Polymorphisms within the Novel Type 2 Diabetes Risk Locus MTNR1B Determine β-Cell Function

Background

Very recently, a novel type 2 diabetes risk gene, i.e., MTNR1B, was identified and reported to affect fasting glycemia. Using our thoroughly phenotyped cohort of subjects at an increased risk for type 2 diabetes, we assessed the association of common genetic variation within the MTNR1B locus with obesity and prediabetes traits, namely impaired insulin secretion and insulin resistance.

Methodology/Principal Findings

We genotyped 1,578 non-diabetic subjects, metabolically characterized by oral glucose tolerance test, for five tagging single nucleotide polymorphisms (SNPs) covering 100% of common genetic variation (minor allele frequency >0.05) within the MTNR1B locus (rs10830962, rs4753426, rs12804291, rs10830963, rs3781638). In a subgroup (N = 513), insulin sensitivity was assessed by hyperinsulinemic-euglycemic clamp, and in a further subgroup (N = 301), glucose-stimulated insulin secretion was determined by intravenous glucose tolerance test. After appropriate adjustment for confounding variables and Bonferroni correction for multiple comparisons, none of the tagging SNPs was reliably associated with measures of adiposity. SNPs rs10830962, rs4753426, and rs10830963 were significantly associated with higher fasting plasma glucose concentrations (p<0.0001) and reduced OGTT- and IVGTT-induced insulin release (p≤0.0007 and p≤0.01, respectively). By contrast, SNP rs3781638 displayed significant association with lower fasting plasma glucose levels and increased OGTT-induced insulin release (p<0.0001 and p≤0.0002, respectively). Moreover, SNP rs3781638 revealed significant association with elevated fasting- and OGTT-derived insulin sensitivity (p≤0.0021). None of the MTNR1B tagging SNPs altered proinsulin-to-insulin conversion.

Conclusions/Significance

In conclusion, common genetic variation within MTNR1B determines glucose-stimulated insulin secretion and plasma glucose concentrations. Their impact on β-cell function might represent the prevailing pathomechanism how MTNR1B variants increase the type 2 diabetes risk.     

A region in domain 1 of CD4 distinct from the primary gp120 binding site is involved in HIV infection and virus-mediated fusion.

The high affinity binding site for human immunodeficiency virus (HIV) envelope glycoprotein gp120 resides within the amino-terminal domain (D1) of CD4. Mutational and antibody epitope analyses have implicated the region encompassing residues 40-60 in D1 as the primary binding site for gp120. Outside of this region, a single residue substitution at position 87 abrogates syncytium formation without affecting gp120 binding. We describe two groups of CD4 monoclonal antibodies (mAbs) which recognize distinct epitopes associated with these regions in D1. These mAbs distinguish between the gp120 binding event and virus infection and virus-induced cell fusion. One cluster of mAbs, which bind at or near the high affinity gp120 binding site, blocked gp120 binding to CD4 and, as expected, also blocked HIV infection of CD4+ cells and virus-induced syncytium formation. A second cluster of mAbs, which recognize the CDR-3 like loop, did not block gp120 binding as demonstrated by their ability to form ternary complexes with CD4 and gp120. Yet, these mAbs strongly inhibited HIV infection of CD4+ cells and HIV-envelope/CD4-mediated syncytium formation. The structure of D1 has recently been solved at atomic resolution and in its general features resembles IgVk regions as predicted from sequence homology and mAb epitopes. In the D1 structure, the regions recognized by these two groups of antibodies correspond to the C'C" (Ig CDR2) and FG (Ig CDR3) hairpin loops, respectively, which are solvent-exposed beta turns protruding in two different directions on a face of D1 distal to the D2 domain. This face is straddled by the longer BC (Ig CDR1) loop which bisects the plain formed by C'C' and FG. This structure is consistent with C'C' and FG forming two distinct epitope clusters within D1. We conclude that the initial interaction between gp120 and CD4 is not sufficient for HIV infection and syncytium formation and that CD4 plays a critical role in the subsequent virus-cell and cell-cell membrane fusion events. We propose that the initial binding of CD4 to gp120 induces conformational changes in gp120 leading to subsequent interactions of the FG loop with other regions in gp120 or with the fusogenic gp41 potion of the envelope gp160 glycoprotein.     

Caloric Restriction Decreases Orthostatic Tolerance Independently from 6° Head-Down Bedrest

Astronauts consume fewer calories during spaceflight and return to earth with an increased risk of orthostatic intolerance. Whether a caloric deficiency modifies orthostatic responses is not understood. Thus, we determined the effects of a hypocaloric diet (25% caloric restriction) during 6° head down bedrest (an analog of spaceflight) on autonomic neural control during lower body negative pressure (LBNP). Nine healthy young men completed a randomized crossover bedrest study, consisting of four (2 weeks each) interventions (normocaloric bedrest, normocaloric ambulatory, hypocaloric bedrest, hypocaloric ambulatory), each separated by 5 months. Muscle sympathetic nerve activity (MSNA) was recorded at baseline following normocaloric and hypocaloric interventions. Heart rate (HR) and arterial pressure were recorded before, during, and after 3 consecutive stages (7 min each) of LBNP (-15, -30, -45 mmHg). Caloric and posture effects during LBNP were compared using two-way ANOVA with repeated measures. There was a strong trend toward reduced basal MSNA following caloric restriction alone (normcaloric vs. hypocaloric: 22±3 vs. 14±4 burst/min, p = 0.06). Compared to the normocaloric ambulatory, both bedrest and caloric restriction were associated with lower systolic blood pressure during LBNP (p<0.01); however, HR responses were directionally opposite (i.e., increase with bedrest, decrease with caloric restriction). Survival analysis revealed a significant reduction in orthostatic tolerance following caloric restriction (normocaloric finishers: 12/16; hypocaloric finishers: 6/16; χ2, p = 0.03). Caloric restriction modifies autonomic responses to LBNP, which may decrease orthostatic tolerance after spaceflight.     

Fast identification of folded human protein domains expressed in <Emphasis Type="Italic">E. coli</Emphasis> suitable for structural analysis

Background  

High-throughput protein structure analysis of individual protein domains requires analysis of large numbers of expression clones to identify suitable constructs for structure determination. For this purpose, methods need to be implemented for fast and reliable screening of the expressed proteins as early as possible in the overall process from cloning to structure determination.     

Comparison of proteomic and genomic analyses of the human breast cancer cell line T47D and the antiestrogen-resistant derivative T47D-r

In search of novel mechanisms leading to the development of antiestrogen-resistance in human breast tumors, we analyzed differences in the gene and protein expression pattern of the human breast carcinoma cell line T47D and its derivative T47D-r, which is resistant toward the pure antiestrogen ZM 182780 (Faslodex trade mark, fulvestrant). Affymetrix DNA chip hybridizations on the commercially available HuGeneFL and Hu95A arrays were carried out in parallel to the proteomics analysis where the total cellular protein content of T47D or T47D-r was separated on two-dimensional gels. Thirty-eight proteins were found to be reproducibly up- or down-regulated more than 2-fold in T47D-r versus T47D in the proteomics analysis. Comparison with differential mRNA analysis revealed that 19 of these were up- or down-regulated in parallel with the corresponding mRNA molecules, among which are the protease cathepsin D, the GTPases Rab11a and MxA, and the secreted protein hAG-2. For 11 proteins, the corresponding mRNA was not found to be differentially expressed, and for eight proteins an inverse regulation was found at the mRNA level. In summary, mRNA expression data, when combined with proteomic information, provide a more detailed picture of how breast cancer cells are altered in their antiestrogen-resistant compared with the antiestrogen-sensitive state.     

Mycoplasma hyorhinis-Contaminated Cell Lines Activate Primary Innate Immune Cells via a Protease-Sensitive Factor

Mycoplasma are a frequent and occult contaminant of cell cultures, whereby these prokaryotic organisms can modify many aspects of cell physiology, rendering experiments that are conducted with such contaminated cells problematic. Chronic Mycoplasma contamination in human monocytic cells lines has been associated with suppressed Toll-like receptor (TLR) function. In contrast, we show here that components derived from a Mycoplasma hyorhinis-infected cell line can activate innate immunity in non-infected primary immune cells. Release of pro-inflammatory cytokines such as IL-6 by dendritic cells in response to Mycoplasma hyorhinis-infected cell components was critically dependent on the adapter protein MyD88 but only partially on TLR2. Unlike canonical TLR2 signaling that is triggered in response to the detection of Mycoplasma infection, innate immune activation by components of Mycoplasma-infected cells was inhibited by chloroquine treatment and sensitive to protease treatment. We further show that in plasmacytoid dendritic cells, soluble factors from Mycoplasma hyorhinis-infected cells induce the production of large amounts of IFN-α. We conclude that Mycoplasma hyorhinis-infected cell lines release protein factors that can potently activate co-cultured innate immune cells via a previously unrecognized mechanism, thus limiting the validity of such co-culture experiments.     

Serum Protein Signatures Differentiating Autoimmune Pancreatitis versus Pancreatic Cancer

Autoimmune pancreatitis (AIP) is defined by characteristic lymphoplasmacytic infiltrate, ductal strictures and a pancreatic enlargement or mass that can mimic pancreatic cancer (PaCa). The distinction between this benign disease and pancreatic cancer can be challenging. However, an accurate diagnosis may pre-empt the misdiagnosis of cancer, allowing the appropriate medical treatment of AIP and, consequently, decreasing the number of unnecessary pancreatic resections.Mass spectrometry (MS) and two-dimensional differential gel electrophoresis (2D-DIGE) have been applied to analyse serum protein alterations associated with AIP and PaCa, and to identify protein signatures indicative of the diseases. Patients'' sera were immunodepleted from the 20 most prominent serum proteins prior to further 2D-DIGE and image analysis. The identity of the most-discriminatory proteins detected, was performed by MS and ELISAs were applied to confirm their expression. Serum profiling data analysis with 2D-DIGE revealed 39 protein peaks able to discriminate between AIP and PaCa. Proteins were purified and further analysed by MALDI-TOF-MS. Peptide mass fingerprinting led to identification of eleven proteins. Among them apolipoprotein A-I, apolipoprotein A-II, transthyretin, and tetranectin were identified and found as 3.0-, 3.5-, 2-, and 1.6-fold decreased in PaCa sera, respectively, whereas haptoglobin and apolipoprotein E were found to be 3.8- and 1.6-fold elevated in PaCa sera. With the exception of haptoglobin the ELISA results of the identified proteins confirmed the 2D-DIGE image analysis characteristics. Integration of the identified serum proteins as AIP markers may have considerable potential to provide additional information for the diagnosis of AIP to choose the appropriate treatment.     

Differential Denaturation of Serum Proteome Reveals a Significant Amount of Hidden Information in Complex Mixtures of Proteins

Recently developed proteomic technologies allow to profile thousands of proteins within a high-throughput approach towards biomarker discovery, although results are not as satisfactory as expected. In the present study we demonstrate that serum proteome denaturation is a key underestimated feature; in fact, a new differential denaturation protocol better discriminates serum proteins according to their electrophoretic mobility as compared to single-denaturation protocols. Sixty nine different denaturation treatments were tested and the 3 most discriminating ones were selected (TRIDENT analysis) and applied to human sera, showing a significant improvement of serum protein discrimination as confirmed by MALDI-TOF/MS and LC-MS/MS identification, depending on the type of denaturation applied. Thereafter sera from mice and patients carrying cutaneous melanoma were analyzed through TRIDENT. Nine and 8 protein bands were found differentially expressed in mice and human melanoma sera, compared to healthy controls (p<0.05); three of them were found, for the first time, significantly modulated: α2macroglobulin (down-regulated in melanoma, p<0.001), Apolipoprotein-E and Apolipoprotein-A1 (both up-regulated in melanoma, p<0.04), both in mice and humans. The modulation was confirmed by immunological methods. Other less abundant proteins (e.g. gelsolin) were found significantly modulated (p<0.05).Conclusions: i) serum proteome contains a large amount of information, still neglected, related to proteins folding; ii) a careful serum denaturation may significantly improve analytical procedures involving complex protein mixtures; iii) serum differential denaturation protocol highlights interesting proteomic differences between cancer and healthy sera.