Miri Stolovich-Rain

Control of pancreatic β cell regeneration by glucose metabolism.

Recent studies revealed a surprising regenerative capacity of insulin-producing β cells in mice, suggesting that regenerative therapy for human diabetes could in principle be achieved. Physiologic β cell regeneration under stressed conditions relies on accelerated proliferation of surviving β cells, but the factors that trigger and control this response remain unclear. Using islet transplantation experiments, we show that β cell mass is controlled systemically rather than by local factors such as tissue damage. Chronic changes in β cell glucose metabolism, rather than blood glucose levels per se, are the main positive regulator of basal and compensatory β cell proliferation in vivo. Intracellularly, genetic and pharmacologic manipulations reveal that glucose induces β cell replication via metabolism by glucokinase, the first step of glycolysis, followed by closure of K(ATP) channels and membrane depolarization. Our data provide a molecular mechanism for homeostatic control of β cell mass by metabolic demand.

LKB1 Regulates Pancreatic β Cell Size, Polarity, and Function

Pancreatic beta cells, organized in the islets of Langerhans, sense glucose and secrete appropriate amounts of insulin. We have studied the roles of LKB1, a conserved kinase implicated in the control of cell polarity and energy metabolism, in adult beta cells. LKB1-deficient beta cells show a dramatic increase in insulin secretion in vivo. Histologically, LKB1-deficient beta cells have striking alterations in the localization of the nucleus and cilia relative to blood vessels, suggesting a shift from hepatocyte-like to columnar polarity. Additionally, LKB1 deficiency causes a 65% increase in beta cell volume. We show that distinct targets of LKB1 mediate these effects. LKB1 controls beta cell size, but not polarity, via the mTOR pathway. Conversely, the precise position of the beta cell nucleus, but not cell size, is controlled by the LKB1 target Par1b. Insulin secretion and content are restricted by LKB1, at least in part, via AMPK. These results expose a molecular mechanism, orchestrated by LKB1, for the coordinated maintenance of beta cell size, form, and function.

The TSC-mTOR Pathway Mediates Translational Activation of TOP mRNAs by Insulin Largely in a Raptor- or Rictor-Independent Manner

ABSTRACT The stimulatory effect of insulin on protein synthesis is due to its ability to activate various translation factors. We now show that insulin can increase protein synthesis capacity also by translational activation of TOP mRNAs encoding various components of the translation machinery. This translational activation involves the tuberous sclerosis complex (TSC), as the knockout of TSC1 or TSC2 rescues TOP mRNAs from translational repression in mitotically arrested cells. Similar results were obtained upon overexpression of Rheb, an immediate TSC1-TSC2 target. The role of mTOR, a downstream effector of Rheb, in translational control of TOP mRNAs has been extensively studied, albeit with conflicting results. Even though rapamycin fully blocks mTOR complex 1 (mTORC1) kinase activity, the response of TOP mRNAs to this drug varies from complete resistance to high sensitivity. Here we show that mTOR knockdown blunts the translation efficiency of TOP mRNAs in insulin-treated cells, thus unequivocally establishing a role for mTOR in this mode of regulation. However, knockout of the raptor or rictor gene has only a slight effect on the translation efficiency of these mRNAs, implying that mTOR exerts its effect on TOP mRNAs through a novel pathway with a minor, if any, contribution of the canonical mTOR complexes mTORC1 and mTORC2. This conclusion is further supported by the observation that raptor knockout renders the translation of TOP mRNAs rapamycin hypersensitive.

Type 2 diabetes and congenital hyperinsulinism cause DNA double-strand breaks and p53 activity in β cells.

β cell failure in type 2 diabetes (T2D) is associated with hyperglycemia, but the mechanisms are not fully understood. Congenital hyperinsulinism caused by glucokinase mutations (GCK-CHI) is associated with β cell replication and apoptosis. Here, we show that genetic activation of β cell glucokinase, initially triggering replication, causes apoptosis associated with DNA double-strand breaks and activation of the tumor suppressor p53. ATP-sensitive potassium channels (KATP channels) and calcineurin mediate this toxic effect. Toxicity of long-term glucokinase overactivity was confirmed by finding late-onset diabetes in older members of a GCK-CHI family. Glucagon-like peptide-1 (GLP-1) mimetic treatment or p53 deletion rescues β cells from glucokinase-induced death, but only GLP-1 analog rescues β cell function. DNA damage and p53 activity in T2D suggest shared mechanisms of β cell failure in hyperglycemia and CHI. Our results reveal membrane depolarization via KATP channels, calcineurin signaling, DNA breaks, and p53 as determinants of β cell glucotoxicity and suggest pharmacological approaches to enhance β cell survival in diabetes.

Recognition and Killing of Human and Murine Pancreatic b Cells by the NK Receptor NKp46

Type 1 diabetes is an incurable disease that is currently treated by insulin injections or in rare cases by islet transplantation. We have recently shown that NKp46, a major killer receptor expressed by NK cells, recognizes an unknown ligand expressed by β cells and that in the absence of NKp46, or when its activity is blocked, diabetes development is inhibited. In this study, we investigate whether NKp46 is involved in the killing of human β cells that are intended to be used for transplantation, and we also thoroughly characterize the interaction between NKp46 and its human and mouse β cell ligands. We show that human β cells express an unknown ligand for NKp46 and are killed in an NKp46-dependent manner. We further demonstrate that the expression of the NKp46 ligand is detected on human β cells already at the embryonic stage and that it appears on murine β cells only following birth. Because the NKp46 ligand is detected on healthy β cells, we wondered why type 1 diabetes does not develop in all individuals and show that NK cells are absent from the vicinity of islets of healthy mice and are detected in situ in proximity with β cells in NOD mice. We also investigate the molecular mechanisms controlling NKp46 interactions with its β cell ligand and demonstrate that the recognition is confined to the membrane proximal domain and stalk region of NKp46 and that two glycosylated residues of NKp46, Thr125 and Asn216, are critical for this recognition.

Pancreatic Beta Cells in Very Old Mice Retain Capacity for Compensatory Proliferation

Background: It has been claimed that the regenerative potential of pancreatic beta cells is lost with advanced age. Results: We demonstrate compensatory replication of beta cells in very old mice, albeit at low levels. Conclusion: The potential for compensatory replication above base line is retained in beta cells of old mice. Significance: We present mouse evidence for retention of regenerative potential in old beta cells. Recent studies suggested that in old mice, beta cells lose their regenerative potential and cannot respond to mitogenic triggers. These studies examined beta cell replication in aged mice under basal conditions and in response to specific stimuli including treatment with the glucagon-like peptide-1 analog exenatide, streptozotocin injection, partial pancreatectomy, and high fat diet. However, it remains possible that the ability to mount a compensatory response of beta cells is retained in old age, but depends on the specific stimulus. Here, we asked whether partial ablation of beta cells in transgenic mice, using doxycycline-inducible expression of diphtheria toxin, triggers a significant compensatory proliferative response in 1–2-year-old animals. Consistent with previous reports, the basal rate of beta cell replication declines dramatically with age, averaging 0.1% in 2-year-old mice. Transient expression of diphtheria toxin in beta cells of old mice resulted in impaired glucose homeostasis and disruption of islet architecture (ratio of beta to alpha cells). Strikingly, the replication rate of surviving beta cells increased 3-fold over basal rate, similarly to the -fold increase in replication rate of beta cells in young transgenic mice. Islet architecture and glucose tolerance slowly normalized, indicating functional significance of compensatory beta cell replication in this setting. Finally, administration of a small molecule glucokinase activator to old mice doubled the frequency of beta cell replication, further showing that old beta cells can respond to the mitogenic trigger of enhanced glycolysis. We conclude that the potential for functionally significant compensatory proliferation of beta cells is retained in old mice, despite a decline in basal replication rate.

Weaning Triggers a Maturation Step of Pancreatic β Cells

Because tissue regeneration deteriorates with age, it is generally assumed that the younger the animal, the better it compensates for tissue damage. We have examined the effect of young age on compensatory proliferation of pancreatic β cells in vivo. Surprisingly, β cells in suckling mice fail to enter the cell division cycle in response to a diabetogenic injury or increased glycolysis. The potential of β cells for compensatory proliferation is acquired following premature weaning to normal chow, but not to a diet mimicking maternal milk. In addition, weaning coincides with enhanced glucose-stimulated oxidative phosphorylation and insulin secretion from islets. Transcriptome analysis reveals that weaning increases the expression of genes involved in replication licensing, suggesting a mechanism for increased responsiveness to the mitogenic activity of high glucose. We propose that weaning triggers a discrete maturation step of β cells, elevating both the mitogenic and secretory response to glucose.

Gastrin: A distinct fate of Neurogenin3 positive progenitor cells in the embryonic pancreas.

Neurogenin3+ (Ngn3+) progenitor cells in the developing pancreas give rise to five endocrine cell types secreting insulin, glucagon, somatostatin, pancreatic polypeptide and ghrelin. Gastrin is a hormone produced primarily by G-cells in the stomach, where it functions to stimulate acid secretion by gastric parietal cells. Gastrin is expressed in the embryonic pancreas and is common in islet cell tumors, but the lineage and regulators of pancreatic gastrin+ cells are not known. We report that gastrin is abundantly expressed in the embryonic pancreas and disappears soon after birth. Some gastrin+ cells in the developing pancreas co-express glucagon, ghrelin or pancreatic polypeptide, but many gastrin+ cells do not express any other islet hormone. Pancreatic gastrin+ cells express the transcription factors Nkx6.1, Nkx2.2 and low levels of Pdx1, and derive from Ngn3+ endocrine progenitor cells as shown by genetic lineage tracing. Using mice deficient for key transcription factors we show that gastrin expression depends on Ngn3, Nkx2.2, NeuroD1 and Arx, but not Pax4 or Pax6. Finally, gastrin expression is induced upon differentiation of human embryonic stem cells to pancreatic endocrine cells expressing insulin. Thus, gastrin+ cells are a distinct endocrine cell type in the pancreas and an alternative fate of Ngn3+ cells.

Glucose metabolism: key endogenous regulator of β-cell replication and survival.

Recent studies in mice have shown that pancreatic β‐cells have a significant potential for regeneration, suggesting that regenerative therapy for diabetes is feasible. Genetic lineage tracing studies indicate that β‐cell regeneration is based on the replication of fully differentiated, insulin‐positive β‐cells. Thus, a major challenge for this field is to identify and enhance the molecular pathways that control β‐cell replication and mass. We review evidence, from human genetics and mouse models, that glucose is a major signal for β‐cell replication. The mitogenic effect of blood glucose is transmitted via glucose metabolism within β‐cells, and through a signalling cascade that resembles the pathway for glucose‐stimulated insulin secretion. We introduce the concept that the individual β‐cell workload, defined as the amount of insulin that an individual β‐cell must secrete to maintain euglycaemia, is the primary determinant of replication, survival and mass. We also propose that a cell‐autonomous pathway, similar to that regulating replication, appears to be responsible for at least some of the toxic effects of glucose on β‐cells. Understanding and uncoupling the mitogenic and toxic effects of glucose metabolism on β‐cells may allow for the development of effective regenerative therapies for diabetes.

PAX6 maintains β cell identity by repressing genes of alternative islet cell types

Type 2 diabetes is thought to involve a compromised &bgr; cell differentiation state, but the mechanisms underlying this dysfunction remain unclear. Here, we report a key role for the TF PAX6 in the maintenance of adult &bgr; cell identity and function. PAX6 was downregulated in &bgr; cells of diabetic db/db mice and in WT mice treated with an insulin receptor antagonist, revealing metabolic control of expression. Deletion of Pax6 in &bgr; cells of adult mice led to lethal hyperglycemia and ketosis that were attributed to loss of &bgr; cell function and expansion of &agr; cells. Lineage-tracing, transcriptome, and chromatin analyses showed that PAX6 is a direct activator of &bgr; cell genes, thus maintaining mature &bgr; cell function and identity. In parallel, we found that PAX6 binds promoters and enhancers to repress alternative islet cell genes including ghrelin, glucagon, and somatostatin. Chromatin analysis and shRNA-mediated gene suppression experiments indicated a similar function of PAX6 in human &bgr; cells. We conclude that reduced expression of PAX6 in metabolically stressed &bgr; cells may contribute to &bgr; cell failure and &agr; cell dysfunction in diabetes.