A multiscale study of the role of dynamin in the regulation of glucose uptake

Glucose uptake in muscle cells in response to insulin is a fundamental mechanism for metabolism. The inability of cells to mobilize the specific glucose transporter GLUT4 is believed to be at least partially accountable for diseases, like diabetes, where cells do not respond to an insulin stimulus. In this work, a microchip is used to detect electrochemically glucose uptake from C2C12 myoblasts cultured on a patch of paper upon exposure to insulin. More importantly, the data suggest a new role for dynamin, a molecular motor which would be involved in GLUT4 translocation by facilitating exocytosis. It is also shown in vivo that dynamin is involved in the response to glucose in a completely distinct organism, namely the nematode Caenorhabditis elegans. The new mechanism for dynamin could therefore be more generally relevant in vivo and may play a role in insulin resistance.

Definition of in Vitro Microenvironments to Characterize and Control Pancreatic Progenitor Expansion and Differentiation

Chiara Greggio

The pancreas plays a central role in metabolism. The exocrine pancreas, composed of ductal and acinar cells, secretes and delivers digestive enzymes into the duodenum where they contribute to food digestion. The endocrine pancreas, constituted by the islets of Langerhans, secretes hormones in the blood to control glucose levels. In particular, insulin is the hormone produced by β-cells that instructs the peripheral tissues to uptake circulating glucose. Diabetes mellitus is a heterogeneous disease characterized by deregulated glucose homeostasis due to an impaired insulin function. The advancements in clinical practice have made of diabetes a chronic, instead of a lethal, disease; yet no definitive cure is available. Beta-cell transplantation offers promising results, especially for type 1 diabetes where β-cells are selectively eliminated by an autoimmune attack. Unfortunately its application suffers from the scarcity of transplantable pancreas/islets from cadaveric donors. Human embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) would constitute unlimited sources of transplantable β-cells, but the available differentiation protocols are not yet optimal, especially with regards to the numbers and the functionality of the generated β-cells. It is anticipated that a stepwise protocol would succeed in generating mature β-cells in vitro if it is capable of fully mimicking the embryonic development of these cells. Many aspects of pancreatic organogenesis have been elucidated and could serve as a guide for driving the commitment to β-cells. Unfortunately, pancreatic progenitors in vitro do not behave as in the intricate context of an embryo. This severely limits both the understanding of pancreatic development at the single-cell level and our ability in manipulating the process. The aim of our work was to develop in vitro methodologies to sustain pancreatic progenitor culture and expansion and to establish conditions to manipulate their progeny. To this aim, we combined an informed approach based on developmental biology and an empirical one. We first observed that pancreatic epithelial explants from embryonic day 10.5 mice could be cultured in presence of Matrigel™ and exogenous FGFs even in the absence of the mesenchyme that normally surrounds the epithelium. Notably, the removal of mesenchyme did not affect the endocrine commitment pattern of pancreatic progenitors. We then defined culture conditions allowing for the expansion of dissociated embryonic pancreatic progenitors in a three-dimensional Matrigel™-based environment. Under these conditions, progenitors proliferated and self-organized to generate pancreatic organoids containing both progenitors and differentiated cells, mostly exocrine, after 7 days of culture. When grafted into recipient pancreatic explants, the cultured progenitors contributed to the three epithelial pancreatic lineages (ductal, endocrine, acinar), integrating seamlessly into the endogenous cellular network. Thus, cultured progenitors retained their potential to differentiate into endocrine cells, but expressed it only in the appropriate niche. Subsequent removal of single components from the culture system led to the identification of some essential requirements for the maintenance/expansion of dissociated pancreatic progenitors, such as a strong FGF signaling and the Rho-associated kinase (ROCK) inhibitor Y-27632. In addition to this, we observed that only small clusters of pancreatic progenitors, but not single cells, were able to generate pancreatic organoids, suggesting a pivotal role for intercellular signaling in progenitor maintenance and expansion. By manipulating the components of the medium, we could affect fate commitment. In particular, the removal of FGF1 increased the yield of endocrine cells generated in vitro. In our search for a full control over the in vitro niche, we explored chemically defined matrices to replace Matrigel™. We showed that pancreatic progenitors could be cultured on laminin-functionalized hydrogels, although less efficiently than in Matrigel™. Moreover, differentiation into exocrine and endocrine cells occurred spontaneously under these conditions. By comparing Matrigel™, hydrogel microwells and two-dimensional culture systems, we uncovered the importance of a tridimensional architecture for pancreatic progenitor maintenance. We showed that pancreatic progenitors lost their identity as they flattened onto 2D surfaces, whereas 3D culture systems maintained the epithelial character of pancreatic progenitors and allowed the acquisition of apical polarization. To conclude, our work provides the first detailed characterization of in vitro culture systems for expansion and manipulation of dissociated embryonic pancreatic progenitors. Further implementation will hopefully allow the establishment of a fully-defined in vitro niche for developing pancreas with potential fundamental implications for the expansion of ES-derived pancreatic progenitors and their differentiation into functional β-cells.

EPFL2011

Autophagy Defect Is Associated with Low Glucose-Induced Apoptosis in 661W Photoreceptor Cells

Pénélope Andreux, Johan Auwerx, Martine Emery

Glucose is an important metabolic substrate of the retina and diabetic patients have to maintain a strict normoglycemia to avoid diabetes secondary effects, including cardiovascular disease, nephropathy, neuropathy and retinopathy. Others and we recently demonstrated the potential role of hypoglycemia in diabetic retinopathy. We showed acute hypoglycemia to induce retinal cell death both in vivo during an hyperinsulinemic/hypoglycemic clamp and in vitro in 661W photoreceptor cells cultured at low glucose concentration. In the present study, we showed low glucose to induce a decrease of BCL2 and BCL-XL anti-apoptotic proteins expression, leading to an increase of free pro-apoptotic BAX. In parallel, we showed that, in retinal cells, low glucose-induced apoptosis is involved in the process of autophagosomes formation through the AMPK/RAPTOR/mTOR pathway. Moreover, the decrease of LAMP2a expression led to a defect in the autophagosome/lysosome fusion process. Specific inhibition of autophagy, either by 3-methyladenine or by down-regulation of ATG5 or ATG7 proteins expression, increased caspase 3 activation and 661W cell death. We show that low glucose modifies the delicate equilibrium between apoptosis and autophagy. Cells struggled against low nutrient condition-induced apoptosis by starting an autophagic process, which led to cell death when inhibited. We conclude that autophagy defect is associated with low glucose-induced 661W cells death that could play a role in diabetic retinopathy. These results could modify the way of addressing negative effects of hypoglycemia. Short-term modulation of autophagy could be envisioned to treat diabetic patients in order to avoid secondary complications of the disease.

Public Library of Science2013

Role of metabolism and mitochondria during glucose sensing and glucose-induced dysfunction in human pancreatic beta-cells

Isabelle Chareyron

Glucose sensing and regulation of insulin secretion are the two main functions performed by the pancreatic beta-cell. Together these processes contribute to the tight control of blood glucose in our body. Compromising the ability of the beta-cells to properly secrete insulin can lead to the development of Type 2 diabetes. Mitochondria play an important role in beta-cell function, by integrating nutrients signals and modulating insulin secretion. In this thesis, we aimed at better understanding the role of metabolism and mitochondria during glucose sensing and glucose-induced dysfunction in human pancreatic beta-cells. To that end, we designed three studies looking at different aspects of beta-cell mitochondrial function. In the first one, we developed an in vitro model of glucose overload in human islet. We report an over activation of the mitochondria when returned to basal conditions after a 4 day culture in elevated glucose. Additionally, these mitochondria only poorly responded to nutrient stimulation. Impaired metabolism secretion coupling and insulin secretion indicated a partial loss of beta-cell function, despite the increased mitochondrial activity. After 4 days of elevated glucose treatment, unusually high levels of glycerol-3-phosphate and pyruvate, even in sub-stimulatory extracellular glucose concentrations, were observed. We propose that the accumulation of these metabolites renders beta-cells blind to subsequent glucose stimulation. After removing the glucose stress for 24 hours the mitochondrial energy metabolism was fully restored and metabolism-secretion coupling was normalized. In the second approach, we measured for the first time the matrix pH in primary human pancreatic beta-cells. We showed that the mitochondrial pH in beta-cell is more acidic than in other cell types. Furthermore, in response to glucose, the beta-cell mitochondrial matrix slightly acidifies, possibly due to uptake and metabolism of pyruvate. Lastly, we observed that adenoviruses used to deliver the genetically encoded pH sensor can have an intrinsic impact on mitochondrial pH and calcium signaling. It must be used at low infection units per cell in the study of primary beta-cells. In the third project, we tried to modify the mitochondrial energy metabolism in beta-cells by inhibiting mitochondrial protein synthesis. We used different antibiotics to achieve this, in particular chloramphenicol, known to interfere with the activity of the mitochondrial ribosome. Surprisingly, we observe that when mitochondrial protein synthesis is inhibited, the respiration of insulin-secreting cells (INS-1E cells, human islets) is not affected. Expression of mtDNA encoded respiratory chain subunits is reduced and a mitochondrial unfolded protein response initiated without reducing respiratory rates. This raises the possibility that limited activation of unfolded protein response has a positive effect on the cells. This doctoral thesis provides novel insights into various aspects of mitochondrial behavior in pancreatic beta-cells, both during physiological and pathophysiological conditions. Additional work is needed to better understand the molecular mechanisms underlying the mitochondrial adaptation to nutrient overload and the associated beta-cell dysfunction leading to the accelerated impairment of glucose homeostasis during the development of Type 2 diabetes.