Lydia Aguilar-Bryan

Neonatal Diabetes Mellitus

An explosion of work over the last decade has produced insight into the multiple hereditary causes of a nonimmunological form of diabetes diagnosed most frequently within the first 6 months of life. These studies are providing increased understanding of genes involved in the entire chain of steps that control glucose homeostasis. Neonatal diabetes is now understood to arise from mutations in genes that play critical roles in the development of the pancreas, of beta-cell apoptosis and insulin processing, as well as the regulation of insulin release. For the basic researcher, this work is providing novel tools to explore fundamental molecular and cellular processes. For the clinician, these studies underscore the need to identify the genetic cause underlying each case. It is increasingly clear that the prognosis, therapeutic approach, and genetic counseling a physician provides must be tailored to a specific gene in order to provide the best medical care.

Channel regulation of glucose sensing in the pancreatic β-cell

Mammalian beta-cells are acutely and chronically regulated by sensing surrounding glucose levels that determine the rate at which insulin is secreted, to maintain euglycemia. Experimental research in vitro and in vivo has shown that, when these cells are exposed to adverse conditions like long periods of hypoglycemia or hyperglycemia, their capability to sense glucose is decreased. Understanding the normal physiology and identifying the main players along this route becomes paramount. In this review, we have taken on the task of looking at the role that ion channels play in the regulation of this process, delineating the different families, and describing the signaling that parallels the glucose sensing process that results in insulin release.

Suppression of KATP channel activity protects murine pancreatic β cells against oxidative stress

The enhanced oxidative stress associated with type 2 diabetes mellitus contributes to disease pathogenesis. We previously identified plasma membrane-associated ATP-sensitive K+ (KATP) channels of pancreatic beta cells as targets for oxidants. Here, we examined the effects of genetic and pharmacologic ablation of KATP channels on loss of mouse beta cell function and viability following oxidative stress. Using mice lacking the sulfonylurea receptor type 1 (Sur1) subunit of KATP channels, we found that, compared with insulin secretion by WT islets, insulin secretion by Sur1-/- islets was less susceptible to oxidative stress induced by the oxidant H2O2. This was likely, at least in part, a result of the reduced ability of H2O2 to hyperpolarize plasma membrane potential and reduce cytosolic free Ca2+ concentration ([Ca2+]c) in the Sur1-/- beta cells. Remarkably, Sur1-/- beta cells were less prone to apoptosis induced by H2O2 or an NO donor than WT beta cells, despite an enhanced basal rate of apoptosis. This protective effect was attributed to upregulation of the antioxidant enzymes SOD, glutathione peroxidase, and catalase. Upregulation of antioxidant enzymes and reduced sensitivity of Sur1-/- cells to H2O2-induced apoptosis were mimicked by treatment with the sulfonylureas tolbutamide and gliclazide. Enzyme upregulation and protection against oxidant-induced apoptosis were abrogated by agents lowering [Ca2+]c. Sur1-/- mice were less susceptible than WT mice to streptozotocin-induced beta cell destruction and subsequent hyperglycemia and death, which suggests that loss of KATP channel activity may protect against streptozotocin-induced diabetes in vivo.

Hypothalamic leucine metabolism regulates liver glucose production.

Amino acids profoundly affect insulin action and glucose metabolism in mammals. Here, we investigated the role of the mediobasal hypothalamus (MBH), a key center involved in nutrient-dependent metabolic regulation. Specifically, we tested the novel hypothesis that the metabolism of leucine within the MBH couples the central sensing of leucine with the control of glucose production by the liver. We performed either central (MBH) or systemic infusions of leucine in Sprague-Dawley male rats during basal pancreatic insulin clamps in combination with various pharmacological and molecular interventions designed to modulate leucine metabolism in the MBH. We also examined the role of hypothalamic ATP-sensitive K+ channels (KATP channels) in the effects of leucine. Enhancing the metabolism of leucine acutely in the MBH lowered blood glucose through a biochemical network that was insensitive to rapamycin but strictly dependent on the hypothalamic metabolism of leucine to α-ketoisocaproic acid and, further, insensitive to acetyl- and malonyl-CoA. Functional KATP channels were also required. Importantly, molecular attenuation of this central sensing mechanism in rats conferred susceptibility to developing hyperglycemia. We postulate that the metabolic sensing of leucine in the MBH is a previously unrecognized mechanism for the regulation of hepatic glucose production required to maintain glucose homeostasis.

Conserved intramolecular disulfide bond is critical to trafficking and fate of ATP-binding cassette (ABC) transporters ABCB6 and sulfonylurea receptor 1 (SUR1)/ABCC8.

The ATP-binding cassette (ABC) transporter ABCB6 is a mitochondrial porphyrin transporter that activates porphyrin biosynthesis. ABCB6 lacks a canonical mitochondrial targeting sequence but reportedly traffics to other cellular compartments such as the plasma membrane. How ABCB6 reaches these destinations is unknown. In this study, we show that endogenous ABCB6 is glycosylated in multiple cell types, indicating trafficking through the endoplasmic reticulum (ER), and has only one atypical site for glycosylation (NXC) in its amino terminus. ABCB6 remained glycosylated when the highly conserved cysteine (Cys-8) was substituted with serine to make a consensus site, NXS. However, this substitution blocked ER exit and produced ABCB6 degradation, which was mostly reversed by the proteasomal inhibitor MG132. The amino terminus of ABCB6 has an additional highly conserved ER luminal cysteine (Cys-26). When Cys-26 was mutated alone or in combination with Cys-8, it also resulted in instability and ER retention. Further analysis revealed that these two cysteines form a disulfide bond. We discovered that other ABC transporters with an amino terminus in the ER had similarly configured conserved cysteines. This analysis led to the discovery of a disease-causing mutation in the sulfonylurea receptor 1 (SUR1)/ABCC8 from a patient with hyperinsulinemic hypoglycemia. The mutant allele only contains a mutation in a conserved amino-terminal cysteine, producing SUR1 that fails to reach the cell surface. These results suggest that for ABC transporters the propensity to form a disulfide bond in the ER defines a unique checkpoint that determines whether a protein is ER-retained.

Activation of the Na+/K+-ATPase by insulin and glucose as a putative negative feedback mechanism in pancreatic beta-cells

Pancreatic beta-cells of sulfonylurea receptor type 1 knock-out (SUR1−/−) mice exhibit an oscillating membrane potential (Vm) demonstrating that hyper-polarisation occurs despite the lack of KATP channels. We hypothesize that glucose activates the Na+/K+-ATPase thus increasing a hyper-polarising current. Elevating glucose in SUR1−/− beta-cells resulted in a transient fall in Vm and [Ca2+]c independent of sarcoplasmic and endoplasmic reticulum Ca2+-activated ATPase (SERCA) activation. This was not affected by K+ channel blockade but inhibited by ATP depletion and by ouabain. Increasing glucose also reduced [Na+]c, an effect reversed by ouabain. Exogenously applied insulin decreased [Na+]c and hyper-polarised Vm. Inhibiting insulin signalling in SUR1−/− beta-cells blunted the glucose-induced decrease of [Ca2+]c. Tolbutamide (1xa0mmol/l) disclosed the SERCA-independent effect of glucose on [Ca2+]c in wild-type beta-cells. The data show that in SUR1−/− beta-cells, glucose activates the Na+/K+-ATPase presumably by increasing [ATP]c. Insulin can also stimulate the pump and potentiate the effect of glucose. Pathways involving the pump may thus serve as potential drug targets in certain metabolic disorders.

The neuronal K + Cl − co-transporter 2 ( Slc12a5 ) modulates insulin secretion

Intracellular chloride concentration ([Cl−]i) in pancreatic β-cells is kept above electrochemical equilibrium due to the predominant functional presence of Cl− loaders such as the Na+K+2Cl− co-transporter 1 (Slc12a2) over Cl−extruders of unidentified nature. Using molecular cloning, RT-PCR, Western blotting, immunolocalization and in vitro functional assays, we establish that the “neuron-specific” K+Cl− co-transporter 2 (KCC2, Slc12a5) is expressed in several endocrine cells of the pancreatic islet, including glucagon secreting α-cells, but particularly in insulin-secreting β-cells, where we provide evidence for its role in the insulin secretory response. Three KCC2 splice variants were identified: the formerly described KCC2a and KCC2b along with a novel one lacking exon 25 (KCC2a-S25). This new variant is undetectable in brain or spinal cord, the only and most abundant known sources of KCC2. Inhibition of KCC2 activity in clonal MIN6 β-cells increases basal and glucose-stimulated insulin secretion and Ca2+ uptake in the presence of glibenclamide, an inhibitor of the ATP-dependent potassium (KATP)-channels, thus suggesting a possible mechanism underlying KCC2-dependent insulin release. We propose that the long-time considered “neuron-specific” KCC2 co-transporter is expressed in pancreatic islet β-cells where it modulates Ca2+-dependent insulin secretion.

Chloride channels and transporters in β-cell physiology

The ability of b-cells to depolarize, regulate [Ca]i, and secrete insulin even in the absence of functional KATP channels strongly suggests the presence of additional ionic cascades of events within the stimulus-secretion coupling. The purpose of this review is to introduce the reader to the role of the long-relegated and largely ignored subject of intracellular Cl concentration ([Cl ]i). The regulation of [Cl ]i by transporters and channels, and their potential involvement in glucose-induced insulin secretion, is also discussed. It is important to keep in mind that, in the last decade, the molecular identification and functional characterization of many diverse regulators of [Cl ]i in b-cells have added to the extraordinary complexity of the b-cell secretory response. We have therefore concentrated on key concepts, and on what we consider may be the most important players involved in the regulation of [Cl ]i in b-cells, but time will tell.

Structural abnormalities in islets from very young children with cystic fibrosis may contribute to cystic fibrosis-related diabetes

Cystic fibrosis (CF)-related diabetes (CFRD) is thought to result from beta-cell injury due in part to pancreas exocrine damage and lipofibrosis. CFRD pancreata exhibit reduced islet density and altered cellular composition. To investigate a possible etiology, we tested the hypothesis that such changes are present in CF pancreata before the development of lipofibrosis. We evaluated pancreas and islet morphology in tissues from very young CF children (<4 years of age), and adult patients with CF and CFRD. The relative number of beta-cells in young CF tissues was reduced by 50% or more when compared to age-matched controls. Furthermore, young CF tissues displayed significantly smaller insulin-positive areas, lower proportion of beta-cells positive for the proliferation marker Ki67 or the ductal marker CK19 vs. control subjects, and islet inflammatory cell infiltrates, independently of the severity of the exocrine lesion and in the absence of amyloid deposits. CFRD pancreata exhibited greater islet injury with further reduction in islet density, decreased relative beta-cell number, and presence of amyloid deposits. Together, these results strongly suggest that an early deficiency in beta-cell number in infants with CF may contribute to the development of glucose intolerance in the CF pediatric population, and to CFRD, later in life.