affinity tyrosine kinase receptors, flt-1 and flk-1, in vivo. Glioma cells were transplanted i.c. or s.c. into syngeneic rats. C6 gliomas exhibit morphological characteristics of human glioblastoma multiforme such as necroses with palisading cells. Immunocytochemistry with von Willebrand factor showed that C6 gliomas are highly vascularized and therefore show another prominent feature of human glioblastoma. GS-9L gliosarcomas were less vascularized. In situ hybridization showed that VEGF is expressed in vivo in rat glioma cells which reside along necrotic areas and therefore closely mimicks the expression pattern of VEGF observed in human glioblastoma. flt-1 and flk-1 are specifically expressed in endothelial cells in the tumor and at the border between tumor and normal brain but are absent from endothelial cells in the normal brain proper. The action of VEGF may therefore be restricted to tumor endothelium. Upregulation of VEGF, but not acid fibroblast growth factor, basic fibroblast growth factor, and platelet-derived growth factor B messenger RNA was observed in hypoxic C6 and GS-9L cells in vitro. These observations are consistent with a role for VEGF in tumor- and hypoxia-induced angiogenesis. Since the expression pattern of VEGF and its receptors in rat glioma appears to be indistinguishable from human glioblastoma multiforme, this model provides an excellent tool to study anti-angiogenic therapy.

High affinity VEGF binding and developmental expression suggest Flk-1 as a major regulator of vasculogenesis and angiogenesis.

Examination of flk-1 receptor tyrosine kinase mRNA expression by in situ hybridization analysis revealed specific association with endothelial cells at all stages of mouse development, including the blood islands in the yolk sac of day 8.5-10.5 embryos, in which the early progenitors of this lineage originate. flk-1 transcripts were abundant in proliferating endothelial cells of vascular sprouts and branching vessels of embryonic and early postnatal brain, but were drastically reduced in adult brain, where proliferation has ceased. Identification of the angiogenic mitogen, vascular endothelial growth factor (VEGF), as the high affinity ligand of Flk-1 and correlation of the temporal and spatial expression pattern of Flk-1 and VEGF suggest a major role of this ligand-receptor signaling system in vasculogenesis and angiogenesis.

Differential effects of carboxy-terminal sequence deletions on platelet-derived growth factor receptor signaling activities and interactions with cellular substrates.

Chimeric receptors composed of the human epidermal growth factor receptor (EGF-R) extracellular domain fused to wild-type and truncated platelet-derived growth factor receptor (PDGF-R) intracellular sequences were stably expressed in NIH 3T3 cells devoid of endogenous EGF-Rs. This experimental system allowed us to investigate the biological activity of PDGF-R cytoplasmic-domain mutants in PDGF-R-responsive NIH 3T3 cells by activating PDGF-specific signaling pathways with EGF. Deletion of 74 carboxy-terminal amino acids severely impaired the ability of the PDGF-R cytoplasmic domain to associate with cellular substrates in vitro. This deletion also inhibited receptor and substrate phosphorylation, reduced the receptor's mitogenic activity, and completely abolished its oncogenic signaling potential. Surprisingly, removal of only six additional amino acids, including Tyr-989, restored substantial receptor and substrate phosphorylation capacity as well as transforming potential and yielded a receptor with wild-type levels of ligand-induced mitogenic activity. However, the ability of this chimera to bind phospholipase C gamma was severely impaired in comparison with the ability of the wild-type receptor, while the association with other cellular proteins was not affected. Further deletion of 35 residues, including Tyr-977, nearly abolished all PDGF-R cytoplasmic-domain biological signaling activities. None of the three C-terminal truncations completely abolished the mitogenic potential of the receptors or had any influence on ligand binding or receptor down regulation. Together, these data implicate the 80 C-terminal-most residues of the PDGF-R, and possibly Tyr-989, in phospholipase C gamma binding, while receptor sequences upstream from Asp-988 appear to be essential for specific interactions with other cellular polypeptides such as ras GTPase-activating protein and phosphatidylinositol 3-kinase. Thus, the mutants described here allow the separation of distinct PDGF-activated signaling pathways and demonstrate that phospholipase C gamma phosphorylation is not required for mitogenesis and transformation.

Analysis of platelet-derived growth factor receptor domain function using a novel chimeric receptor approach.

A chimeric receptor consisting of an epidermal growth factor (EGF) receptor ligand-binding domain and platelet-derived growth factor (PDGF) receptor transmembrane and cytoplasmic signalling domains has been constructed and shown to be fully functional in phosphorylation, mitogenesis, transformation, Ca2+ release, and pH change assays. Expression of this receptor in EGF receptor-deficient, PDGF-responsive NIH 3T3 cells allows the activation of PDGF signalling pathways by EGF. This system was used to examine the function of kinase insertion sequences (KIS). While a mutant with a KIS deletion of 83 amino acids displayed a significant but reduced ability to induce mitogenic, transforming, and Ca2+ release responses in transfected cells, deletion of 20 additional amino acids resulted in abolishment of such activities. This differential loss of signalling potential correlated with the reduced or abolished potential of these receptor mutants to phosphorylate cellular substrates such as PLC gamma. Our results suggest an integral role for KIS in PDGF receptor cytoplasmic domain conformation and an involvement in substrate interaction, but provide no evidence for an exclusive role of KIS in the mediation of biological signals.