Insulin Signaling in Type 2 Diabetes: EXPERIMENTAL AND MODELING ANALYSES REVEAL MECHANISMS OF INSULIN RESISTANCE IN HUMAN ADIPOCYTES*

Type 2 diabetes originates in an expanding adipose tissue that for unknown reasons becomes insulin resistant. Insulin resistance reflects impairments in insulin signaling, but mechanisms involved are unclear because current research is fragmented. We report a systems level mechanistic understanding of insulin resistance, using systems wide and internally consistent data from human adipocytes. Based on quantitative steady-state and dynamic time course data on signaling intermediaries, normally and in diabetes, we developed a dynamic mathematical model of insulin signaling. The model structure and parameters are identical in the normal and diabetic states of the model, except for three parameters that change in diabetes: (i) reduced concentration of insulin receptor, (ii) reduced concentration of insulin-regulated glucose transporter GLUT4, and (iii) changed feedback from mammalian target of rapamycin in complex with raptor (mTORC1). Modeling reveals that at the core of insulin resistance in human adipocytes is attenuation of a positive feedback from mTORC1 to the insulin receptor substrate-1, which explains reduced sensitivity and signal strength throughout the signaling network. Model simulations with inhibition of mTORC1 are comparable with experimental data on inhibition of mTORC1 using rapamycin in human adipocytes. We demonstrate the potential of the model for identification of drug targets, e.g. increasing the feedback restores insulin signaling, both at the cellular level and, using a multilevel model, at the whole body level. Our findings suggest that insulin resistance in an expanded adipose tissue results from cell growth restriction to prevent cell necrosis.     

Dyslipidemia,but not hyperglycemia and insulin resistance,is associated with marked alterations in the HDL lipidome in type 2 diabetic subjects in the DIWA cohort: Impact on small HDL particles

In this study we have used mass spectrometry in order to characterize the HDL lipidome in three groups of women from the DIWA cohort; one control group, plus two groups with type 2 diabetes with insulin resistance; one dyslipidemic and one normolipidemic. The aim was to investigate whether dyslipidemia is required in addition to insulin resistance for the occurrence of an altered HDL lipidome, which in turn might impact HDL functionality. The dyslipidemic type 2 diabetic subjects were distinguished by obesity, hypertriglyceridemia with elevated apoC3, low HDL-cholesterol and chronic low grade inflammation. In a stepwise multivariate linear regression analysis, including biomarkers of dyslipidemia and insulin resistance as independent variables, only dyslipidemia showed a significant correlation with HDL lipid classes. Small HDL-particles predominated in dyslipidemic subjects in contrast to the normolipidemic diabetic and control groups, and were enriched in lysophosphatidylcholine (+ 13%), a product of proinflammatory phospholipases, and equally in two core lipids, palmitate-rich triacylglycerols and diacylglycerols (+ 77 %), thereby reflecting elevated CETP activity. Dyslipidemic small HDL particles were further distinguished not only as the primary carrier of ceramides, which promote inflammation and insulin resistance, but also by a subnormal plasmalogen/apoAI ratio, consistent with elevated oxidative stress typical of type 2 diabetes. From these data we conclude that in type 2 diabetes, dyslipidemia predominates relative to hyperglycemia for the occurrence of an altered HDL lipidome. Furthermore, dyslipidemia alters the cargo of bioactive lipids, with implications for HDL function.     

Synthesis of C-1′-Branched Acyclic Nucleosides

Abstract

Two C-1′-branched acyclic thymine derivatives, 1-[2-hydroxy-1-(2-hydroxyethoxy)ethyl]thymine and 1-[3-hydroxy-1-(2-hydroxyethoxy)-propyl]thymine were synthesized by a novel iodine-activated reaction of a tolylthio derivative with ethylene glycol. This synthetic method provides a potentially versatile synthetic entry to C-1′-branched acyclic nucleosides.     

Dipeptidyl Peptidase IV Inhibition Activates CREB and Improves Islet Vascularization through VEGF-A/VEGFR-2 Signaling Pathway

Substitution of pancreatic islets is a potential therapy to treat diabetes and it depends on reconstitution of islet’s capillary network. In this study, we addressed the question whether stabilization of Glucagon-Like-Peptide-1 (GLP-1) by inhibiting Dipeptidyl Peptidase-IV (DPP-IV) increases β-cell mass by modulating vascularization. Mouse or porcine donor islets were implanted under kidney capsule of diabetic mice treated with DPP-IV inhibitor sitagliptin. Grafts were analyzed for insulin production, β-cell proliferation and vascularization. In addition, the effect of sitagliptin on sprouting and Vascular Endothelial Growth Factor (VEGF)-A expression was examined ex vivo. The cAMP response element-binding (CREB) and VEGF-A/ Vascular Endothelial Growth Factor Receptor (VEGFR)-2 signaling pathway leading to islet vascularization was explored. Sitagliptin increased mean insulin content of islet grafts and area of insulin-positive tissue as well as β-cell proliferation. Interestingly, sitagliptin treatment also markedly increased endothelial cell proliferation, microvessel density and blood flow. Finally, GLP-1 (7-36) stimulated sprouting and VEGF expression, which was significantly enhanced by sitagliptin- mediated inhibition of DPP-IV. Our in vivo data demonstrate that sitagliptin treatment phosphorylated CREB and induced islet vascularization through VEGF-A/VEGFR-2 signaling pathway. This study paves a new pathway for improvement of islet transplantation in treating diabetes mellitus.     

Immunogenicity of the carcinoembryonic antigen derived peptide 694 in HLA-A2 healthy donors and colorectal carcinoma patients

Carcinoembryonic antigen (CEACAM5) is commonly overexpressed in human colon cancer. Several antigenic peptides recognized by cytolytic CD8+ T-cells have been identified and used in colon cancer phase-I vaccination clinical trials. The HLA-A*0201-binding CEA_694–702 peptide was recently isolated from acid eluted MHC-I associated peptides from a human colon tumor cell line. However, the immunogenicity of this peptide in humans remains unknown. We found that the peptide CEA_694–702 binds weakly to HLA-A*0201 molecules and is ineffective at inducing specific CD8+ T-cell responses in healthy donors. Immunogenic-altered peptide ligands with increased affinity for HLA-A*0201 were identified. Importantly, the elicited cytolytic T lymphocyte (CTL) lines and clones cross-reacted with the wild-type CEA_694–702 peptide. Tumor cells expressing CEA were recognized in a peptide and HLA-A*0201 restricted fashion, but high-CEA expression levels appear to be required for CTL recognition. Finally, CEA-specific T-cell precursors could be readily expanded by in vitro stimulation of peripheral blood mononuclear cell (PBMC) from colon cancer patients with altered CEA peptide. However, the CEA-specific CD8+ T-cell clones derived from cancer patients revealed low-functional avidity and impaired tumor-cell recognition. Together, using T-cells to demonstrate the processing and presentation of the peptide CEA694-702, we were able to corroborate its presentation by tumor cells. However, the low avidity of the specific CTLs generated from cancer patients as well as the high-antigen expression levels required for CTL recognition pose serious concerns for the use of CEA694-702 in cancer immunotherapy.     

Age-related changes in conducted vasodilation: effects of exercise training and role in functional hyperemia

Conducted vasodilation may coordinate blood flow in microvascular networks during skeletal muscle contraction. We tested the hypotheses that 1) exercise training enhances conducted vasodilation and 2) age-related changes in the capacity for conduction affect muscle perfusion during contractions. To address hypothesis 1, young (4-5 mo), adult (12-14 mo), and old (19-21 mo) C57BL6 male mice were sedentary or given access to running wheels for 8 wk. Voluntary running distances were significantly different (in km/day): young = 5.8 +/- 0.1, adult = 3.9 +/- 0.1, old = 2.2 +/- 0.1 (P < 0.05). In gluteus maximus muscles, conducted vasodilation was greater in adult than in young or old mice (P < 0.05) and greater in young sedentary than in old sedentary mice but was not affected by exercise training. Citrate synthase activity was greater with exercise training at all ages (P < 0.05). mRNA for endothelial nitric oxide synthase did not differ among ages, but endothelial nitric oxide synthase protein expression was greater in adult and old mice with exercise training (P < 0.05). Connexin 37, connexin 40, and connexin 43 mRNA were not affected by exercise training and did not differ by age. To address hypothesis 2, perfusion of the gluteus maximus muscle during light to severe workloads was assessed by Doppler microprobe at 3-26 mo of age. Maximum perfusion decreased linearly across the lifespan. Perfusion at the highest workload, absolute and relative to maximum, decreased across the lifespan, with a steeper decline beyond approximately 20 mo of age. In this model, 1) exercise training does not alter conducted vasodilation and 2) muscle perfusion is maintained up to near maximum workloads despite age-related changes in conducted vasodilation.     

Agrobacterium-mediated high-frequency transformation of an elite commercial maize (Zea mays L.) inbred line

Key message

An improved Agrobacterium -mediated transformation protocol is described for a recalcitrant commercial maize elite inbred with optimized media modifications and AGL1. These improvements can be applied to other commercial inbreds.

Abstract

This study describes a significantly improved Agrobacterium-mediated transformation protocol in a recalcitrant commercial maize elite inbred, PHR03, using optimal co-cultivation, resting and selection media. The use of green regenerative tissue medium components, high copper and 6-benzylaminopurine, in resting and selection media dramatically increased the transformation frequency. The use of glucose in resting medium further increased transformation frequency by improving the tissue induction rate, tissue survival and tissue proliferation from immature embryos. Consequently, an optimal combination of glucose, copper and cytokinin in the co-cultivation, resting and selection media resulted in significant improvement from 2.6 % up to tenfold at the T₀ plant level using Agrobacterium strain LBA4404 in transformation of PHR03. Furthermore, we evaluated four different Agrobacterium strains, LBA4404, AGL1, EHA105, and GV3101 for transformation frequency and event quality. AGL1 had the highest transformation frequency with up to 57.1 % at the T₀ plant level. However, AGL1 resulted in lower quality events (defined as single copy for transgenes without Agrobacterium T-DNA backbone) when compared to LBA4404 (30.1 vs 25.6 %). We propose that these improvements can be applied to other recalcitrant commercial maize inbreds.     

Cadmium Exposure and Incidence of Diabetes Mellitus - Results from the Malm? Diet and Cancer Study

Background

Cadmium is a pollutant with multiple adverse health effects: renal dysfunction, osteoporosis and fractures, cancer, and probably cardiovascular disease. Some studies have reported associations between cadmium and impaired fasting glucose and diabetes. However, this relationship is controversial and there is a lack of longitudinal studies.

Objectives

To examine prospectively whether cadmium in blood is associated with incidence of diabetes mellitus.

Methods

The study population consists of 4585 subjects without history of diabetes (aged 46 to 67 years, 60% women), who participated in the Malmö Diet and Cancer study during 1991–1994. Blood cadmium levels were estimated from hematocrit and cadmium concentrations in erythrocytes. Incident cases of diabetes were identified from national and local diabetes registers.

Results

Cadmium concentrations in blood were not associated with blood glucose and insulin levels at the baseline examination. However, cadmium was positively associated with HbA1c in former smokers and current smokers. During a mean follow-up of 15.2±4.2 years, 622 (299 men and 323 women) were diagnosed with new-onset of diabetes. The incidence of diabetes was not significantly associated with blood cadmium level at baseline, neither in men or women. The hazard ratio (4^th vs 1^st quartile) was 1.11 (95% confidence interval 0.82–1.49), when adjusted for potential confounders.

Conclusions

Elevated blood cadmium levels are not associated with increased incidence of diabetes. The positive association between HbA1c and blood cadmium levels has a likely explanation in mechanisms related to erythrocyte turnover and smoking.