Latif Rachdi

Disruption of Tsc2 in pancreatic β cells induces β cell mass expansion and improved glucose tolerance in a TORC1-dependent manner

Regulation of pancreatic β cell mass and function is a major determinant for the development of diabetes. Growth factors and nutrients are important regulators of β cell mass and function. The signaling pathways by which these growth signals modulate these processes have not been completely elucidated. Tsc2 is an attractive candidate to modulate these processes, because it is a converging point for growth factor and nutrient signals. In these experiments, we generated mice with conditional deletion of Tsc2 in β cells (βTsc2−/−). These mice exhibited decreased glucose levels and hyperinsulinemia in the fasting and fed state. Improved glucose tolerance in these mice was observed as early as 4 weeks of age and was still present in 52-week-old mice. Deletion of Tsc2 in β cells induced expansion of β cell mass by increased proliferation and cell size. Rapamycin treatment reversed the metabolic changes in βTsc2−/− mice by induction of insulin resistance and reduction of β cell mass. The reduction of β cell mass in βTsc2−/− mice by inhibition of the mTOR/Raptor (TORC1) complex with rapamycin treatment suggests that TORC1 mediates proliferative and growth signals induced by deletion of Tsc2 in β cells. These studies uncover a critical role for the Tsc2/mTOR pathway in regulation of β cell mass and carbohydrate metabolism in vivo.

Development of a conditionally immortalized human pancreatic β cell line

Diabetic patients exhibit a reduction in β cells, which secrete insulin to help regulate glucose homeostasis; however, little is known about the factors that regulate proliferation of these cells in human pancreas. Access to primary human β cells is limited and a challenge for both functional studies and drug discovery progress. We previously reported the generation of a human β cell line (EndoC-βH1) that was generated from human fetal pancreas by targeted oncogenesis followed by in vivo cell differentiation in mice. EndoC-βH1 cells display many functional properties of adult β cells, including expression of β cell markers and insulin secretion following glucose stimulation; however, unlike primary β cells, EndoC-βH1 cells continuously proliferate. Here, we devised a strategy to generate conditionally immortalized human β cell lines based on Cre-mediated excision of the immortalizing transgenes. The resulting cell line (EndoC-βH2) could be massively amplified in vitro. After expansion, transgenes were efficiently excised upon Cre expression, leading to an arrest of cell proliferation and pronounced enhancement of β cell-specific features such as insulin expression, content, and secretion. Our data indicate that excised EndoC-βH2 cells are highly representative of human β cells and should be a valuable tool for further analysis of human β cells.

Regulation of β‐cell mass and function by the Akt/protein kinase B signalling pathway

The insulin receptor substrate‐2/phosphoinositide 3‐kinase (PI3K) pathway plays a critical role in the regulation of β‐cell mass and function, demonstrated both in vitro and in vivo. The serine threonine kinase Akt is one of the promising downstream molecules of this pathway that has been identified as a potential target to regulate function and induce proliferation and survival of β cells. Here we summarize some of the molecular mechanisms, downstream signalling pathways and critical components involved in the regulation of β‐cell mass and function by Akt.

L-Leucine Alters Pancreatic β-Cell Differentiation and Function via the mTor Signaling Pathway

Leucine (Leu) is an essential branched-chain amino acid, which activates the mammalian target of rapamycin (mTOR) signaling pathway. The effect of Leu on cell differentiation during embryonic development is unknown. Here, we show that Leu supplementation during pregnancy significantly increased fetal body weight, caused fetal hyperglycemia and hypoinsulinemia, and decreased the relative islet area. We also used rat embryonic pancreatic explant culture for elucidating the mechanism of Leu action on β-cell development. We found that in the presence of Leu, differentiation of pancreatic duodenal homeobox-1–positive progenitor cells into neurogenin3-positive endocrine progenitor cells was inefficient and resulted in decreased β-cell formation. Mechanistically, Leu increases the intracellular levels of hypoxia-inducible factor 1-α, a repressor of endocrine fate in the pancreas, by activating the mTOR complex 1 signaling pathway. Collectively, our findings indicate that Leu supplementation during pregnancy could potentially increase the risk of type 2 diabetes mellitus by inhibiting the differentiation of pancreatic endocrine progenitor cells during a susceptible period of fetal life.

Differential effects of p27 in regulation of β-cell mass during development, neonatal period, and adult life

β-Cell cycle progression and proliferation are critical to maintain β-cell mass in adult mice. Of the cell cycle inhibitors, p27Kip1 is thought to be the primary modulator of the proliferative status in most cell types. p27 plays a role in β-cell adaptation in genetic models of insulin resistance. To study the role of p27 in β-cells during physiological conditions and at different stages of β-cell differentiation, we studied mice deficient of or overexpressing p27. Experiments in p27-deficient mice showed improved glucose tolerance and hyperinsulinemia. These changes were associated with increased islet mass and proliferation. The experiments overexpressing p27 in β-cells were performed using a doxycycline-inducible model. Interestingly, overexpression of p27 for 16 weeks in β-cells from adult mice had no effect on glucose tolerance, β-cell mass, or proliferation. In contrast, induction of p27 expression during β-cell development or early neonatal period resulted in severe glucose intolerance and reduced β-cell mass by decreased proliferation. These changes were reversible upon discontinuation of doxycycline. These experiments suggest that p27 is a critical molecule for β-cell proliferation during β-cell development and early postnatal life but not for maintenance of adult mass.

Peptide-mediated activation of Akt and extracellular regulated kinase signaling prevents lymphocyte apoptosis

Lymphocyte apoptosis is a hallmark of sepsis and contributes to disease mortality. In other acute injuries, such as myocardial and cerebral ischemia/reperfusion, apoptosis plays a significant role in disease‐associated morbidity and mortality. We previously showed that constitutive activation of the potent antiapoptotic Akt/protein kinase B signaling pathway in lymphocytes both reduces sepsis‐induced lymphocyte apoptosis and confers a significant survival advantage compared to wild‐type littermates. Here, we demonstrate a therapeutic approach to acutely augment Akt activity in a wild‐type animal. A cell‐permeable peptide conjugated to the Akt‐binding domain of the endogenous Akt coactivator, Tcl‐1, prolongs Akt activity, activates extracellular regulated kinase (ERK) signaling and protects lymphocytes from numerous apoptotic stimuli both in vitro and in vivo. Molecular approaches to activate the antiapoptotic Akt and ERK signaling pathways may provide a novel tool to study these signaling pathways, as well as a new antiapoptotic strategy for the treatment of sepsis and other acute injuries. McDunn, J. E., Muenzer, J. T., Rachdi, L., Chang, K. C., Davis, C. G., Dunne, W. M., Piwnica‐Worms, D., Mizrachi, E.‐B., Hotchkiss, R. S. Peptidemediated activation of Akt and ERK signaling prevents lymphocyte apoptosis. FASEB J. 22, 561−568 (2008)