miR-10b and miR-223-3p in serum microvesicles signal progression from prediabetes to type 2 diabetes.

PURPOSE: Type 2 diabetes frequently remains undiagnosed for years, whereas early detection of affected individuals would facilitate the implementation of timely and cost-effective therapies, hence decreasing morbidity. With the intention of identifying novel diagnostic biomarkers, we characterized the miRNA profile of microvesicles isolated from retroactive serum samples of normoglycemic individuals and two groups of subjects with prediabetes that in the following 4 years either progressed to overt diabetes or remained stable. METHODS: We profiled miRNAs in serum microvesicles of a selected group of control and prediabetic individuals participating in the PREDAPS cohort study. Half of the subjects with prediabetes were diagnosed with diabetes during the 4 years of follow-up, while the glycemic status of the other half remained unchanged. RESULTS: We identified two miRNAs, miR-10b and miR-223-3p, which target components of the insulin signaling pathway and whose ratio discriminates between these two subgroups of prediabetic individuals at a stage at which other features, including glycemia, are less proficient at separating them. In global, the profile of miRNAs in microvesicles of prediabetic subjects primed to progress to overt diabetes was more similar to that of diabetic patients than the profile of prediabetic subjects who did not progress. CONCLUSION: We have identified a miRNA signature in serum microvesicles that can be used as a new screening biomarker to identify subjects with prediabetes at high risk of developing diabetes, hence allowing the implementation of earlier, and probably more effective, therapeutic interventions.

Obesity rather than regional fat depots marks the metabolomic pattern of adipose tissue: an untargeted metabolomic approach.

OBJECTIVE: This study compares the patterns of visceral (VIS) and subcutaneous (SC) adipose tissue (AT)-derived metabolites from non-obese (BMI 24-26 kg/m2) and obese subjects (BMI > 40 kg/m2) with no major metabolic risk factors other than BMI. METHODS: SC- and VIS- AT obtained from obese (Ob) and non-obese (NOb) subjects during surgery were incubated to obtain their metabolites. Differences related to obesity or anatomical provenances of AT were assessed using an untargeted metabolomics approach based on gas chromatography-mass spectrometry. RESULTS: The overall effect of obesity on the metabolite profile resulted more remarkable than the effect of regional AT. Only the depletion of 2-ketoisocaproic (2-KIC) acid reached statistical significance for the SC-AT alone, although it was observed in both depots. Obesity induced more significant changes in several amino acids levels of the VIS-AT metabolites. On the one hand, higher released levels of glutamine and alanine were detected in the VIS- obese AT, whereas on the other, the VIS- obese AT presented a diminished uptake of essential amino acids (methionine, threonine, lysine), BCAAs, leucine, and serine. CONCLUSION: This study shows that obesity markedly affects the amino acid metabolic signature of the AT before the clinical onset of other significant metabolic alterations aside from BMI.

Translational evidence of endothelial damage in obese individuals: inflammatory and prothrombotic responses.

BACKGROUND: Obesity is associated with an increased atherothrombotic morbidity/mortality risk. However, there is no direct evidence of subclinical activation of the endothelium in obese subjects without other major cardiometabolic risk factors. OBJECTIVES: We applied a translational approach to investigate endothelial activation occurring in response to the components secreted by visceral and subcutaneous adipose tissue and their corresponding cell fractions obtained from obese subjects without other major cardiometabolic risk factors, as compared with non-obese controls. METHODS: Fat pads and cell fractions were incubated with serum-free medium to obtain their secretomes, which were analyzed by protein arrays. Endothelial cells (ECs) were exposed to the different secretomes to evaluate changes in gene expression, composition and reactivity of the extracellular matrix (ECM), and cell growth and viability. RESULTS: ECs incubated in the presence of obese secretomes displayed increased proliferation, altered cell morphology, augmented expression of VCAM-1, ICAM-1, and von Willebrand factor, and higher ECM reactivity towards circulating platelets. The visceral secretomes, especially the stromal one, induced the strongest expression of these markers, together with a more reactive ECM. These changes occurred through nuclear factor-κB (NF-κB) activation. CONCLUSION: This is the first translational study demonstrating that the cytokines secreted by the adipose tissue from obese individuals without other major cardiometabolic complications have a hazardous effect on the endothelium, through activation of the NF-κB pathway.

Rare variants identified in the HNF- 4 alpha beta-cell-specific promoter and alternative exon 1 lack biological significance in maturity onset diabetes of the young and young onset Type II diabetes.

AIMS/HYPOTHESIS: The recently identified alternative promoter (P2) of HNF-4 alpha is the major HNF-4 alpha transcription start site in pancreatic beta cells. The significance of the P2 promoter was shown by the identification of a mutation in the IPF-1 binding site of the alternative promoter which cosegregated with diabetes in a large MODY family. The role of the P2 promoter and the associated alternative exon 1 in both MODY and polygenic Type II (non-insulin-dependent) diabetes mellitus is not known. Linkage to this region in studies of Type II diabetes makes the P2 region a strong candidate for a role in Type II diabetes susceptibility. METHODS: To assess the role of the P2 region we screened MODY, young-onset Type II diabetic subjects, and probands from Type II diabetes families linked to chromosome 20 for variants of the P2 promoter and associated exon of HNF-4 alpha. RESULTS: Two variants were found that were not present in the control subjects. The -79 C/T substitution was present in a MODY family but did not perfectly cosegregate with diabetes. A -276 G/T substitution was identified in two UK young-onset diabetes probands but did not co-segregate with diabetes. Reporter gene studies did not indicate changes in transcriptional activity caused by either the -79 C/T or -276 G/T single nucleotide substitutions. CONCLUSION/INTERPRETATION: We found no evidence to suggest that variation in the P2 proximal promoter region and associated alternative exon 1 of HNF-4 alpha contribute to young onset Type II diabetes susceptibility in Northern Europeans.

A transcription factor regulatory circuit in differentiated pancreatic cells.

Mutations in the human genes encoding hepatocyte nuclear factors (HNF) 1alpha, 1beta, 4alpha, and IPF1(PDX1/IDX1/STF1) result in pancreatic beta cell dysfunction and diabetes mellitus. In hepatocytes, hnf4alpha controls the transcription of hnf1alpha, suggesting that this same interaction may operate in beta cells and thus account for the common diabetic phenotype. We show that, in pancreatic islet and exocrine cells, hnf4alpha expression unexpectedly depends on hnf1alpha. This effect is tissue-specific and mediated through direct occupation by hnf1alpha of an alternate promoter located 45.6 kb from the previously characterized hnf4alpha promoter. Hnf1alpha also exerts direct control of pancreatic-specific expression of hnf4gamma and hnf3gamma. Hnf1alpha dependence of hnf4alpha, hnf4gamma, hnf3gamma, and two previously characterized distal targets (glut2 and pklr) is established only after differentiated cells arise during pancreatic embryonic development. These studies define an unexpected hierarchical regulatory relationship between two genes involved in human monogenic diabetes in the cells, which are relevant to its pathophysiology. Furthermore, they indicate that hnf1alpha is an essential component of a transcription factor circuit whose role may be to maintain differentiated functions of pancreatic cells.

Hepatic nuclear factor 1-alpha directs nucleosomal hyperacetylation to its tissue-specific transcriptional targets.

Mutations in the gene encoding hepatic nuclear factor 1-alpha (HNF1-alpha) cause a subtype of human diabetes resulting from selective pancreatic beta-cell dysfunction. We have analyzed mice lacking HNF1-alpha to study how this protein controls beta-cell-specific transcription in vivo. We show that HNF1-alpha is essential for the expression of glut2 glucose transporter and L-type pyruvate kinase (pklr) genes in pancreatic insulin-producing cells, whereas in liver, kidney, or duodenum tissue, glut2 and pklr expression is maintained in the absence of HNF1-alpha. HNF1-alpha nevertheless occupies the endogenous glut2 and pklr promoters in both pancreatic islet and liver cells. However, it is indispensable for hyperacetylation of histones in glut2 and pklr promoter nucleosomes in pancreatic islets but not in liver cells, where glut2 and pklr chromatin remains hyperacetylated in the absence of HNF1-alpha. In contrast, the phenylalanine hydroxylase promoter requires HNF1-alpha for transcriptional activity and localized histone hyperacetylation only in liver tissue. Thus, different HNF1-alpha target genes have distinct requirements for HNF1-alpha in either pancreatic beta-cells or liver cells. The results indicate that HNF1-alpha occupies target gene promoters in diverse tissues but plays an obligate role in transcriptional activation only in cellular- and promoter-specific contexts in which it is required to recruit histone acetylase activity. These findings provide genetic evidence based on a live mammalian system to establish that a single activator can be essential to direct nucleosomal hyperacetylation to transcriptional targets.

Characterization of insulin and insulin-like growth factor-I ovarian receptors during the reproductive cycle of carp (Cyprinus carpio).

Insulin and insulin-like growth factor-I (IGF-I) receptors were characterized in glycoprotein fractions prepared by wheat germ agglutinin-agarose affinity chromatography from the ovaries of carp. Insulin-specific overall binding in carp ovaries was 6- to 11-fold lower than IGF-I binding (2.7 +/- 0.48% vs. 22.8 +/- 3.6% per 20 microg glycoprotein). Cold IGF-I displaced radiolabeled IGF-I binding in doses 1000- to 3000-fold lower than cold insulin. On the other hand, cold insulin displaced radiolabeled insulin binding at concentrations 5- to 30-fold lower than cold IGF-I. The alpha-subunit molecular masses of carp insulin and IGF-I receptors were smaller than the alpha-subunit molecular mass of rat insulin receptor (125 and 120 vs. 135 kDa, respectively). Autophosphorylation of carp beta-subunit insulin and IGF-I receptors showed similar molecular masses that did not differ from the molecular mass of rat insulin beta subunit. Receptor tyrosine kinase activity was stimulated in a dose-dependent manner by insulin and IGF-I. Insulin and IGF-I stimulated tyrosine kinase activity and reached a maximum, respectively, of 224 +/- 14% and 279 +/- 7% of basal phosphorylation. Insulin and IGF-I binding characteristics were measured through different stages of follicular development. High specific binding of both peptides in primary oocyte growth (5.6 +/- 0.8% and 50 +/- 10% per 20 microg glycoprotein for insulin and IGF-I, respectively) decreased to a minimum at the end of vitellogenesis, followed by a slight increase later, in the preovulatory stage. The presence of insulin and IGF-I receptors in carp ovaries and the changes in percentage of binding throughout the reproductive cycle suggest that, in carp, the roles of insulin and IGF-I depend on the ovarian maturation stage.

The proto-oncogene Crk-II enhances apoptosis by a Ras-dependent, Raf-1/MAP kinase-independent pathway.

Human embryonic kidney 293 cells and 293 cells overexpressing different amounts of the adaptor protein Crk-II (ranging from 3- to 10-fold higher levels than the parental cell line) were examined for their ability to undergo apoptosis when maintained in control and serum-free (SF) medium. Parental 293 cells undergo apoptosis only when deprived of serum for prolonged periods of time (24-48 h). On the other hand, 293 cells overexpressing different levels of Crk-II present detectable levels of apoptosis as measured by DNA fragmentation when grown in control medium, with a marked increase when they are deprived of serum for 12-48 h. To determine the pathways involved in Crk-II-induced apoptosis, Crk-II overexpressing cells were transiently transfected with a dominant-negative Ras construct (N17-Ras). Compared to cells transfected with control vectors, the cells overexpressing N17-Ras presented lower levels of apoptosis when maintained in SF-medium. On the other hand, transient transfection of a dominant-negative Raf-1 construct (K375W-Raf-1) did not decrease apoptosis; slightly increasing DNA fragmentation levels were seen. Similar results were obtained when the cells were incubated in the presence of a MEK1 inhibitor. The results presented here suggest that overexpression of Crk-II induces apoptosis via a Ras-dependent, Raf-1/MEK1/ERK-independent pathway.

Specific inhibition of insulin-like growth factor-1 and insulin receptor tyrosine kinase activity and biological function by tyrphostins.

A series of the synthetic protein tyrosine kinase inhibitors known as tyrphostins were studied for their effect on insulin-like growth factor-1 and insulin-stimulated cellular proliferation on NIH-3T3 fibroblasts overexpressing either receptor, as well as for their ability to inhibit ligand-stimulated receptor autophosphorylation and tyrosine kinase activity toward exogenous substrates. Several of the tyrphostins tested demonstrated a dramatic effect by inhibiting hormone-stimulated cell proliferation, with IC50s in the submicromolar range, while being unable to block serum-stimulated cell proliferation. The tyrphostins also inhibited receptor autophosphorylation and tyrosine kinase activity, with a higher IC50, in the micromolar range. Most of the tyrphostins tested presented no clear preference for either receptor, although two of them (AG1024 and AG1034) showed significantly lower IC50s for IGF-1 than for insulin receptors. These results suggest that, in spite of the high homology of the kinase regions of both receptors, it could be possible to design and synthesize small molecules capable of discriminating between them. The synthesis of such specific inhibitors could be an excellent tool to establish the precise signalling mechanisms that distinguish between the different effects of these two hormones.