Noncanonical CDK4 signaling rescues diabetes in a mouse model by promoting ß cell differentiation.

Expanding ß cell mass is a critical goal in the fight against diabetes. CDK4, an extensively characterized cell cycle activator, is required to establish and maintain ß cell number. ß cell failure in the IRS2-deletion mouse type 2 diabetes model is, in part, due to loss of CDK4 regulator cyclin D2. We set out to determine whether replacement of endogenous CDK4 with the inhibitor-resistant mutant CDK4-R24C rescued the loss of ß cell mass in IRS2-deficient mice. Surprisingly, not only ß cell mass but also ß cell dedifferentiation was effectively rescued, despite no improvement in whole body insulin sensitivity. Ex vivo studies in primary islet cells revealed a mechanism in which CDK4 intervened downstream in the insulin signaling pathway to prevent FOXO1-mediated transcriptional repression of critical ß cell transcription factor Pdx1. FOXO1 inhibition was not related to E2F1 activity, to FOXO1 phosphorylation, or even to FOXO1 subcellular localization, but rather was related to deacetylation and reduced FOXO1 abundance. Taken together, these results demonstrate a differentiation-promoting activity of the classical cell cycle activator CDK4 and support the concept that ß cell mass can be expanded without compromising function.

CDKN2A/B T2D Genome-Wide Association Study Risk SNPs Impact Locus Gene Expression and Proliferation in Human Islets.

Genome-wide association studies link the CDKN2A/B locus with type 2 diabetes (T2D) risk, but mechanisms increasing risk remain unknown. The CDKN2A/B locus encodes cell cycle inhibitors p14, p15, and p16; MTAP; and ANRIL, a long noncoding RNA. The goal of this study was to determine whether CDKN2A/B T2D risk SNPs impact locus gene expression, insulin secretion, or ß-cell proliferation in human islets. Islets from donors without diabetes (n = 95) were tested for SNP genotype (rs10811661, rs2383208, rs564398, and rs10757283), gene expression (p14, p15, p16, MTAP, ANRIL, PCNA, KI67, and CCND2), insulin secretion (n = 61), and ß-cell proliferation (n = 47). Intriguingly, locus genes were coregulated in islets in two physically overlapping cassettes: p14-p16-ANRIL, which increased with age, and MTAP-p15, which did not. Risk alleles at rs10811661 and rs2383208 were differentially associated with expression of ANRIL, but not p14, p15, p16, or MTAP, in age-dependent fashion, such that younger homozygous risk donors had higher ANRIL expression, equivalent to older donor levels. We identified several risk SNP combinations that may impact locus gene expression, suggesting possible mechanisms by which SNPs impact locus biology. Risk allele carriers at ANRIL coding SNP rs564398 had reduced ß-cell proliferation index. In conclusion, CDKN2A/B locus SNPs may impact T2D risk by modulating islet gene expression and ß-cell proliferation.

PKCζ Is Essential for Pancreatic ß-Cell Replication During Insulin Resistance by Regulating mTOR and Cyclin-D2.

Adaptive ß-cell replication occurs in response to increased metabolic demand during insulin resistance. The intracellular mediators of this compensatory response are poorly defined and their identification could provide significant targets for ß-cell regeneration therapies. Here we show that glucose and insulin in vitro and insulin resistance in vivo activate protein kinase C ζ (PKCζ) in pancreatic islets and ß-cells. PKCζ is required for glucose- and glucokinase activator-induced proliferation of rodent and human ß-cells in vitro. Furthermore, either kinase-dead PKCζ expression (KD-PKCζ) or disruption of PKCζ in mouse ß-cells blocks compensatory ß-cell replication when acute hyperglycemia/hyperinsulinemia is induced. Importantly, KD-PKCζ inhibits insulin resistance-mediated mammalian target of rapamycin (mTOR) activation and cyclin-D2 upregulation independent of Akt activation. In summary, PKCζ activation is key for early compensatory ß-cell replication in insulin resistance by regulating the downstream signals mTOR and cyclin-D2. This suggests that alterations in PKCζ expression or activity might contribute to inadequate ß-cell mass expansion and ß-cell failure leading to type 2 diabetes.

Glucose Induces Mouse ß-Cell Proliferation via IRS2, MTOR, and Cyclin D2 but Not the Insulin Receptor.

An important goal in diabetes research is to understand the processes that trigger endogenous ß-cell proliferation. Hyperglycemia induces ß-cell replication, but the mechanism remains debated. A prime candidate is insulin, which acts locally through the insulin receptor. Having previously developed an in vivo mouse hyperglycemia model, we tested whether glucose induces ß-cell proliferation through insulin signaling. By using mice lacking insulin signaling intermediate insulin receptor substrate 2 (IRS2), we confirmed that hyperglycemia-induced ß-cell proliferation requires IRS2 both in vivo and ex vivo. Of note, insulin receptor activation was not required for glucose-induced proliferation, and insulin itself was not sufficient to drive replication. Glucose and insulin caused similar acute signaling in mouse islets, but chronic signaling differed markedly, with mammalian target of rapamycin (MTOR) and extracellular signal-related kinase (ERK) activation by glucose and AKT activation by insulin. MTOR but not ERK activation was required for glucose-induced proliferation. Cyclin D2 was necessary for glucose-induced ß-cell proliferation. Cyclin D2 expression was reduced when either IRS2 or MTOR signaling was lost, and restoring cyclin D2 expression rescued the proliferation defect. Human islets shared many of these regulatory pathways. Taken together, these results support a model in which IRS2, MTOR, and cyclin D2, but not the insulin receptor, mediate glucose-induced proliferation.

Insulin demand regulates ß cell number via the unfolded protein response.

Although stem cell populations mediate regeneration of rapid turnover tissues, such as skin, blood, and gut, a stem cell reservoir has not been identified for some slower turnover tissues, such as the pancreatic islet. Despite lacking identifiable stem cells, murine pancreatic ß cell number expands in response to an increase in insulin demand. Lineage tracing shows that new ß cells are generated from proliferation of mature, differentiated ß cells; however, the mechanism by which these mature cells sense systemic insulin demand and initiate a proliferative response remains unknown. Here, we identified the ß cell unfolded protein response (UPR), which senses insulin production, as a regulator of ß cell proliferation. Using genetic and physiologic models, we determined that among the population of ß cells, those with an active UPR are more likely to proliferate. Moreover, subthreshold endoplasmic reticulum stress (ER stress) drove insulin demand-induced ß cell proliferation, through activation of ATF6. We also confirmed that the UPR regulates proliferation of human ß cells, suggesting that therapeutic UPR modulation has potential to expand ß cell mass in people at risk for diabetes. Together, this work defines a stem cell-independent model of tissue homeostasis, in which differentiated secretory cells use the UPR sensor to adapt organ size to meet demand.

Adaptive ß-cell proliferation increases early in high-fat feeding in mice, concurrent with metabolic changes, with induction of islet cyclin D2 expression.

Type 2 diabetes (T2D) is caused by relative insulin deficiency, due in part to reduced ß-cell mass (11, 62). Therapies aimed at expanding ß-cell mass may be useful to treat T2D (14). Although feeding rodents a high-fat diet (HFD) for an extended period (3-6 mo) increases ß-cell mass by inducing ß-cell proliferation (16, 20, 53, 54), evidence suggests that adult human ß-cells may not meaningfully proliferate in response to obesity. The timing and identity of the earliest initiators of the rodent compensatory growth response, possible therapeutic targets to drive proliferation in refractory human ß-cells, are not known. To develop a model to identify early drivers of ß-cell proliferation, we studied mice during the first week of HFD exposure, determining the onset of proliferation in the context of diet-related physiological changes. Within the first week of HFD, mice consumed more kilocalories, gained weight and fat mass, and developed hyperglycemia, hyperinsulinemia, and glucose intolerance due to impaired insulin secretion. The ß-cell proliferative response also began within the first week of HFD feeding. Intriguingly, ß-cell proliferation increased before insulin resistance was detected. Cyclin D2 protein expression was increased in islets by day 7, suggesting it may be an early effector driving compensatory ß-cell proliferation in mice. This study defines the time frame and physiology to identify novel upstream regulatory signals driving mouse ß-cell mass expansion, in order to explore their efficacy, or reasons for inefficacy, in initiating human ß-cell proliferation.

ChREBP mediates glucose-stimulated pancreatic ß-cell proliferation.

Glucose stimulates rodent and human ß-cell replication, but the intracellular signaling mechanisms are poorly understood. Carbohydrate response element-binding protein (ChREBP) is a lipogenic glucose-sensing transcription factor with unknown functions in pancreatic ß-cells. We tested the hypothesis that ChREBP is required for glucose-stimulated ß-cell proliferation. The relative expression of ChREBP was determined in liver and ß-cells using quantitative RT-PCR (qRT-PCR), immunoblotting, and immunohistochemistry. Loss- and gain-of-function studies were performed using small interfering RNA and genetic deletion of ChREBP and adenoviral overexpression of ChREBP in rodent and human ß-cells. Proliferation was measured by 5-bromo-2'-deoxyuridine incorporation, [(3)H]thymidine incorporation, and fluorescence-activated cell sorter analysis. In addition, the expression of cell cycle regulatory genes was measured by qRT-PCR and immunoblotting. ChREBP expression was comparable with liver in mouse pancreata and in rat and human islets. Depletion of ChREBP decreased glucose-stimulated proliferation in ß-cells isolated from ChREBP(-/-) mice, in INS-1-derived 832/13 cells, and in primary rat and human ß-cells. Furthermore, depletion of ChREBP decreased the glucose-stimulated expression of cell cycle accelerators. Overexpression of ChREBP amplified glucose-stimulated proliferation in rat and human ß-cells, with concomitant increases in cyclin gene expression. In conclusion, ChREBP mediates glucose-stimulated proliferation in pancreatic ß-cells.

The expression and distribution of Wnt and Wnt receptor mRNAs during early sea urchin development.

The protein beta-catenin plays a critically important role in establishing axial polarity during early animal development. In many organisms, beta-catenin is degraded preferentially on one side of the cleavage stage embryo. On the opposite side of the embryo, beta-catenin is stabilized and accumulates in the nucleus, where it functions in concert with members of the LEF/TCF family to activate the transcription of diverse target genes. Genes that are activated by beta-catenin play an essential role in the specification of endomesoderm and in the establishment of key signaling centers in the early embryo. In several organisms, the asymmetric distribution of maternal components of the canonical Wnt pathway has been shown to be responsible for the polarized stabilization of beta-catenin. In this study, we identified all Wnt and Wnt receptor mRNAs that are present in unfertilized sea urchin eggs and early embryos and analyzed their distributions along the primary (AV) axis. Our findings indicate that the asymmetric distribution of a maternal Wnt or Wnt receptor mRNA is unlikely to be a primary determinant of the polarized stabilization of beta-catenin along the AV axis. This contrasts sharply with findings in other organisms and points to remarkable evolutionary flexibility in the molecular mechanisms that underlie this otherwise very highly conserved patterning process.