Possible mechanisms involved in improved beta cell function in pregnant women with type 1 diabetes.

Pregnancy is known to be associated with an increased demand for insulin that is normally compensated by an increased beta cell mass and insulin secretion. Recent studies have suggested enhanced beta cell function during pregnancy in women with type 1 diabetes (T1D). To explore the possible mechanisms behind enhanced beta cell function during pregnancy in women with T1D we investigated the impact of circulating factors in serum from nine women from each group of pregnant women with and without T1D, after pregnancy and non-diabetic non-pregnant women on rat islet cell proliferation and apoptosis, and on T-lymphocyte activation. In addition, circulating levels of pancreatic hormones and selected cytokines and adipokines were measured. Rat islet cell proliferation was higher in serum from pregnant women with T1D (p < 0.05) compared to T1D women after pregnancy. Apoptosis in INS-1E cell was lower (p < 0.05) in serum from pregnant women with T1D compared to T1D women after pregnancy. T-lymphocyte cell (Jurkat) proliferation was reduced by serum from pregnant women without T1D only (p < 0.05). Higher C-peptide levels and lower levels of ghrelin, IL-6, MCP-1, IL-8 and adipsin were observed in pregnant women with T1D compared to T1D women after pregnancy. In conclusion, the improved beta cell function in women with T1D during pregnancy may be due to lower levels of proinflammatory cytokines and/or higher levels of pregnancy-associated growth factors.

An Abbreviated Protocol for In Vitro Generation of Functional Human Embryonic Stem Cell-Derived Beta-Like Cells.

The ability to yield glucose-responsive pancreatic beta-cells from human pluripotent stem cells in vitro will facilitate the development of the cell replacement therapies for the treatment of Type 1 Diabetes. Here, through the sequential in vitro targeting of selected signaling pathways, we have developed an abbreviated five-stage protocol (25-30 days) to generate human Embryonic Stem Cell-Derived Beta-like Cells (ES-DBCs). We showed that Geltrex, as an extracellular matrix, could support the generation of ES-DBCs more efficiently than that of the previously described culture systems. The activation of FGF and Retinoic Acid along with the inhibition of BMP, SHH and TGF-beta led to the generation of 75% NKX6.1+/NGN3+ Endocrine Progenitors. The inhibition of Notch and tyrosine kinase receptor AXL, and the treatment with Exendin-4 and T3 in the final stage resulted in 35% mono-hormonal insulin positive cells, 1% insulin and glucagon positive cells and 30% insulin and NKX6.1 co-expressing cells. Functionally, ES-DBCs were responsive to high glucose in static incubation and perifusion studies, and could secrete insulin in response to successive glucose stimulations. Mitochondrial metabolic flux analyses using Seahorse demonstrated that the ES-DBCs could efficiently metabolize glucose and generate intracellular signals to trigger insulin secretion. In conclusion, targeting selected signaling pathways for 25-30 days was sufficient to generate ES-DBCs in vitro. The ability of ES-DBCs to secrete insulin in response to glucose renders them a promising model for the in vitro screening of drugs, small molecules or genes that may have potential to influence beta-cell function.

A Predictive Metabolic Signature for the Transition From Gestational Diabetes Mellitus to Type 2 Diabetes.

Gestational diabetes mellitus (GDM) affects 3-14% of pregnancies, with 20-50% of these women progressing to type 2 diabetes (T2D) within 5 years. This study sought to develop a metabolomics signature to predict the transition from GDM to T2D. A prospective cohort of 1,035 women with GDM pregnancy were enrolled at 6-9 weeks postpartum (baseline) and were screened for T2D annually for 2 years. Of 1,010 women without T2D at baseline, 113 progressed to T2D within 2 years. T2D developed in another 17 women between 2 and 4 years. A nested case-control design used 122 incident case patients matched to non-case patients by age, prepregnancy BMI, and race/ethnicity. We conducted metabolomics with baseline fasting plasma and identified 21 metabolites that significantly differed by incident T2D status. Machine learning optimization resulted in a decision tree modeling that predicted T2D incidence with a discriminative power of 83.0% in the training set and 76.9% in an independent testing set, which is far superior to measuring fasting plasma glucose levels alone. The American Diabetes Association recommends T2D screening in the early postpartum period via oral glucose tolerance testing after GDM, which is a time-consuming and inconvenient procedure. Our metabolomics signature predicted T2D incidence from a single fasting blood sample. This study represents the first metabolomics study of the transition from GDM to T2D validated in an independent testing set, facilitating early interventions.

Digested wheat gluten inhibits binding between leptin and its receptor.

BACKGROUND: Leptin resistance is considered a primary risk factor for obesity. It has been hypothesized that dietary cereal grain protein could cause leptin resistance by preventing leptin from binding to its receptor. Non-degraded dietary wheat protein has been found in human serum at a mean level of 41 ng/mL. Here, we report our findings from testing whether enzymatically digested gluten from wheat prevents leptin from binding to the leptin receptor in vitro. Gluten from wheat was digested with pepsin and trypsin under physiological conditions. Pepsin and trypsin activity was removed from the gluten digest with a 10 kDa spin-filter or by heat treatment at 100°C for 30 min. Binding to the leptin receptor of leptin mixed with gluten digest at a series of concentrations was measured using surface plasmon resonance technology. RESULTS: Binding of the gluten digest to the leptin receptor was not detected. Spin-filtered gluten digest inhibited binding of leptin to the leptin receptor, with 50% inhibition at a gluten digest concentration of ~10 ng/mL. Heat-treated gluten digest did not inhibit leptin binding. CONCLUSIONS: Digested wheat gluten inhibits binding of leptin to the leptin receptor, with half-maximal inhibition at 10 ng/mL. The inhibition is significant at clinically relevant concentrations and could therefore serve as a novel pathway to investigate to understand the molecular basis of leptin resistance, obesity and associated disorders.

Implications for the offspring of circulating factors involved in beta cell adaptation in pregnancy.

OBJECTIVE: Several studies have shown an increase in beta cell mass during pregnancy. Somatolactogenic hormones are known to stimulate the proliferation of existing beta cells in rodents whereas the mechanism in humans is still unclear. We hypothesize that in addition to somatolactogenic hormones there are other circulating factors involved in beta cell adaptation to pregnancy. This study aimed at screening for potential pregnancy-associated circulating beta cell growth factors. SAMPLES: Serum samples from nonpregnant and pregnant women. METHODS: The effect of serum from pregnant women on the proliferation of rat beta cells was studied using [3H]thymidine incorporation and 5-ethynyl-2'-deoxyuridine proliferation assays. In addition, serum from pregnant and nonpregnant women was fractionated by gel filtration and high performance liquid chromatography. The fractionated serum was screened for mitogenic activity in INS-1E cells. Proteins and peptides in mitogenic active serum fractions were identified by amino acid sequencing and mass spectrometry. MAIN OUTCOME MEASURES: Presence of circulating beta cell proliferating factors. RESULTS: Late gestational pregnancy serum significantly increased proliferation of rat beta cells compared with early pregnancy and nonpregnancy. The mitogenic active serum fractions contained proteins and peptides derived from kininogen-1, fibrinogen-α, α1-antitrypsin, apolipoprotein-A1, placental lactogen, angiotensinogen and serum albumin. CONCLUSION: Pregnancy serum is able to stimulate proliferation of rat beta cells. We have identified several circulating factors that may contribute to beta cell adaptation to pregnancy. Further studies are needed to elucidate their possible role in glucose homeostasis in the mother and her offspring.

Impact of fetal and neonatal environment on beta cell function and development of diabetes.

The global epidemic of diabetes is a serious threat against health and healthcare expenses. Although genetics is important it does not explain the dramatic increase in incidence, which must involve environmental factors. Two decades ago the concept of the thrifty phenotype was introduced, stating that the intrauterine environment during pregnancy has an impact on the gene expression that may persist until adulthood and cause metabolic diseases like obesity and type 2 diabetes. As the pancreatic beta cells are crucial in the regulation of metabolism this article will describe the influence of normal pregnancy on the beta cells in both the mother and the fetus and how various conditions like diabetes, obesity, overnutrition and undernutrition during and after pregnancy may influence the ability of the offspring to adapt to changes in insulin demand later in life. The influence of environmental factors including nutrients and gut microbiota on appetite regulation, mitochondrial activity and the immune system that may affect beta cell growth and function directly and indirectly is discussed. The possible role of epigenetic changes in the transgenerational transmission of the adverse programming may be the most threatening aspect with regard to the global diabetes epidemics. Finally, some suggestions for intervention are presented.

Co-ordinated regulation of neurogenin-3 expression in the maternal and fetal pancreas during pregnancy.

OBJECTIVE: Several studies have shown increased beta cell mass during pregnancy in both rodents and humans. Proliferation of existing beta cells seems to be the predominant mechanism in rodents, whereas the mechanism in humans is unclear. We hypothesized that neogenesis contributes to the increased beta cell mass in pregnancy and that circulating factors are involved. SAMPLES: Pancreatic tissue from mice and rat and serum from pregnant women. METHOD: Morphometric analysis of pancreas of pregnant and nonpregnant mice was carried out by immunocytochemical staining for the neogenic marker neurogenin-3. Messenger RNA levels of neurogenin-3 and the transcription factor musculoaponeurotic fibrosarcoma oncogene family protein B in fetal rat pancreas cells, cultured with serum from pregnant women, were measured by quantitative polymerase chain reaction. MAIN OUTCOME MEASURES: The number of neurogenin-3-positive cells present in pregnant mice was increased compared with nonpregnant mice. Neurogenin-3 and musculoaponeurotic fibrosarcoma oncogene family protein B mRNA was detected in fetal rat pancreas exposed to serum from pregnant women. RESULTS: In pregnant mice we found a 3.6-fold increase in beta cell volume at day 18 compared with nonpregnant mice and a 3.5-fold increase in neurogenin-3 volume at day 14, mainly located in the acinar compartment where it was eightfold higher than in nonpregnant mice. In fetal rat pancreatic cells exposed to serum from pregnant women we found a marked increase in both neurogenin-3 and musculoaponeurotic fibrosarcoma oncogene family protein B mRNA levels in fibroblast-like cells. CONCLUSION: These results suggest that neogenesis contributes to the increased beta cell mass in pregnancy and that circulating factors are involved in beta cell formation in both the maternal and fetal pancreas during pregnancy.

(111)Indium Labelling of Recombinant Activated Coagulation Factor VII: In Vitro and Preliminary In Vivo Studies in Healthy Rats.

The aim of this study is to investigate whether (111)Indium-labelled recombinant FVIIa (rFVIIa) could be a potential radiopharmaceutical for localization of bleeding sources. DTPA-conjugated rFVIIa was radiolabelled with (111)In chloride. In vitro binding efficiency of (111)In-DTPA-rFVIIa to F1A2-Mab-sepharose was 99% in buffer, while it was 88-82% in serum. The binding efficiency of (111)In-DTPA-rFVIIa to TF (1-209)-sepharose was 48% in buffer whereas 39%-36% in serum, respectively. In vivo experiment was conducted in healthy rats, and gamma camera images were taken immediately after iv. administration of 1.6-1.8 MBq (111)In-DTPA-rFVIIa up to 120-130 min. Five min after administration of (111)In-DTPA-rFVIIa, percentage of (111)In activity was 6.0% in the cardiac region and 24.5% in the liver region. After 2 hours activity was decreased to 3.3% in heart while it had increased to 42.0% in the liver. The (111)In-DTPA-rFVIIa might be a potential radiopharmaceutical for visualisation of tissues with significant TF expression such as acute bleeding lesions in the gastrointestinal tract.

Easy and efficient (111)indium labeling of long-term stored DTPA conjugated protein.

UNLABELLED: The labelling efficiency of long-term stored DTPA-conjugates has not been reported previously even though DTPA has been in extensive use as metal chelator in the development of radiopharmaceuticals and contrast agents. DTPA is often used as a bifunctional chelating agent conjugated to tumor targeting vehicles such as monoclonal antibodies and receptor directed peptides. The purpose of this study was to monitor the labelling efficiency of a DTPA-conjugate on long-term storage using 111In-chloride at two different temperatures and incubation times for the In-labelling. METHOD: Cyclic-diethylene-triamine-pentaacetic acid (cDTAP) was conjugated to a polyclonal immunoglobulin-G (IgG) in borate buffer, pH 8.2 at +4?C for 4 hours. Then the DTPA-conjugate was dialyzed against 50 mmol/l sodium citrate buffer saline, pH 6.0 and stored at -80° C in aliquots of 1 mg/0.5 ml. The DTPA-conjugate was labeled with 111In-chloride in citrate buffer, pH 6. The labelling reaction was incubated at room temperature (RT) for 30 min and at +4?C for 90 min. Determination of labelling efficiency was performed using ITLC and an instant chromatography scanner equipped with a NaI crystal. The labelling efficiency of the DTPA-conjugate was monitored every third month for 12 months. RESULTS: The median labelling efficiencies varied between 92 and 96% during the whole period. The two combinations of incubation times and temperatures (30 min at RT and 90 min at +4°C) had no affect on labelling efficiency of the DTPA-conjugate, stored for 12 months. CONCLUSION: Our study shows that 111In-labelling can easily be performed within 30 min at RT for thermo-stable proteins like polyclonal, DTPA-conjugated IgG stored long-term at -80°C with a high 111In-labelling efficiency.

Angiogenesis in vestibular schwannomas: expression of extracellular matrix factors MMP-2, MMP-9, and TIMP-1.

OBJECTIVES/HYPOTHESIS: Vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMPs) are potent mediators of tumor angiogenesis. It has been demonstrated that vestibular schwannoma VEGF expression correlates with tumor growth pattern, whereas knowledge on the expression of MMPs is lacking. This study targets the angiogenic process by investigation of tumor expression of MMP-2, MMP-9, and tissue inhibitors of metalloproteinase (TIMP)-1. A possible correlation with gender, patient age, symptom duration, tumor size, and the absolute and relative growth rate is explored. STUDY DESIGN: Prospective vestibular schwannoma tissue sampling for ELISA and immunohistochemical determination of MMP-2, MMP-9 and TIMP-1. METHODS: Thirty-four patients with a sporadic, noncystic, vestibular schwannoma were selected prospectively. Repeated, preoperative magnetic resonance imaging determined the tumor growth pattern. Following translabyrinthine resection, an enzyme-linked immunosorbent assay was used for determination of the MMP-2, MMP-9, and TIMP-1 concentration in tumor sample homogenates. Immunohistochemical labeling was performed in 12 randomly selected tumors. RESULTS: : All tumor homogenates expressed measurable MMP-9, MMP-2, and TIMP-1. Immunolabeling localized MMP-9 expression to the tumor cells, whereas MMP-2 and TIMP-1 was found interstitially. A significant correlation existed between the concentration MMP-9 and absolute tumor growth rate, whereas a weak correlation occurred for the relative growth rate. CONCLUSIONS: Vestibular schwannomas express MMP-2, MMP-9, and TIMP-1 and the tumor concentration of MMP-9 correlates with absolute tumor growth rate, but not with age, gender, symptom duration, or preoperative tumor size. No correlations existed between any clinical parameter and MMP-2 or TIMP-1 expression. We conclude that MMP-9 appears to be involved in the growth of vestibular schwannomas.

VEGF and VEGF receptor-1 concentration in vestibular schwannoma homogenates correlates to tumor growth rate.

OBJECTIVE: Vascular endothelial growth factor (VEGF) is one of the most potent mediators of angiogenesis, which is a mandatory process during tumor growth. Immunohistochemical studies have demonstrated VEGF expression in vestibular schwannomas (VS), and a semi-quantitation of staining intensity indicated a correlation between tumor growth rate and VEGF expression. The present objectives were to determine the concentration of VEGF and the high-affinity receptor VEGFR-1 in VS homogenates and to examine a possible correlation with symptom duration, tumor size, or growth rate. STUDY DESIGN, PATIENTS, AND METHODS: Prospective selection of 27 patients with VS growth determined by repeated magnetic resonance imaging. Patient files were reviewed for symptom duration and all magnetic resonance images reviewed for determination of tumor size and growth rate. ELISA was used for determination of the VEGF and VEGFR-1 concentration in tumor homogenates. SETTING: Tertiary University Hospital Clinic. RESULTS: All tumor homogenates contained VEGF and VEGFR-1. A significant correlation existed between the concentration of both VEGF and VEGFR-1 and tumor growth rate but not symptom duration or tumor size. CONCLUSION: The concentration of VEGF and VEGFR-1 in VS homogenates correlates with tumor growth rate but not with tumor size or symptom duration. We conclude that VEGF and VEGFR-1 appear to be directly involved in the growth pattern of VS.