Fabrizio Thorel

Conversion of adult pancreatic α-cells to β-cells after extreme β-cell loss

Pancreatic insulin-producing β-cells have a long lifespan, such that in healthy conditions they replicate little during a lifetime. Nevertheless, they show increased self-duplication after increased metabolic demand or after injury (that is, β-cell loss). It is not known whether adult mammals can differentiate (regenerate) new β-cells after extreme, total β-cell loss, as in diabetes. This would indicate differentiation from precursors or another heterologous (non-β-cell) source. Here we show β-cell regeneration in a transgenic model of diphtheria-toxin-induced acute selective near-total β-cell ablation. If given insulin, the mice survived and showed β-cell mass augmentation with time. Lineage-tracing to label the glucagon-producing α-cells before β-cell ablation tracked large fractions of regenerated β-cells as deriving from α-cells, revealing a previously disregarded degree of pancreatic cell plasticity. Such inter-endocrine spontaneous adult cell conversion could be harnessed towards methods of producing β-cells for diabetes therapies, either in differentiation settings in vitro or in induced regeneration.

Conversion of adult pancreatic alpha-cells to beta-cells after extreme beta-cell loss.

Pancreatic insulin-producing β-cells have a long lifespan, such that in healthy conditions they replicate little during a lifetime. Nevertheless, they show increased self-duplication after increased metabolic demand or after injury (that is, β-cell loss). It is not known whether adult mammals can differentiate (regenerate) new β-cells after extreme, total β-cell loss, as in diabetes. This would indicate differentiation from precursors or another heterologous (non-β-cell) source. Here we show β-cell regeneration in a transgenic model of diphtheria-toxin-induced acute selective near-total β-cell ablation. If given insulin, the mice survived and showed β-cell mass augmentation with time. Lineage-tracing to label the glucagon-producing α-cells before β-cell ablation tracked large fractions of regenerated β-cells as deriving from α-cells, revealing a previously disregarded degree of pancreatic cell plasticity. Such inter-endocrine spontaneous adult cell conversion could be harnessed towards methods of producing β-cells for diabetes therapies, either in differentiation settings in vitro or in induced regeneration.

Diabetes recovery by age-dependent conversion of pancreatic δ-cells into insulin producers

Total or near-total loss of insulin-producing β-cells occurs in type 1 diabetes. Restoration of insulin production in type 1 diabetes is thus a major medical challenge. We previously observed in mice in which β-cells are completely ablated that the pancreas reconstitutes new insulin-producing cells in the absence of autoimmunity. The process involves the contribution of islet non-β-cells; specifically, glucagon-producing α-cells begin producing insulin by a process of reprogramming (transdifferentiation) without proliferation. Here we show the influence of age on β-cell reconstitution from heterologous islet cells after near-total β-cell loss in mice. We found that senescence does not alter α-cell plasticity: α-cells can reprogram to produce insulin from puberty through to adulthood, and also in aged individuals, even a long time after β-cell loss. In contrast, before puberty there is no detectable α-cell conversion, although β-cell reconstitution after injury is more efficient, always leading to diabetes recovery. This process occurs through a newly discovered mechanism: the spontaneous en masse reprogramming of somatostatin-producing δ-cells. The juveniles display ‘somatostatin-to-insulin’ δ-cell conversion, involving dedifferentiation, proliferation and re-expression of islet developmental regulators. This juvenile adaptability relies, at least in part, upon the combined action of FoxO1 and downstream effectors. Restoration of insulin producing-cells from non-β-cell origins is thus enabled throughout life via δ- or α-cell spontaneous reprogramming. A landscape with multiple intra-islet cell interconversion events is emerging, offering new perspectives for therapy.

Context-specific α-to-β-cell reprogramming by forced Pdx1 expression

Using single transcription factors to reprogram cells could produce important insights into the epigenetic mechanisms that direct normal differentiation, or counter inappropriate plasticity, or even provide new ways of manipulating normal ontogeny in vitro to control lineage diversification and differentiation. We enforced Pdx1 expression from the Neurogenin-3-expressing endocrine commitment point onward and found during the embryonic period a minor increased β-cell allocation with accompanying reduced α-cell numbers. More surprisingly, almost all remaining Pdx1-containing glucagon/Arx-producing cells underwent a fairly rapid conversion at postnatal stages, through glucagon-insulin double positivity, to a state indistinguishable from normal β cells, resulting in complete α-cell absence. This α-to-β conversion was not caused by activating Pdx1 in the later glucagon-expressing state. Our findings reveal that Pdx1 can work single-handedly as a potent context-dependent autonomous reprogramming agent, and suggest a postnatal differentiation evaluation stage involved in normal endocrine maturation.

Nkx6.1 controls a gene regulatory network required for establishing and maintaining pancreatic Beta cell identity.

All pancreatic endocrine cell types arise from a common endocrine precursor cell population, yet the molecular mechanisms that establish and maintain the unique gene expression programs of each endocrine cell lineage have remained largely elusive. Such knowledge would improve our ability to correctly program or reprogram cells to adopt specific endocrine fates. Here, we show that the transcription factor Nkx6.1 is both necessary and sufficient to specify insulin-producing beta cells. Heritable expression of Nkx6.1 in endocrine precursors of mice is sufficient to respecify non-beta endocrine precursors towards the beta cell lineage, while endocrine precursor- or beta cell-specific inactivation of Nkx6.1 converts beta cells to alternative endocrine lineages. Remaining insulin+ cells in conditional Nkx6.1 mutants fail to express the beta cell transcription factors Pdx1 and MafA and ectopically express genes found in non-beta endocrine cells. By showing that Nkx6.1 binds to and represses the alpha cell determinant Arx, we identify Arx as a direct target of Nkx6.1. Moreover, we demonstrate that Nkx6.1 and the Arx activator Isl1 regulate Arx transcription antagonistically, thus establishing competition between Isl1 and Nkx6.1 as a critical mechanism for determining alpha versus beta cell identity. Our findings establish Nkx6.1 as a beta cell programming factor and demonstrate that repression of alternative lineage programs is a fundamental principle by which beta cells are specified and maintained. Given the lack of Nkx6.1 expression and aberrant activation of non-beta endocrine hormones in human embryonic stem cell (hESC)–derived insulin+ cells, our study has significant implications for developing cell replacement therapies.

Normal Glucagon Signaling and β-Cell Function After Near-Total α-Cell Ablation in Adult Mice

OBJECTIVE To evaluate whether healthy or diabetic adult mice can tolerate an extreme loss of pancreatic α-cells and how this sudden massive depletion affects β-cell function and blood glucose homeostasis. RESEARCH DESIGN AND METHODS We generated a new transgenic model allowing near-total α-cell removal specifically in adult mice. Massive α-cell ablation was triggered in normally grown and healthy adult animals upon diphtheria toxin (DT) administration. The metabolic status of these mice was assessed in 1) physiologic conditions, 2) a situation requiring glucagon action, and 3) after β-cell loss. RESULTS Adult transgenic mice enduring extreme (98%) α-cell removal remained healthy and did not display major defects in insulin counter-regulatory response. We observed that 2% of the normal α-cell mass produced enough glucagon to ensure near-normal glucagonemia. β-Cell function and blood glucose homeostasis remained unaltered after α-cell loss, indicating that direct local intraislet signaling between α- and β-cells is dispensable. Escaping α-cells increased their glucagon content during subsequent months, but there was no significant α-cell regeneration. Near-total α-cell ablation did not prevent hyperglycemia in mice having also undergone massive β-cell loss, indicating that a minimal amount of α-cells can still guarantee normal glucagon signaling in diabetic conditions. CONCLUSIONS An extremely low amount of α-cells is sufficient to prevent a major counter-regulatory deregulation, both under physiologic and diabetic conditions. We previously reported that α-cells reprogram to insulin production after extreme β-cell loss and now conjecture that the low α-cell requirement could be exploited in future diabetic therapies aimed at regenerating β-cells by reprogramming adult α-cells.

Pancreatic Inactivation of c-Myc Decreases Acinar Mass and Transdifferentiates Acinar Cells Into Adipocytes in Mice

BACKGROUND & AIMS The pancreatic mass is determined by the coordinated expansion and differentiation of progenitor cells and is maintained via tight control of cell replacement rates. The basic helix-loop-helix transcription factor c-Myc is one of the main regulators of these processes in many organs. We studied the requirement of c-Myc in controlling the generation and maintenance of pancreatic mass. METHODS We conditionally inactivated c-Myc in Pdx1+ pancreatic progenitor cells. Pancreata of mice lacking c-Myc (c-Myc(P-/-) mice) were analyzed during development and ageing. RESULTS Pancreatic growth in c-Myc(P-/-) mice was impaired starting on E12.5, in early primordia, because of decreased proliferation and altered differentiation of exocrine progenitors; islet progenitors were spared. Acinar cell maturation was defective in the adult hypotrophic pancreas, which hampered exocrine mass maintenance in aged animals. From 2 to 10 months of age, the c-Myc(P-/-) pancreas was progressively remodeled without inflammatory injury. Loss of acinar cells increased with time, concomitantly with adipose tissue accumulation. Using a genetic cell lineage tracing analysis, we demonstrated that pancreatic adipose cells were derived directly from transdifferentiating acinar cells. This epithelial-to-mesenchyme transition was also observed in normal aged specimens and in pancreatitis. CONCLUSIONS These results provide evidence indicating that c-Myc activity is required for growth and maturation of the exocrine pancreas, and sheds new light on the ontogeny of pancreatic adipose cells in processes of organ degenerescence and tissue involution.

Leptin engages a hypothalamic neurocircuitry to permit survival in the absence of insulin

The dogma that life without insulin is incompatible has recently been challenged by results showing the viability of insulin-deficient rodents undergoing leptin monotherapy. Yet, the mechanisms underlying these actions of leptin are unknown. Here, the metabolic outcomes of intracerebroventricular (i.c.v.) administration of leptin in mice devoid of insulin and lacking or re-expressing leptin receptors (LEPRs) only in selected neuronal groups were assessed. Our results demonstrate that concomitant re-expression of LEPRs only in hypothalamic γ-aminobutyric acid (GABA) and pro-opiomelanocortin (POMC) neurons is sufficient to fully mediate the lifesaving and antidiabetic actions of leptin in insulin deficiency. Our analyses indicate that enhanced glucose uptake by brown adipose tissue and soleus muscle, as well as improved hepatic metabolism, underlies these effects of leptin. Collectively, our data elucidate a hypothalamic-dependent pathway enabling life without insulin and hence pave the way for developing better treatments for diseases of insulin deficiency.

Definition of a Short Region of XPG Necessary for TFIIH Interaction and Stable Recruitment to Sites of UV Damage

ABSTRACT XPG is the human endonuclease that cuts 3′ to DNA lesions during nucleotide excision repair. Missense mutations in XPG can lead to xeroderma pigmentosum (XP), whereas truncated or unstable XPG proteins cause Cockayne syndrome (CS), normally yielding life spans of <7 years. One XP-G individual who had advanced XP/CS symptoms at 28 years has been identified. The genetic, biochemical, and cellular defects in this remarkable case provide insight into the onset of XP and CS, and they reveal a previously unrecognized property of XPG. Both of this individuals XPG alleles produce a severely truncated protein, but an infrequent alternative splice generates an XPG protein lacking seven internal amino acids, which can account for his very slight cellular UV resistance. Deletion of XPG amino acids 225 to 231 does not abolish structure-specific endonuclease activity. Instead, this region is essential for interaction with TFIIH and for the stable recruitment of XPG to sites of local UV damage after the prior recruitment of TFIIH. These results define a new functional domain of XPG, and they demonstrate that recruitment of DNA repair proteins to sites of damage does not necessarily lead to productive repair reactions. This observation has potential implications that extend beyond nucleotide excision repair.

Progenitor cell domains in the developing and adult pancreas

Unlike organs with defined stem cell compartments, such as the intestine, the pancreas has limited capacity to regenerate. The question of whether the adult pancreas harbors facultative stem/progenitor cells has been a prime subject of debate. Cumulative evidence from recent genetic lineage tracing studies, in which specific cell populations were marked and traced in adult mice, suggests that endocrine and acinar cells are no longer generated from progenitors in the adult pancreas. These studies further indicate that adult pancreatic ductal cells are not a source for endocrine cells following pancreatic injury, as previously suggested. Our own studies have shown that adult ductal cells reinitiate expression of some endocrine progenitor markers, including Ngn3, after injury by partial duct ligation (PDL), but that these cells do not undergo endocrine cell differentiation. Here, we present additional evidence that endocrine cells do not arise from ducts following b-cell ablation by streptozotocin or by a diphtheria toxin-expressing transgene or when b-cell ablation is combined with PDL. In this review, we discuss findings from recent lineage tracing studies of embryonic and adult pancreatic ductal cells. Based upon the combined evidence from these studies, we propose that multipotency is associated with a specific transcriptional signature.