Oscillations in K(ATP) conductance drive slow calcium oscillations in pancreatic ß-cells.

ATP-sensitive K+ (K(ATP)) channels were first reported in the ß-cells of pancreatic islets in 1984, and it was soon established that they are the primary means by which the blood glucose level is transduced to cellular electrical activity and consequently insulin secretion. However, the role that the K(ATP) channels play in driving the bursting electrical activity of islet ß-cells, which drives pulsatile insulin secretion, remains unclear. One difficulty is that bursting is abolished when several different ion channel types are blocked pharmacologically or genetically, making it challenging to distinguish causation from correlation. Here, we demonstrate a means for determining whether activity-dependent oscillations in K(ATP) conductance play the primary role in driving electrical bursting in ß-cells. We use mathematical models to predict that if K(ATP) is the driver, then contrary to intuition, the mean, peak, and nadir levels of ATP/ADP should be invariant to changes in glucose within the concentration range that supports bursting. We test this in islets using Perceval-HR to image oscillations in ATP/ADP. We find that mean, peak, and nadir levels are indeed approximately invariant, supporting the hypothesis that oscillations in K(ATP) conductance are the main drivers of the slow bursting oscillations typically seen at stimulatory glucose levels in mouse islets. In conclusion, we provide, for the first time to our knowledge, causal evidence for the role of K(ATP) channels not only as the primary target for glucose regulation but also for their role in driving bursting electrical activity and pulsatile insulin secretion.

Chop/Ddit3 depletion in ß cells alleviates ER stress and corrects hepatic steatosis in mice.

Type 2 diabetes (T2D) is a metabolic disorder characterized by hyperglycemia, hyperinsulinemia, and insulin resistance (IR). During the early phase of T2D, insulin synthesis and secretion by pancreatic ß cells is enhanced, which can lead to proinsulin misfolding that aggravates endoplasmic reticulum (ER) protein homeostasis in ß cells. Moreover, increased circulating insulin may contribute to fatty liver disease. Medical interventions aimed at alleviating ER stress in ß cells while maintaining optimal insulin secretion are therefore an attractive therapeutic strategy for T2D. Previously, we demonstrated that germline Chop gene deletion preserved ß cells in high-fat diet (HFD)-fed mice and in leptin receptor-deficient db/db mice. In the current study, we further investigated whether targeting Chop/Ddit3 specifically in murine ß cells conferred therapeutic benefits. First, we showed that Chop deletion in ß cells alleviated ß cell ER stress and delayed glucose-stimulated insulin secretion (GSIS) in HFD-fed mice. Second, ß cell-specific Chop deletion prevented liver steatosis and hepatomegaly in aged HFD-fed mice without affecting basal glucose homeostasis. Third, we provide mechanistic evidence that Chop depletion reduces ER Ca2+ buffering capacity and modulates glucose-induced islet Ca2+ oscillations, leading to transcriptional changes of ER chaperone profile ("ER remodeling"). Last, we demonstrated that a GLP1-conjugated Chop antisense oligonucleotide strategy recapitulated the reduction in liver triglycerides and pancreatic insulin content. In summary, our results demonstrate that Chop depletion in ß cells provides a therapeutic strategy to alleviate dysregulated insulin secretion and consequent fatty liver disease in T2D.

Differentiation of human umbilical cord blood-derived mononuclear cells to endocrine pancreatic lineage.

Generation of insulin-producing cells remains a major limitation for cellular replacement therapy in treatment of diabetes. To understand the potential of human umbilical cord blood (hUCB)-derived mononuclear cells (MNCs) in cell replacement therapy for diabetes, we studied MNCs isolated from 270 human umbilical cord blood samples. We characterized these by immunostaining and real-time PCR and studied their ability to differentiate into insulin-producing cells. We observe that freshly isolated MNCs as well as mesenchymal-like cells grown out by in vitro culture of isolated MNCs express key pancreatic transcription factors: pdx1, ngn3, isl1, brn4 and pax6. However, after 32-fold expansion, MNCs show decreased abundance of pdx1 and ngn3, indicating that islet/pancreatic progenitors detected in freshly isolated MNCs die or are diluted out during in vitro expansion. We therefore transplanted freshly isolated MNCs in NOD/SCID (immuno-incompetent) or FVB/NJ (immuno-competent) mice to check their ability to differentiate into insulin-producing cells. We observe that after 9 weeks of transplantation, approximately 25% grafts exhibit human insulin-producing (16% immunopositive) cells. The number and abundance of pro-insulin transcript-containing cells increased when the animals underwent partial pancreatectomy, 15 days after transplantation. Our results indicate that such hUCB-derived MNC population contains a subset of "pancreas-committed" cells that have the potential to differentiate into insulin-producing cells in vivo. Further studies in understanding the differentiation potential of this subset of pancreas-committed hUCB-derived MNCs will provide us with an autologous source of "lineage-committed" progenitors for cell replacement therapy in diabetes.

Mitophagy protects ß cells from inflammatory damage in diabetes.

Inflammatory damage contributes to ß cell failure in type 1 and 2 diabetes (T1D and T2D, respectively). Mitochondria are damaged by inflammatory signaling in ß cells, resulting in impaired bioenergetics and initiation of proapoptotic machinery. Hence, the identification of protective responses to inflammation could lead to new therapeutic targets. Here, we report that mitophagy serves as a protective response to inflammatory stress in both human and rodent ß cells. Utilizing in vivo mitophagy reporters, we observed that diabetogenic proinflammatory cytokines induced mitophagy in response to nitrosative/oxidative mitochondrial damage. Mitophagy-deficient ß cells were sensitized to inflammatory stress, leading to the accumulation of fragmented dysfunctional mitochondria, increased ß cell death, and hyperglycemia. Overexpression of CLEC16A, a T1D gene and mitophagy regulator whose expression in islets is protective against T1D, ameliorated cytokine-induced human ß cell apoptosis. Thus, mitophagy promotes ß cell survival and prevents diabetes by countering inflammatory injury. Targeting this pathway has the potential to prevent ß cell failure in diabetes and may be beneficial in other inflammatory conditions.

IAPP toxicity activates HIF1α/PFKFB3 signaling delaying ß-cell loss at the expense of ß-cell function.

The islet in type 2 diabetes (T2D) is characterized by amyloid deposits derived from islet amyloid polypeptide (IAPP), a protein co-expressed with insulin by ß-cells. In common with amyloidogenic proteins implicated in neurodegeneration, human IAPP (hIAPP) forms membrane permeant toxic oligomers implicated in misfolded protein stress. Here, we establish that hIAPP misfolded protein stress activates HIF1α/PFKFB3 signaling, this increases glycolysis disengaged from oxidative phosphorylation with mitochondrial fragmentation and perinuclear clustering, considered a protective posture against increased cytosolic Ca2+ characteristic of toxic oligomer stress. In contrast to tissues with the capacity to regenerate, ß-cells in adult humans are minimally replicative, and therefore fail to execute the second pro-regenerative phase of the HIF1α/PFKFB3 injury pathway. Instead, ß-cells in T2D remain trapped in the pro-survival first phase of the HIF1α injury repair response with metabolism and the mitochondrial network adapted to slow the rate of cell attrition at the expense of ß-cell function.

Publisher Correction: IAPP toxicity activates HIF1α/PFKFB3 signaling delaying ß-cell loss at the expense of ß-cell function.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

MicroRNA profiling of developing and regenerating pancreas reveal post-transcriptional regulation of neurogenin3.

The mammalian pancreas is known to show a remarkable degree of regenerative ability. Several studies until now have demonstrated that the mammalian pancreas can regenerate in normal as well as diabetic conditions. These studies illustrate that pancreatic transcription factors that are seen to be expressed in a temporal fashion during development are re-expressed during regeneration. The only known exception to this is Neurogenin3 (NGN3). Though NGN3 protein, which marks all the pro-endocrine cells during development, is not seen during mouse pancreas regeneration, functional neo-islets are generated by 4 weeks after 70% pancreatectomy. We observed that pancreatic transcription factors upstream of ngn3 showed similar gene expression patterns during development and regeneration. However, gene transcripts of transcription factors immediately downstream of ngn3 (neuroD and nkx2.2) did not show such similarities in expression. Since NGN3 protein was not detected at any time point during regeneration, we reasoned that post-transcriptional silencing of ngn3 by microRNAs may be a possible mechanism. We carried out microRNA analysis of 283 known and validated mouse microRNAs during different stages of pancreatic development and regeneration and identified that 4 microRNAs; miR-15a, miR-15b, miR-16 and miR-195, which can potentially bind to ngn3 transcript, are expressed at least 200-fold higher in the regenerating mouse pancreas as compared to embryonic day (e) 10.5 or e 16.5 developing mouse pancreas. Inhibition of these miRNAs in regenerating pancreatic cells using anti-sense miRNA-specific inhibitors, induces expression of NGN3 and its downstream players: neuroD and nkx2.2. Similarly, overexpression of miRNAs targeting ngn3 during pancreas development shows reduction in the number of hormone-producing cells. It appears that during pancreatic regeneration in mice, increased expression of these microRNAs allows endocrine regeneration via an alternate pathway that does not involve NGN3 protein. Our studies on microRNA profiling of developing and regenerating pancreas provide us with better understanding of mechanisms that regulate post-natal islet neogenesis.

New pancreas from old: microregulators of pancreas regeneration.

MicroRNAs (miRNAs) are 18-22 nucleotide RNA molecules that mediate post-transcriptional gene silencing, primarily by binding to the 3' untranslated region of their target mRNA. Several studies have demonstrated the role of miRNAs in mouse pancreas development (miR-124a, miR-503, miR-541, miR-214) as well as in insulin secretion (miR-375, miR-9). Pancreatic transcription factors that are temporally expressed during early pancreas development are re-expressed during pancreas regeneration following pancreatectomy in mice. The only exception to this is Neurogenin3 (NGN3). Here, we discuss recent evidence for miRNA-mediated silencing of ngn3, which inhibits endocrine cell development via the classical 'stem cell pathway' during mouse pancreatic regeneration, thereby favoring beta-cell regeneration.

Asna1/TRC40 Controls ß-Cell Function and Endoplasmic Reticulum Homeostasis by Ensuring Retrograde Transport.

Type 2 diabetes (T2D) is characterized by insulin resistance and ß-cell failure. Insulin resistance per se, however, does not provoke overt diabetes as long as compensatory ß-cell function is maintained. The increased demand for insulin stresses the ß-cell endoplasmic reticulum (ER) and secretory pathway, and ER stress is associated with ß-cell failure in T2D. The tail recognition complex (TRC) pathway, including Asna1/TRC40, is implicated in the maintenance of endomembrane trafficking and ER homeostasis. To gain insight into the role of Asna1/TRC40 in maintaining endomembrane homeostasis and ß-cell function, we inactivated Asna1 in ß-cells of mice. We show that Asna1(ß-/-) mice develop hypoinsulinemia, impaired insulin secretion, and glucose intolerance that rapidly progresses to overt diabetes. Loss of Asna1 function leads to perturbed plasma membrane-to-trans Golgi network and Golgi-to-ER retrograde transport as well as to ER stress in ß-cells. Of note, pharmacological inhibition of retrograde transport in isolated islets and insulinoma cells mimicked the phenotype of Asna1(ß-/-) ß-cells and resulted in reduced insulin content and ER stress. These data support a model where Asna1 ensures retrograde transport and, hence, ER and insulin homeostasis in ß-cells.