Philippe Klee

Islet-cell-to-cell communication as basis for normal insulin secretion.

The emergence of pancreatic islets has necessitated the development of a signalling system for the intra‐ and inter‐islet coordination of β cells. With evolution, this system has evolved into a complex regulatory network of partially cross‐talking pathways, whereby individual cells sense the state of activity of their neighbours and, accordingly, regulate their own level of functioning. A consistent feature of this network in vertebrates is the expression of connexin (Cx)‐36‐made cell‐to‐cell channels, which cluster at gap junction domains of the cell membrane, and which adjacent β cells use to share cytoplasmic ions and small metabolites within individual islets. This chapter reviews what is known about Cx36, and the mechanism whereby this β‐cell connexin significantly regulates insulin secretion. It further outlines other less established functions of the protein and evaluates its potential relevance for the development of novel therapeutic approaches to diabetes.

Modeling intrauterine growth retardation in rodents: impact on pancreas development and glucose homeostasis.

Fetal adverse environment, such as insufficient maternal nutrition, placental insufficiency and stress, alters organ development and leads to poor fetal growth, also called intrauterine growth retardation (IUGR). IUGR is associated with an increased risk of perinatal mortality and morbidity as well as late-onset metabolic diseases, such as obesity, diabetes and hypertension in adulthood. In the rodent model, IUGR can be induced by fetal caloric restriction, fetal protein restriction, by exposure to high levels of glucocorticoids or by restricted placental blood supply. Such experimental IUGR models show a decreased beta cell mass and lower pancreatic insulin content. Recent research has provided an insight into the mechanisms responsible for the loss of beta cells. Here we review models that give further details about the molecular determinants of fetal and postnatal pancreatic islet development that are required to understand the consequences of fetal insults.

Connexins protect mouse pancreatic β cells against apoptosis

Type 1 diabetes develops when most insulin-producing β cells of the pancreas are killed by an autoimmune attack. The in vivo conditions modulating the sensitivity and resistance of β cells to this attack remain largely obscure. Here, we show that connexin 36 (Cx36), a trans-membrane protein that forms gap junctions between β cells in the pancreatic islets, protects mouse β cells against both cytotoxic drugs and cytokines that prevail in the islet environment at the onset of type 1 diabetes. We documented that this protection was at least partially dependent on intercellular communication, which Cx36 and other types of connexin channels establish within pancreatic islets. We further found that proinflammatory cytokines decreased expression of Cx36 and that experimental reduction or augmentation of Cx36 levels increased or decreased β cell apoptosis, respectively. Thus, we conclude that Cx36 is central to β cell protection from toxic insults.

Connexin36 and pancreatic β-cell functions

Abstract Most cell types are functionally coupled by connexin (Cx) channels, i.e. exchange cytoplasmic ions and small metabolites through gap junction domains of their membrane. This form of direct cell-to-cell communication occurs in all existing animals, whatever their position in the phylogenetic scale, and up to humans. Pancreatic β-cells are no exception, and normally cross-talk with their neighbors via channels made of Cx36. These exchanges importantly contribute to coordinate and synchronize the function of individual cells within pancreatic islets, particularly in the context of glucose-induced insulin secretion. Compelling evidence now indicates that Cx36-mediated coupling, and/or the Cx36 protein per se, play significant regulatory roles in various β-cell functions, ranging from the biosynthesis, storage and release of insulin. Recent preliminary data further suggest that the protein may also be implicated in the balance of β-cell growth versus necrosis and apoptosis, and in the regulated expression of specific genes. Here, we review this evidence, discuss the possible involvement of Cx36 in the pathophysiology of diabetes, and evaluate the relevance of this connexin in the therapeutic approaches to the disease.

Connexin-dependent signaling in neuro-hormonal systems.

The advent of multicellular organisms was accompanied by the development of short- and long-range chemical signalling systems, including those provided by the nervous and endocrine systems. In turn, the cells of these two systems have developed mechanisms for interacting with both adjacent and distant cells. With evolution, such mechanisms have diversified to become integrated in a complex regulatory network, whereby individual endocrine and neuro-endocrine cells sense the state of activity of their neighbors and, accordingly, regulate their own level of functioning. A consistent feature of this network is the expression of connexin-made channels between the (neuro)hormone-producing cells of all endocrine glands and secretory regions of the central nervous system so far investigated in vertebrates. This review summarizes the distribution of connexins in the mammalian (neuro)endocrine systems, and what we know about the participation of these proteins on hormone secretion, the life of the producing cells, and the action of (neuro)hormones on specific targets. The data gathered since the last reviews on the topic are summarized, with particular emphasis on the roles of Cx36 in the function of the insulin-producing beta cells of the endocrine pancreas, and of Cx40 in that of the renin-producing juxta-glomerular epithelioid cells of the kidney cortex. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics.

Connexin implication in the control of the murine beta-cell mass

Diabetes develops when the insulin needs of peripheral cells exceed the availability or action of the hormone. This situation results from the death of most beta-cells in type 1 diabetes, and from an inability of the beta-cell mass to adapt to increasing insulin needs in type 2 and gestational diabetes. We analyzed several lines of transgenic mice and showed that connexins (Cxs), the transmembrane proteins that form gap junctions, are implicated in the modulation of the beta-cell mass. Specifically, we found that the native Cx36 does not alter islet size or insulin content, whereas the Cx43 isoform increases both parameters, and Cx32 has a similar effect only when combined with GH. These findings open interesting perspectives for the in vitro and in vivo regulation of the beta-cell mass.

Connexin36 contributes to INS-1E cells survival through modulation of cytokine-induced oxidative stress, ER stress and AMPK activity

Cell-to-cell communication mediated by gap junctions made of Connexin36 (Cx36) contributes to pancreatic β-cell function. We have recently demonstrated that Cx36 also supports β-cell survival by a still unclear mechanism. Using specific Cx36 siRNAs or adenoviral vectors, we now show that Cx36 downregulation promotes apoptosis in INS-1E cells exposed to the pro-inflammatory cytokines (IL-1β, TNF-α and IFN-γ) involved at the onset of type 1 diabetes, whereas Cx36 overexpression protects against this effect. Cx36 overexpression also protects INS-1E cells against endoplasmic reticulum (ER) stress-mediated apoptosis, and alleviates the cytokine-induced production of reactive oxygen species, the depletion of the ER Ca2+ stores, the CHOP overexpression and the degradation of the anti-apoptotic protein Bcl-2 and Mcl-1. We further show that cytokines activate the AMP-dependent protein kinase (AMPK) in a NO-dependent and ER-stress-dependent manner and that AMPK inhibits Cx36 expression. Altogether, the data suggest that Cx36 is involved in Ca2+ homeostasis within the ER and that Cx36 expression is downregulated following ER stress and subsequent AMPK activation. As a result, cytokine-induced Cx36 downregulation elicits a positive feedback loop that amplifies ER stress and AMPK activation, leading to further Cx36 downregulation. The data reveal that Cx36 plays a central role in the oxidative stress and ER stress induced by cytokines and the subsequent regulation of AMPK activity, which in turn controls Cx36 expression and mitochondria-dependent apoptosis of insulin-producing cells.

Early metabolic defects in dexamethasone-exposed and undernourished intrauterine growth restricted rats.

Poor fetal growth, also known as intrauterine growth restriction (IUGR), is a worldwide health concern. IUGR is commonly associated with both an increased risk in perinatal mortality and a higher prevalence of developing chronic metabolic diseases later in life. Obesity, type 2 diabetes or metabolic syndrome could result from noxious “metabolic programming.” In order to better understand early alterations involved in metabolic programming, we modeled IUGR rat pups through either prenatal exposure to synthetic glucocorticoid (dams infused with dexamethasone 100 µg/kg/day, DEX) or prenatal undernutrition (dams feeding restricted to 30% of ad libitum intake, UN). Physiological (glucose and insulin tolerance), morphometric (automated tissue image analysis) and transcriptomic (quantitative PCR) approaches were combined during early life of these IUGR pups with a special focus on their endocrine pancreas and adipose tissue development. In the absence of catch-up growth before weaning, DEX and UN IUGR pups both presented basal hyperglycaemia, decreased glucose tolerance, and pancreatic islet atrophy. Other early metabolic defects were model-specific: DEX pups presented decreased insulin sensitivity whereas UN pups exhibited lowered glucose-induced insulin secretion and more marked alterations in gene expression of pancreatic islet and adipose tissue development regulators. In conclusion, these results show that before any catch-up growth, IUGR rats present early physiologic, morphologic and transcriptomic defects, which can be considered as initial mechanistic basis of metabolic programming.

Connexin Modulators of Endocrine Function

The emergence of multicellular organisms has necessitated the specialization of short- and long-range chemical signaling systems, including that provided by the endocrine system. Conversely, the existence of an endocrine system conceptually demands a multicellular organism, to which proper signaling usually also imposes a multicellular gland. Accordingly, the secretory cells of all endocrine glands have developed mechanisms for interacting with adjacent and distant cells. With evolution, such mechanisms have diversified and have been progressively integrated in a complex regulatory network, whereby individual endocrine cells sense the state of activity of their neighbors and regulate accordingly their own level of functioning. A consistent feature of this network is the expression of connexin-made channels between the hormone-producing cells of all glands so far investigated in vertebrates. In a few instances, these channels have also been documented between the endocrine cells and nearby target cells. Here, we have reviewed the distribution of connexins in the mammalian endocrine system, and have discussed the recent evidence pointing to the participation of these proteins in the functioning of endocrine cells, and on the action of hormones on specific target cells.

Gap Junctions and Insulin Secretion

About 30 years ago, pancreatic beta cells were shown to be connected by gap junctions and to exhibit glucose-induced oscillatory electrical activity, two features that were hypothetically linked to insulin secretion. Since then, gap junctions have been shown to be an obligatory feature of beta cells in all species and all physiological conditions studied. They are composed of connexin proteins and allow for beta-cell to beta-cell exchanges of current-carrying ions and other small cytosolic metabolites which synchronize the electrical and metabolic activity of beta cells, and recruit these cells for insulin biosynthesis and release. Together, these effects account for the significant contribution of gap junction-dependent signaling to the control of insulin secretion. More recent data suggest that gap junctions, either via the expression of connexin proteins and/or of the intercellular communications that these proteins permit, also significantly influence beta-cell growth, apoptosis, and the resistance of islets to immune attack. The mechanism(s) whereby gap junction signaling exerts these multiple effects is still obscure. Understanding this mechanism is relevant both to our understanding of the physiology of pancreatic islets and to the pathophysiology of beta-cell dysfunction in both type 1 and type 2 diabetes. Furthermore, appropriate expression of gap junctions may be a prerequisite for the engineering of surrogate insulin-producing cells and their proper three-dimensional packaging, which may be important for using these cells as a cell-based therapy for the treatment of diabetic patients. Here, we review the current status of our knowledge in this field and its exciting perspectives.