Extensive elimination of acinar cells during normal postnatal pancreas growth.

While programmed cell death plays important roles during morphogenetic stages of development, post-differentiation organ growth is considered an efficient process whereby cell proliferation increases cell number. Here we demonstrate that early postnatal growth of the pancreas unexpectedly involves massive acinar cell elimination. Measurements of cell proliferation and death in the human pancreas in comparison to the actual increase in cell number predict daily elimination of 0.7% of cells, offsetting 88% of cell formation over the first year of life. Using mouse models, we show that death is associated with mitosis, through a failure of dividing cells to generate two viable daughters. In p53-deficient mice, acinar cell death and proliferation are reduced, while organ size is normal, suggesting that p53-dependent developmental apoptosis triggers compensatory proliferation. We propose that excess cell turnover during growth of the pancreas, and presumably other organs, facilitates robustness to perturbations and supports maintenance of tissue architecture.

Glycemic control releases regenerative potential of pancreatic beta cells blocked by severe hyperglycemia.

Diabetogenic ablation of beta cells in mice triggers a regenerative response whereby surviving beta cells proliferate and euglycemia is regained. Here, we identify and characterize heterogeneity in response to beta cell ablation. Efficient beta cell elimination leading to severe hyperglycemia (>28 mmol/L), causes permanent diabetes with failed regeneration despite cell cycle engagement of surviving beta cells. Strikingly, correction of glycemia via insulin, SGLT2 inhibition, or a ketogenic diet for about 3 weeks allows partial regeneration of beta cell mass and recovery from diabetes, demonstrating regenerative potential masked by extreme glucotoxicity. We identify gene expression changes in beta cells exposed to extremely high glucose levels, pointing to metabolic stress and downregulation of key cell cycle genes, suggesting failure of cell cycle completion. These findings reconcile conflicting data on the impact of glucose on beta cell regeneration and identify a glucose threshold converting glycemic load from pro-regenerative to anti-regenerative.

Intramuscular mRNA BNT162b2 vaccine against SARS-CoV-2 induces neutralizing salivary IgA.

Intramuscularly administered vaccines stimulate robust serum neutralizing antibodies, yet they are often less competent in eliciting sustainable "sterilizing immunity" at the mucosal level. Our study uncovers a strong temporary neutralizing mucosal component of immunity, emanating from intramuscular administration of an mRNA vaccine. We show that saliva of BNT162b2 vaccinees contains temporary IgA targeting the receptor-binding domain (RBD) of severe acute respiratory syndrome coronavirus-2 spike protein and demonstrate that these IgAs mediate neutralization. RBD-targeting IgAs were found to associate with the secretory component, indicating their bona fide transcytotic origin and their polymeric multivalent nature. The mechanistic understanding of the high neutralizing activity provided by mucosal IgA, acting at the first line of defense, will advance vaccination design and surveillance principles and may point to novel treatment approaches and new routes of vaccine administration and boosting.

Targeted in situ cross-linking mass spectrometry and integrative modeling reveal the architectures of three proteins from SARS-CoV-2.

Atomic structures of several proteins from the coronavirus family are still partial or unavailable. A possible reason for this gap is the instability of these proteins outside of the cellular context, thereby prompting the use of in-cell approaches. In situ cross-linking and mass spectrometry (in situ CLMS) can provide information on the structures of such proteins as they occur in the intact cell. Here, we applied targeted in situ CLMS to structurally probe Nsp1, Nsp2, and nucleocapsid (N) proteins from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and obtained cross-link sets with an average density of one cross-link per 20 residues. We then employed integrative modeling that computationally combined the cross-linking data with domain structures to determine full-length atomic models. For the Nsp2, the cross-links report on a complex topology with long-range interactions. Integrative modeling with structural prediction of individual domains by the AlphaFold2 system allowed us to generate a single consistent all-atom model of the full-length Nsp2. The model reveals three putative metal binding sites and suggests a role for Nsp2 in zinc regulation within the replication-transcription complex. For the N protein, we identified multiple intra- and interdomain cross-links. Our integrative model of the N dimer demonstrates that it can accommodate three single RNA strands simultaneously, both stereochemically and electrostatically. For the Nsp1, cross-links with the 40S ribosome were highly consistent with recent cryogenic electron microscopy structures. These results highlight the importance of cellular context for the structural probing of recalcitrant proteins and demonstrate the effectiveness of targeted in situ CLMS and integrative modeling.

The core clock transcription factor BMAL1 drives circadian ß-cell proliferation during compensatory regeneration of the endocrine pancreas.

Circadian clocks in pancreatic islets participate in the regulation of glucose homeostasis. Here we examined the role of these timekeepers in ß-cell regeneration after the massive ablation of ß cells by doxycycline-induced expression of diphtheria toxin A (DTA) in Insulin-rtTA/TET-DTA mice. Since we crossed reporter genes expressing α- and ß-cell-specific fluorescent proteins into these mice, we could follow the fate of α- and ß cells separately. As expected, DTA induction resulted in an acute hyperglycemia, which was accompanied by dramatic changes in gene expression in residual ß cells. In contrast, only temporal alterations of gene expression were observed in α cells. Interestingly, ß cells entered S phase preferentially during the nocturnal activity phase, indicating that the diurnal rhythm also plays a role in the orchestration of ß-cell regeneration. Indeed, in arrhythmic Bmal1-deficient mice, which lack circadian clocks, no compensatory ß-cell proliferation was observed, and the ß-cell ablation led to aggravated hyperglycemia, hyperglucagonemia, and fatal diabetes.

Inhibition of mTORC1 by ER stress impairs neonatal ß-cell expansion and predisposes to diabetes in the Akita mouse.

Unresolved ER stress followed by cell death is recognized as the main cause of a multitude of pathologies including neonatal diabetes. A systematic analysis of the mechanisms of ß-cell loss and dysfunction in Akita mice, in which a mutation in the proinsulin gene causes a severe form of permanent neonatal diabetes, showed no increase in ß-cell apoptosis throughout life. Surprisingly, we found that the main mechanism leading to ß-cell dysfunction is marked impairment of ß-cell growth during the early postnatal life due to transient inhibition of mTORC1, which governs postnatal ß-cell growth and differentiation. Importantly, restoration of mTORC1 activity in neonate ß-cells was sufficient to rescue postnatal ß-cell growth, and to improve diabetes. We propose a scenario for the development of permanent neonatal diabetes, possibly also common forms of diabetes, where early-life events inducing ER stress affect ß-cell mass expansion due to mTOR inhibition.

Postnatal Exocrine Pancreas Growth by Cellular Hypertrophy Correlates with a Shorter Lifespan in Mammals.

Developmental processes in different mammals are thought to share fundamental cellular mechanisms. We report a dramatic increase in cell size during postnatal pancreas development in rodents, accounting for much of the increase in organ size after birth. Hypertrophy of pancreatic acinar cells involves both higher ploidy and increased biosynthesis per genome copy; is maximal adjacent to islets, suggesting endocrine to exocrine communication; and is partly driven by weaning-related processes. In contrast to the situation in rodents, pancreas cell size in humans remains stable postnatally, indicating organ growth by pure hyperplasia. Pancreatic acinar cell volume varies 9-fold among 24 mammalian species analyzed, and shows a striking inverse correlation with organismal lifespan. We hypothesize that cellular hypertrophy is a strategy for rapid postnatal tissue growth, entailing life-long detrimental effects.

PAX6 maintains ß cell identity by repressing genes of alternative islet cell types.

Type 2 diabetes is thought to involve a compromised ß 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 ß cell identity and function. PAX6 was downregulated in ß 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 ß cells of adult mice led to lethal hyperglycemia and ketosis that were attributed to loss of ß cell function and expansion of α cells. Lineage-tracing, transcriptome, and chromatin analyses showed that PAX6 is a direct activator of ß cell genes, thus maintaining mature ß 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 ß cells. We conclude that reduced expression of PAX6 in metabolically stressed ß cells may contribute to ß cell failure and α cell dysfunction in diabetes.

Phosphorylated Ribosomal Protein S6 Is Required for Akt-Driven Hyperplasia and Malignant Transformation, but Not for Hypertrophy, Aneuploidy and Hyperfunction of Pancreatic ß-Cells.

Constitutive expression of active Akt (Akttg) drives hyperplasia and hypertrophy of pancreatic ß-cells, concomitantly with increased insulin secretion and improved glucose tolerance, and at a later stage the development of insulinoma. To determine which functions of Akt are mediated by ribosomal protein S6 (rpS6), an Akt effector, we generated mice that express constitutive Akt in ß-cells in the background of unphosphorylatable ribosomal protein S6 (rpS6P-/-). rpS6 phosphorylation deficiency failed to block Akttg-induced hypertrophy and aneuploidy in ß-cells, as well as the improved glucose homeostasis, indicating that Akt carries out these functions independently of rpS6 phosphorylation. In contrast, rpS6 phosphorylation deficiency efficiently restrained the reduction in nuclear localization of the cell cycle inhibitor p27, as well as the development of Akttg-driven hyperplasia and tumor formation in ß-cells. In vitro experiments with Akttg and rpS6P-/-;Akttg fibroblasts demonstrated that rpS6 phosphorylation deficiency leads to reduced translation fidelity, which might underlie its anti-tumorigenic effect in the pancreas. However, the role of translation infidelity in tumor suppression cannot simply be inferred from this heterologous experimental model, as rpS6 phosphorylation deficiency unexpectedly elevated the resistance of Akttg fibroblasts to proteotoxic, genotoxic as well as autophagic stresses. In contrast, rpS6P-/- fibroblasts exhibited a higher sensitivity to these stresses upon constitutive expression of oncogenic Kras. The latter result provides a possible mechanistic explanation for the ability of rpS6 phosphorylation deficiency to enhance DNA damage and protect mice from Kras-induced neoplastic transformation in the exocrine pancreas. We propose that Akt1 and Kras exert their oncogenic properties through distinct mechanisms, even though both show addiction to rpS6 phosphorylation.

Weaning triggers a maturation step of pancreatic ß 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 ß cells in vivo. Surprisingly, ß cells in suckling mice fail to enter the cell division cycle in response to a diabetogenic injury or increased glycolysis. The potential of ß 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 ß cells, elevating both the mitogenic and secretory response to glucose.

Reassessment of the role of TSC, mTORC1 and microRNAs in amino acids-meditated translational control of TOP mRNAs.

TOP mRNAs encode components of the translational apparatus, and repression of their translation comprises one mechanism, by which cells encountering amino acid deprivation downregulate the biosynthesis of the protein synthesis machinery. This mode of regulation involves TSC as knockout of TSC1 or TSC2 rescued TOP mRNAs translation in amino acid-starved cells. The involvement of mTOR in translational control of TOP mRNAs is demonstrated by the ability of constitutively active mTOR to relieve the translational repression of TOP mRNA upon amino acid deprivation. Consistently, knockdown of this kinase as well as its inhibition by pharmacological means blocked amino acid-induced translational activation of these mRNAs. The signaling of amino acids to TOP mRNAs involves RagB, as overexpression of active RagB derepressed the translation of these mRNAs in amino acid-starved cells. Nonetheless, knockdown of raptor or rictor failed to suppress translational activation of TOP mRNAs by amino acids, suggesting that mTORC1 or mTORC2 plays a minor, if any, role in this mode of regulation. Finally, miR10a has previously been suggested to positively regulate the translation of TOP mRNAs. However, we show here that titration of this microRNA failed to downregulate the basal translation efficiency of TOP mRNAs. Moreover, Drosha knockdown or Dicer knockout, which carries out the first and second processing steps in microRNAs biosynthesis, respectively, failed to block the translational activation of TOP mRNAs by amino acid or serum stimulation. Evidently, these results are questioning the positive role of microRNAs in this mode of regulation.

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

ß 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 ß cell replication and apoptosis. Here, we show that genetic activation of ß 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 ß cells from glucokinase-induced death, but only GLP-1 analog rescues ß cell function. DNA damage and p53 activity in T2D suggest shared mechanisms of ß cell failure in hyperglycemia and CHI. Our results reveal membrane depolarization via KATP channels, calcineurin signaling, DNA breaks, and p53 as determinants of ß cell glucotoxicity and suggest pharmacological approaches to enhance ß cell survival in diabetes.

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.

Pancreatic beta cells in very old mice retain capacity for compensatory proliferation.

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.

Recognition and killing of human and murine pancreatic beta 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 ß 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 ß cells that are intended to be used for transplantation, and we also thoroughly characterize the interaction between NKp46 and its human and mouse ß cell ligands. We show that human ß 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 ß cells already at the embryonic stage and that it appears on murine ß cells only following birth. Because the NKp46 ligand is detected on healthy ß 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 ß cells in NOD mice. We also investigate the molecular mechanisms controlling NKp46 interactions with its ß 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, Thr(125) and Asn(216), are critical for this recognition.

Control of pancreatic ß cell regeneration by glucose metabolism.

Recent studies revealed a surprising regenerative capacity of insulin-producing ß cells in mice, suggesting that regenerative therapy for human diabetes could in principle be achieved. Physiologic ß cell regeneration under stressed conditions relies on accelerated proliferation of surviving ß cells, but the factors that trigger and control this response remain unclear. Using islet transplantation experiments, we show that ß cell mass is controlled systemically rather than by local factors such as tissue damage. Chronic changes in ß cell glucose metabolism, rather than blood glucose levels per se, are the main positive regulator of basal and compensatory ß cell proliferation in vivo. Intracellularly, genetic and pharmacologic manipulations reveal that glucose induces ß 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 ß cell mass by metabolic demand.

LKB1 regulates pancreatic beta 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.

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.

Ribosomal protein S6 phosphorylation is a determinant of cell size and glucose homeostasis.

The regulated phosphorylation of ribosomal protein (rp) S6 has attracted much attention since its discovery in 1974, yet its physiological role has remained obscure. To directly address this issue, we have established viable and fertile knock-in mice, whose rpS6 contains alanine substitutions at all five phosphorylatable serine residues (rpS6(P-/-)). Here we show that contrary to the widely accepted model, this mutation does not affect the translational control of TOP mRNAs. rpS6(P-/-) mouse embryo fibroblasts (MEFs) display an increased rate of protein synthesis and accelerated cell division, and they are significantly smaller than rpS6(P+/+) MEFs. This small size reflects a growth defect, rather than a by-product of their faster cell division. Moreover, the size of rpS6(P-/-) MEFs, unlike wild-type MEFs, is not further decreased upon rapamycin treatment, implying that the rpS6 is a critical downstream effector of mTOR in regulation of cell size. The small cell phenotype is not confined to embryonal cells, as it also selectively characterizes pancreatic beta-cells in adult rpS6(P-/-) mice. These mice suffer from diminished levels of pancreatic insulin, hypoinsulinemia, and impaired glucose tolerance.