Dario F. De Jesus

SerpinB1 Promotes Pancreatic β Cell Proliferation

Although compensatory islet hyperplasia in response to insulin resistance is a recognized feature in diabetes, the factor(s) that promote β cell proliferation have been elusive. We previously reported that the liver is a source for such factors in the liver insulin receptor knockout (LIRKO) mouse, an insulin resistance model that manifests islet hyperplasia. Using proteomics we show that serpinB1, a protease inhibitor, which is abundant in the hepatocyte secretome and sera derived from LIRKO mice, is the liver-derived secretory protein that regulates β cell proliferation in humans, mice, and zebrafish. Small-molecule compounds, that partially mimic serpinB1 effects of inhibiting elastase activity, enhanced proliferation of β cells, and mice lacking serpinB1 exhibit attenuated β cell compensation in response to insulin resistance. Finally, SerpinB1 treatment of islets modulated proteins in growth/survival pathways. Together, these data implicate serpinB1 as an endogenous protein that can potentially be harnessed to enhance functional β cell mass in patients with diabetes.

Soluble factors secreted by T-cells promote β-cell proliferation

Type 1 diabetes is characterized by infiltration of pancreatic islets with immune cells, leading to insulin deficiency. Although infiltrating immune cells are traditionally considered to negatively impact β-cells by promoting their death, their contribution to proliferation is not fully understood. Here we report that islets exhibiting insulitis also manifested proliferation of β-cells that positively correlated with the extent of lymphocyte infiltration. Adoptive transfer of diabetogenic CD4+ and CD8+ T cells, but not B cells, selectively promoted β-cell proliferation in vivo independent from the effects of blood glucose or circulating insulin or by modulating apoptosis. Complementary to our in vivo approach, coculture of diabetogenic CD4+ and CD8+ T cells with NOD.RAG1−/− islets in an in vitro transwell system led to a dose-dependent secretion of candidate cytokines/chemokines (interleukin-2 [IL-2], IL-6, IL-10, MIP-1α, and RANTES) that together enhanced β-cell proliferation. These data suggest that soluble factors secreted from T cells are potential therapeutic candidates to enhance β-cell proliferation in efforts to prevent and/or delay the onset of type 1 diabetes.

Maternal insulin resistance and transient hyperglycemia impact the metabolic and endocrine phenotypes of offspring

Studies in both humans and rodents suggest that maternal diabetes leads to a higher risk of the fetus developing impaired glucose tolerance and obesity during adulthood. However, the impact of hyperinsulinemia in the mother on glucose homeostasis in the offspring has not been fully explored. We aimed to determine the consequences of maternal insulin resistance on offspring metabolism and endocrine pancreas development using the LIRKO mouse model, which exhibits sustained hyperinsulinemia and transient increase in blood glucose concentrations during pregnancy. We examined control offspring born to either LIRKO or control mothers on embryonic days 13.5, 15.5, and 17.5 and postpartum days 0, 4, and 10. Control offspring born to LIRKO mothers displayed low birth weights and subsequently rapidly gained weight, and their blood glucose and plasma insulin concentrations were higher than offspring born to control mothers in early postnatal life. In addition, concentrations of plasma leptin, glucagon, and active GLP-1 were higher in control pups from LIRKO mothers. Analyses of the endocrine pancreas revealed significantly reduced β-cell area in control offspring of LIRKO mothers shortly after birth. β-Cell proliferation and total islet number were also lower in control offspring of LIRKO mothers during early postnatal days. Together, these data indicate that maternal hyperinsulinemia and the transient hyperglycemia impair endocrine pancreas development in the control offspring and induce multiple metabolic alterations in early postnatal life. The relatively smaller β-cell mass/area and β-cell proliferation in these control offspring suggest cell-autonomous epigenetic mechanisms in the regulation of islet growth and development.

Epigenetic modifiers of islet function and mass.

Type 2 diabetes (T2D) is associated with insulin resistance in target tissues including the β-cell, leading to significant β-cell loss and secretory dysfunction. T2D is also associated with aging, and the underlying mechanisms that increase susceptibility of an individual to develop the disease implicate epigenetics: interactions between susceptible loci and the environment. In this review, we discuss the effects of aging on β-cell function and adaptation, besides the significance of mitochondria in islet bioenergetics and epigenome. We highlight three important modulators of the islet epigenome, namely: metabolites, hormones, and the nutritional state. Unraveling the signaling pathways that regulate the islet epigenome during aging will help to better understand the development of disease progression and to design novel therapies for diabetes prevention.

Compensatory islet response to insulin resistance revealed by quantitative proteomics

Compensatory islet response is a distinct feature of the prediabetic insulin-resistant state in humans and rodents. To identify alterations in the islet proteome that characterize the adaptive response, we analyzed islets from 5 month old male control, high-fat diet fed (HFD), or obese ob/ob mice by LC-MS/MS and quantified ~1100 islet proteins (at least two peptides) with a false discovery rate < 1%. Significant alterations in abundance were observed for ~350 proteins among groups. The majority of alterations were common to both models, and the changes of a subset of ~40 proteins and 12 proteins were verified by targeted quantification using selected reaction monitoring and western blots, respectively. The insulin-resistant islets in both groups exhibited reduced expression of proteins controlling energy metabolism, oxidative phosphorylation, hormone processing, and secretory pathways. Conversely, an increased expression of molecules involved in protein synthesis and folding suggested effects in endoplasmic reticulum stress response, cell survival, and proliferation in both insulin-resistant models. In summary, we report a unique comparison of the islet proteome that is focused on the compensatory response in two insulin-resistant rodent models that are not overtly diabetic. These data provide a valuable resource of candidate proteins to the scientific community to undertake further studies aimed at enhancing β-cell mass in patients with diabetes. The data are available via the MassIVE repository, under accession no. MSV000079093.

Excessive Cellular Proliferation Negatively Impacts Reprogramming Efficiency of Human Fibroblasts

The impact of somatic cell proliferation rate on induction of pluripotent stem cells remains controversial. Herein, we report that rapid proliferation of human somatic fibroblasts is detrimental to reprogramming efficiency when reprogrammed using a lentiviral vector expressing OCT4, SOX2, KLF4, and cMYC in insulin‐rich defined medium. Human fibroblasts grown in this medium showed higher proliferation, enhanced expression of insulin signaling and cell cycle genes, and a switch from glycolytic to oxidative phosphorylation metabolism, but they displayed poor reprogramming efficiency compared with cells grown in normal medium. Thus, in contrast to previous studies, our work reveals an inverse correlation between the proliferation rate of somatic cells and reprogramming efficiency, and also suggests that upregulation of proteins in the growth factor signaling pathway limits the ability to induce pluripotency in human somatic fibroblasts.

Insulin receptor-mediated signaling regulates pluripotency markers and lineage differentiation

Objectives Insulin receptor (IR)-mediated signaling is involved in the regulation of pluripotent stem cells; however, its direct effects on regulating the maintenance of pluripotency and lineage development are not fully understood. The main objective of this study is to understand the role of IR signaling in pluripotency and lineage development. Methods To explore the role of IR signaling, we generated IR knock-out (IRKO) mouse induced pluripotent stem cells (miPSCs) from E14.5 mouse embryonic fibroblasts (MEFs) of global IRKO mice using a cocktail of four reprogramming factors: Oct4, Sox2, Klf4, cMyc. We performed pluripotency characterization and directed the differentiation of control and IRKO iPSCs into neural progenitors (ectoderm), adipocyte progenitors (mesoderm), and pancreatic beta-like cells (endoderm). We mechanistically confirmed these findings via phosphoproteomics analyses of control and IRKO iPSCs. Results Interestingly, expression of pluripotency markers including Klf4, Lin28a, Tbx3, and cMyc were upregulated, while abundance of Oct4 and Nanog were enhanced by 4-fold and 3-fold, respectively, in IRKO iPSCs. Analyses of signaling pathways demonstrated downregulation of phospho-STAT3, p-mTor and p-Erk and an increase in the total mTor and Erk proteins in IRKO iPSCs in the basal unstimulated state. Stimulation with leukemia inhibitory factor (LIF) showed a ∼33% decrease of phospho-ERK in IRKO iPSCs. On the contrary, Erk phosphorylation was increased during in vitro spontaneous differentiation of iPSCs lacking IRs. Lineage-specific directed differentiation of the iPSCs revealed that cells lacking IR showed enhanced expression of neuronal lineage markers (Pax6, Tubb3, Ascl1 and Oligo2) while exhibiting a decrease in adipocyte (Fas, Acc, Pparγ, Fabp4, C/ebpα, and Fsp27) and pancreatic beta cell markers (Ngn3, Isl1, and Sox9). Further molecular characterization by phosphoproteomics confirmed the novel IR-mediated regulation of the global pluripotency network including several key proteins involved in diverse aspects of growth and embryonic development. Conclusion We report, for the first time to our knowledge, the phosphoproteome of insulin, IGF1, and LIF stimulation in mouse iPSCs to reveal the importance of insulin receptor signaling for the maintenance of pluripotency and lineage determination.

A Novel Role for SerpinB1, a Protease Inhibitor, in Hepatic Biology

SerpinB1, a protease inhibitor that is secreted from the liver in response to insulin resistance, was recently reported to promote pancreatic β cell proliferation. We focused on investigating the significance of SerpinB1 in the liver to gain insights into its role in regulating whole body metabolism. We determined SerpinB1 gene expression in multiple metabolic tissues harvested under fasting or fed states from ∼34-week-old male Liver-specific insulin receptor knockout (LIRKO) mice and littermate controls. While in controls SerpinB1 expression was reduced by ∼50% after feeding when compared to the fasting state, (P=0.02, n=4-5), the LIRKOs, in contrast, exhibited a 2.3-fold higher (P=0.01, n=5-6) expression in the fed vs. fasting states and the higher levels were consistent with earlier observations in the LIRKO livers. We next took advantage of the availability of liver samples from 18 to 24-week-old male SerpinB1 KO mice and littermate controls and subjected them to RNA-sequencing. As expected, SerpinB1 gene expression was absent in the KO samples, and interestingly, was associated with a significant decrease in gene expression related to cell cycle, NF-kappa B signaling, and inflammatory pathways. Independent validation of these data by RT-qPCR confirmed a decrease in cell cycle-related genes such as Ccna2 (-60%, P=0.04), Ccnb2 (-54%, P=0.04), Ccnd1 (-46%, P=0.03), and Ccne1 (-51%, P=0.07), and the cell proliferation marker, Mki67 (-60%, P=0.00) (n=5). Furthermore, knockdown of SerpinB1 in the human hepatocyte cell line, HepG2, exhibited significantly decreased expression of Ccnb1 (-36%, P=0.01), Ccnb2 (-44%, P=0.00), Ccne1 (-21%, P=0.00), MKi67 (-30%, P=0.00), and MCM2 (-29%, P=0.00) genes, and increased expression of P21 (+165%, P=0.00) (n=3 for control vs. knockdown groups). These novel data implicate endogenous SerpinB1 in cell proliferation in hepatocytes and warrant further investigation of its role in hepatocyte function and the overall impact on glucose homeostasis. Disclosure K. Orime: None. D.F. De Jesus: None. R. Kulkarni: None.