A human antibody against pathologic IAPP aggregates protects beta cells in type 2 diabetes models

In patients with type 2 diabetes, pancreatic beta cells progressively degenerate and gradually lose their ability to produce insulin and regulate blood glucose. Beta cell dysfunction and loss is associated with an accumulation of aggregated forms of islet amyloid polypeptide (IAPP) consisting of soluble prefibrillar IAPP oligomers as well as insoluble IAPP fibrils in pancreatic islets. Here, we describe a human monoclonal antibody selectively targeting IAPP oligomers and neutralizing IAPP aggregate toxicity by preventing membrane disruption and apoptosis in vitro. Antibody treatment in male rats and mice transgenic for human IAPP, and human islet-engrafted mouse models of type 2 diabetes triggers clearance of IAPP oligomers resulting in beta cell protection and improved glucose control. These results provide new evidence for the pathological role of IAPP oligomers and suggest that antibody-mediated removal of IAPP oligomers could be a pharmaceutical strategy to support beta cell function in type 2 diabetes

Retrospective evaluation of whole exome and genome mutation calls in 746 cancer samples

Funder: NCI U24CA211006Abstract: The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) curated consensus somatic mutation calls using whole exome sequencing (WES) and whole genome sequencing (WGS), respectively. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2,658 cancers across 38 tumour types, we compare WES and WGS side-by-side from 746 TCGA samples, finding that ~80% of mutations overlap in covered exonic regions. We estimate that low variant allele fraction (VAF < 15%) and clonal heterogeneity contribute up to 68% of private WGS mutations and 71% of private WES mutations. We observe that ~30% of private WGS mutations trace to mutations identified by a single variant caller in WES consensus efforts. WGS captures both ~50% more variation in exonic regions and un-observed mutations in loci with variable GC-content. Together, our analysis highlights technological divergences between two reproducible somatic variant detection efforts

T cells accumulate in non-diabetic islets during ageing

Background: The resident immune population of pancreatic islets has roles in islet development, beta cell physiology, and the pathology of diabetes. These roles have largely been attributed to islet macrophages, comprising 90% of islet immune cells (in the absence of islet autoimmunity), and, in the case of type 1 diabetes, to infiltrating autoreactive T cells. In adipose, tissue-resident and recruited T and B cells have been implicated in the development of insulin resistance during diet-induced obesity and ageing, but whether this is paralleled in the pancreatic islets is not known. Here, we investigated the non-macrophage component of resident islet immune cells in islets isolated from C57BL/6 J male mice during ageing (3 to 24 months of age) and following similar weight gain achieved by 12 weeks of 60% high fat diet. Immune cells were also examined by flow cytometry in cadaveric non-diabetic human islets. Results: Immune cells comprised 2.7 ± 1.3% of total islet cells in non-diabetic mouse islets, and 2.3 ± 1.7% of total islet cells in non-diabetic human islets. In 3-month old mice on standard diet, B and T cells each comprised approximately 2–4% of the total islet immune cell compartment, and approximately 0.1% of total islet cells. A similar amount of T cells were present in non-diabetic human islets. The majority of islet T cells expressed the αβ T cell receptor, and were comprised of CD8-positive, CD4-positive, and regulatory T cells, with a minor population of γδ T cells. Interestingly, the number of islet T cells increased linearly (R² = 0.9902) with age from 0.10 ± 0.05% (3 months) to 0.38 ± 0.11% (24 months) of islet cells. This increase was uncoupled from body weight, and was not phenocopied by a degree similar weight gain induced by high fat diet in mice. Conclusions: This study reveals that T cells are a part of the normal islet immune population in mouse and human islets, and accumulate in islets during ageing in a body weight-independent manner. Though comprising only a small subset of the immune cells within islets, islet T cells may play a role in the physiology of islet ageing.Medicine, Faculty ofOther UBCNon UBCSurgery, Department ofReviewedFacult

Impaired Ca(2+) signaling in β-cells lacking leptin receptors by Cre-loxP recombination.

Obesity is a major risk factor for diabetes and is typically associated with hyperleptinemia and a state of leptin resistance. The impact of chronically elevated leptin levels on the function of insulin-secreting β-cells has not been elucidated. We previously generated mice lacking leptin signaling in β-cells by using the Cre-loxP strategy and showed that these animals develop increased body weight and adiposity, hyperinsulinemia, impaired glucose-stimulated insulin secretion and insulin resistance. Here, we performed several in vitro studies and observed that β-cells lacking leptin signaling in this model are capable of properly metabolizing glucose, but show impaired intracellular Ca(2+) oscillations and lack of synchrony within the islets in response to glucose, display reduced response to tolbutamide and exhibit morphological abnormalities including increased autophagy. Defects in intracellular Ca(2+) signaling were observed even in neonatal islets, ruling out the possible contribution of obesity to the β-cell irregularities observed in adults. In parallel, we also detected a disrupted intracellular Ca(2+) pattern in response to glucose and tolbutamide in control islets from adult transgenic mice expressing Cre recombinase under the rat insulin promoter, despite these animals being glucose tolerant and secreting normal levels of insulin in response to glucose. This unexpected observation impeded us from discerning the consequences of impaired leptin signaling as opposed to long-term Cre expression in the function of insulin-secreting cells. These findings highlight the need to generate improved Cre-driver mouse models or new tools to induce Cre recombination in β-cells

Decreased amplitude in intracellular Ca²⁺ responses to tolbutamide in <i>Lepr^flox/flox RIP-Cre</i> adult islets.

<p>A: Representative recordings from a control <i>Lepr^flox/flox</i> islet (solid line) and a <i>Lepr^flox/flox RIP-Cre</i> islet (dotted line) in response to tolbutamide. B: Graph plotting ΔFmax-Fmin of each peak in response to tolbutamide in the population of islets that showed two peaks. Data are expressed as mean ± SEM. Statistical analysis was performed using Student t test, *** p<0.0001. Responses are representative of 22 islets from 4 mice per group.</p

<i>Lepr^flox/flox RIP-Cre</i> pancreatic β-cells display impaired intracellular Ca²⁺ oscillations in response to glucose.

<p>A: [Ca²⁺]_i recordings of a <i>Lepr^flox/flox</i> islet (left panel) and <i>Lepr^flox/flox RIP-Cre</i> islet (right panel) in response to increasing glucose (G) concentrations and potassium chloride (KCl) from adult mice. B and C: Representative [Ca²⁺]_i recordings showing three different regions per islet of a <i>Lepr^flox/flox</i> islet (left panel) and a <i>Lepr^flox/flox RIP-Cre</i> islet (right panel) from adult (B) and neonatal (C) mice. Graphs are representative of 17–20 islets from 3 neonatal mice per group, and 37–38 islets from 3–4 adult mice per group.</p

Transmission electron microscopy reveals autophagy within <i>Lepr^flox/flox RIP-Cre</i> β-cells.

<p>Pancreas sections from <i>Lepr^flox/flox</i> (A) and <i>Lepr^flox/flox RIP-Cre</i> (B and D) mice were analyzed by transmission electron microscopy (magnification 9300X and 11000X) and quantified (C). Multigranular bodies were numerous in <i>Lepr^flox/flox RIP-Cre</i> β-cells compared to <i>Lepr^flox/flox</i> β-cells (white squares). Events of macroautophagy (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071075#pone-0071075-g004" target="_blank">Figure 4D</a>, upper inset) and microautophagy (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071075#pone-0071075-g004" target="_blank">Figure 4D</a>, bottom inset) were captured in <i>Lepr^flox/flox RIP-Cre</i> β-cells. Scale bar = 2 µm (A and B) and 0.5 µm (D). Micrographs are representative of 3 pancreata analyzed per group. Data are expressed as mean ± SEM. Statistical analysis was performed using Student t test, ** p<0.01.</p

<i>Lepr^+/+ RIP-Cre</i> mice present impaired β-cell Ca²⁺ signaling in response to glucose and tolbutamide.

<p>A: Representative [Ca²⁺]_i recordings of a <i>Lepr^+/+</i> islet (left panel) and <i>Lepr^+/+ RIP-Cre</i> islet (right panel) in response to increasing glucose (G) concentrations and potassium chloride (KCl) from 5–6 week old mice. B: Representative [Ca²⁺]_i recordings of a <i>Lepr^+/+</i> islet (solid line) and <i>Lepr^+/+ RIP-Cre</i> islet (dotted line) in response to tolbutamide. C: Graph plotting the AUC of the Ca²⁺ transients during the stimulation with tolbutamide. Data are expressed as mean ± SEM. Statistical analysis was performed using Student t test. *** p<0.0001. Graphs are representative of 18–20 islets from 3 mice per group (in response to glucose) and 16–27 islets from 2–3 mice per group (in response to tolbutamide).</p