Glucagon receptor (GcgR) antagonism decrease α-cell proliferation.

<p>(A) Immunofluorescent staining of GcgR on αTC1 cells. (B) Proliferative response of α cells after 72 hours (left panel) or 24 hours (right panel) of GcgR antagonist II (14 µM) +/− insulin (2 nM) treatment. The cell proliferation was assessed by both ELISA-based relative proliferation assay (left panel) and by counting the cells in S phase (propidium iodide staining for DNA cell cycle analysis) (right panel). (C) db/db mice were treated with GcgR antagonist II for up to 17 days. Blood glucose measurements in non-treated db/db (n = 4, open diamond) and treated db/db (n = 5, closed diamond) mice. (D) Fasting plasma insulin levels of db/db mice measured after 17days of GcgR antagonist treatment. For control db/db mice n = 4, treated db/db mice n = 5, non-diabetic mice n = 7. (E) Fasting plasma glucagon levels of db/db mice measured after 17days of GcgR antagonist II treatment. For control db/db mice n = 4, treated db/db mice n = 5, non-diabetic mice n = 7. (F) α-cell number in islets from db/db mice was counted (control n = 71 islets, treated n = 84 islets). (G) Immunofluorescent staining of glucagon and insulin in frozen sections of pancreata from treated and non-treated db/db mice. Data are expressed as means ±SEM (n = 6); ** p<0.005, *** p<0.0005.</p

IRs signal through IRS2 and AKT and activate mTOR, resulting in α-cell proliferation.

<p>(A) Western blot analysis of insulin receptor signaling molecules in α cells. αTC1 cells were treated with increasing concentration of insulin for 10 minutes and whole cell lysate was used for western blotting. (B) Immunoprecipitation of IRS1 and IRS2 in pancreatic cells. αTC1 cells were treated with increasing concentration of insulin for 10 minutes and whole cell lysates was collected. IRS1 and IRS2 proteins were immunoprecipitated, and immunoblotted with an antibody that recognizes phosphorylated IRS1/2 (Tyr612). Protein levels of IRS1 (black arrows) and IRS2 (red arrows) in αTC1 and MIN6 cells. The densitometry represents relative expression of IRS2 protein levels. (C) αTC1 cells were treated with increasing concentration of insulin for 10 minutes and whole cell lysate was blotted for phosphorylated and total mTOR. (D) α-TC1 cells were treated for 72 hours (left panel) or 24 hours (right panel) with insulin (2 nM) +/− rapamycin (20 mg/ml). The cell proliferation was assessed by both ELISA-based relative proliferation assay (left panel: data are expressed as means ±SEM [n = 6]; ** p<0.005, *** p<0.0005) and by counting the cells in S phase (propidium iodide staining for DNA cell cycle analysis) (right panel).</p

Culturing α cells with MIN6 β cells or insulin increases α-cell proliferation.

<p>(A) αTC1 cells were co-cultured with MIN6 β cells (n = 16), αTC1 (n = 16), CHO-NEO (n = 12) and medium (n = 12) in transwell plates, as shown. Results show relative proliferation rates of α-TC1 cells. (B) and (C), glucagon and insulin levels in medium from αTC1 and MIN6 cells, assayed at the times shown. (D) αTC1 cells were treated with insulin for 5 days at the concentration shown. EC50 = 2 nM, n = 6 separate experiments. All data are expressed as means± SEM; ***p<0.0005.</p

α-cell numbers are increased in db/db and streptozotocin (STZ)-treated mice.

<p>(A) Immunofluorescent staining of insulin and glucagon in mice at ages shown. Glucagon-positive cells are on the rim of islets in non-diabetic mice (arrows). (B) Mean glucagon-positive area in islets over time, in non-diabetic and db/db mice. (C) Random plasma glucagon levels in non-diabetic and db/db mice. (D) Random plasma insulin levels, over time, in db/db and non-diabetic mice. (E) Immunofluorescent staining of insulin and glucagon and quantification of glucagon-positive area in islets of STZ-treated CD1 mice. (F) PCNA-positive nuclei in glucagon-positive cells in islets from four different db/db mice. (G) Immunofluorescent staining of insulin and glucagon in human pancreatic paraffin sections, demonstrating lack of stereotypy in the distribution of α and β cells in human islets.</p

Genome-Wide Significant SNPs from the Sex-Combined Multi-Ethnic Meta-Analysis.

<p>The novel loci identified using Multi-Ethnic Meta-analysis (that were not identified in the European only analysis) are listed in <b>bold</b>.</p>*<p>When possible, plausible biological candidate genes have been listed; otherwise, the closest gene is designated.</p>‡<p>Lead SNP is the SNP with the lowest <i>p</i>-value for each locus.</p>†<p>Positions are relative to Human Genome NCBI Build 36.</p>§<p>log₁₀ Bayes factor (BF) from the MANTRA analysis. A log₁₀ BF of 6 and higher was considered as a conservative threshold for genome-wide significance.</p>††<p>The posterior probability of heterogeneity between studies.</p>¶<p>EA: effect allele, NEA: non-effect allele.</p>¶¶<p>EAF: Frequency of effect allele in CEU, East Asian, and AA, populations respectively.</p

The Association of Lead Genome-Wide Significant SNPs for Adiponectin with mRNA Levels of Their Nearest Gene.

‡<p>Lead SNP is the SNP with the lowest <i>p</i>-value for each gene in gene expression data.</p>‡‡<p>Lead SNP is the SNP with the lowest <i>p</i>-value for each locus in meta-analysis from discovery phase.</p>¶<p>EA: Effect allele.</p>¶¶<p>EAF: Frequency of effect allele.</p>§<p>Betas are estimated expression levels of the genes.</p>*<p>P value for lead SNP is the SNP in gene expression data.</p>**<p>P value for lead SNP in meta-analysis from discovery phase.</p>$<p>r² LD between lead SNP from expression and lead SNP from meta-analysis.</p

Regional plots of eight newly discovered genome-wide significant chromosomal regions associated with adiponectin concentrations in European populations.

<p>A) chromosome 16q23.2, B) chromosome 19 q13.11 C) Chromosome 3p21.1, D) two loci on chromosome 12q24.31, E) chromosome 8q24.13, F) chromosome 6p21.1, and G) chromosome 1q41. In each panel, purple diamonds indicate the top SNPs, which have the strongest evidence of association. Each circle shows a SNP with a color scale relating the r² value for that SNP and the top SNP from HapMap CEU. Blue lines indicate estimated recombination rates from HapMap. The bottom panels illustrate the relative position of genes near each locus. Candidate genes are indicated by red ovals.</p

Flow chart of study design.

<p>Flow chart of study design.</p

The Association of mRNA Levels from Genes in Candidate Loci in Human Adipocytes with Circulating Adiponectin Levels.

§<p>Betas are estimated from log transformed and quantile-quantile normalized values.</p>*<p>These two loci are independent loci.</p

Lead SNP per Locus for Genome-Wide Significant SNPs Arising from the Sex-Combined Meta-Analysis in European Populations.

<p> <i>All SNPs achieving genome-wide significance in the joint analysis phase are marked in italics.</i></p>*<p>Joint analysis indicates results from the meta-analysis of discovery and follow-up <i>in-silico</i> and <i>de-novo</i> phases.</p>**<p>When possible, plausible biological candidate genes have been listed; otherwise, the closest gene is designated.</p>‡<p>Lead SNP is the SNP with the lowest <i>p</i>-value for each locus.</p>§<p>Betas are estimated from models using the natural log transformed adiponectin.</p>¶<p>EA: Effect allele, NEA: Non-effect allele.</p>¶¶<p>EAF: Effect allele frequency.</p