Leslie S. Satin

SphK1 and SphK2, sphingosine kinase isoenzymes with opposing functions in sphingolipid metabolism

The potent sphingolipid metabolite sphingosine 1-phosphate is produced by phosphorylation of sphingosine catalyzed by sphingosine kinase (SphK) types 1 and 2. In contrast to pro-survival SphK1, the putative BH3-only protein SphK2 inhibits cell growth and enhances apoptosis. Here we show that SphK2 catalytic activity also contributes to its ability to induce apoptosis. Overexpressed SphK2 also increased cytosolic free calcium induced by serum starvation. Transfer of calcium to mitochondria was required for SphK2-induced apoptosis, as cell death and cytochrome c release was abrogated by inhibition of the mitochondrial Ca2+ transporter. Serum starvation increased the proportion of SphK2 in the endoplasmic reticulum and targeting SphK1 to the endoplasmic reticulum converted it from anti-apoptotic to pro-apoptotic. Overexpression of SphK2 increased incorporation of [3H]palmitate, a substrate for both serine palmitoyltransferase and ceramide synthase, into C16-ceramide, whereas SphK1 decreased it. Electrospray ionizationmass spectrometry/mass spectrometry also revealed an opposite effect on ceramide mass levels. Importantly, specific down-regulation of SphK2 reduced conversion of sphingosine to ceramide in the recycling pathway and conversely, down-regulation of SphK1 increased it. Our results demonstrate that SphK1 and SphK2 have opposing roles in the regulation of ceramide biosynthesis and suggest that the location of sphingosine 1-phosphate production dictates its functions.

Total insulin and IGF-I resistance in pancreatic β cells causes overt diabetes

An appropriate β cell mass is pivotal for the maintenance of glucose homeostasis. Both insulin and IGF-1 are important in regulation of β cell growth and function (reviewed in ref. 2). To define the roles of these hormones directly, we created a mouse model lacking functional receptors for both insulin and IGF-1 only in β cells (βDKO), as the hormones have overlapping mechanisms of action and activate common downstream proteins. Notably, βDKO mice were born with a normal complement of islet cells, but 3 weeks after birth, they developed diabetes, in contrast to mild phenotypes observed in single mutants. Normoglycemic 2-week-old βDKO mice manifest reduced β cell mass, reduced expression of phosphorylated Akt and the transcription factor MafA, increased apoptosis in islets and severely compromised β cell function. Analyses of compound knockouts showed a dominant role for insulin signaling in regulating β cell mass. Together, these data provide compelling genetic evidence that insulin and IGF-I–dependent pathways are not critical for development of β cells but that a loss of action of these hormones in β cells leads to diabetes. We propose that therapeutic improvement of insulin and IGF-I signaling in β cells might protect against type 2 diabetes.

Reduction of Voltage-Dependent Mg2+ Blockade of NMDA Current in Mechanically Injured Neurons

Activation of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptors is implicated in the pathophysiology of traumatic brain injury. Here, the effects of mechanical injury on the voltage-dependent magnesium (Mg2+) block of NMDA currents in cultured rat cortical neurons were examined. Stretch-induced injury was found to reduce the Mg2+ blockade, resulting in significantly larger ionic currents and increases in intracellular free calcium (Ca2+) concentration after NMDA stimulation of injured neurons. The Mg2+ blockade was partially restored by increased extracellular Mg2+ concentration or by pretreatment with the protein kinase C inhibitor calphostin C. These findings could account for the secondary pathological changes associated with traumatic brain injury.

A Homozygous Mutation in a Novel Zinc-Finger Protein, ERIS, Is Responsible for Wolfram Syndrome 2

A single missense mutation was identified in a novel, highly conserved zinc-finger gene, ZCD2, in three consanguineous families of Jordanian descent with Wolfram syndrome (WFS). It had been shown that these families did not have mutations in the WFS1 gene (WFS1) but were mapped to the WFS2 locus at 4q22-25. A G-->C transversion at nucleotide 109 predicts an amino acid change from glutamic acid to glutamine (E37Q). Although the amino acid is conserved and the mutation is nonsynonymous, the pathogenesis for the disorder is because the mutation also causes aberrant splicing. The mutation was found to disrupt messenger RNA splicing by eliminating exon 2, and it results in the introduction of a premature stop codon. Mutations in WFS1 have also been found to cause low-frequency nonsyndromic hearing loss, progressive hearing loss, and isolated optic atrophy associated with hearing loss. Screening of 377 probands with hearing loss did not identify mutations in the WFS2 gene. The WFS1-encoded protein, Wolframin, is known to localize to the endoplasmic reticulum and plays a role in calcium homeostasis. The ZCD2-encoded protein, ERIS (endoplasmic reticulum intermembrane small protein), is also shown to localize to the endoplasmic reticulum but does not interact directly with Wolframin. Lymphoblastoid cells from affected individuals show a significantly greater rise in intracellular calcium when stimulated with thapsigargin, compared with controls, although no difference was observed in resting concentrations of intracellular calcium.

The Ca2+ Dynamics of Isolated Mouse β-Cells and Islets: Implications for Mathematical Models

[Ca(2+)](i) and electrical activity were compared in isolated beta-cells and islets using standard techniques. In islets, raising glucose caused a decrease in [Ca(2+)](i) followed by a plateau and then fast (2-3 min(-1)), slow (0.2-0.8 min(-1)), or a mixture of fast and slow [Ca(2+)](i) oscillations. In beta-cells, glucose transiently decreased and then increased [Ca(2+)](i), but no islet-like oscillations occurred. Simultaneous recordings of [Ca(2+)](i) and electrical activity suggested that differences in [Ca(2+)](i) signaling are due to differences in islet versus beta-cell electrical activity. Whereas islets exhibited bursts of spikes on medium/slow plateaus, isolated beta-cells were depolarized and exhibited spiking, fast-bursting, or spikeless plateaus. These electrical patterns in turn produced distinct [Ca(2+)](i) patterns. Thus, although isolated beta-cells display several key features of islets, their oscillations were faster and more irregular. beta-cells could display islet-like [Ca(2+)](i) oscillations if their electrical activity was converted to a slower islet-like pattern using dynamic clamp. Islet and beta-cell [Ca(2+)](i) changes followed membrane potential, suggesting that electrical activity is mainly responsible for the [Ca(2+)] dynamics of beta-cells and islets. A recent model consisting of two slow feedback processes and passive endoplasmic reticulum Ca(2+) release was able to account for islet [Ca(2+)](i) responses to glucose, islet oscillations, and conversion of single cell to islet-like [Ca(2+)](i) oscillations. With minimal parameter variation, the model could also account for the diverse behaviors of isolated beta-cells, suggesting that these behaviors reflect natural cell heterogeneity. These results support our recent model and point to the important role of beta-cell electrical events in controlling [Ca(2+)](i) over diverse time scales in islets.

Neurotransmitters and their receptors in the islets of Langerhans of the pancreas: What messages do acetylcholine, glutamate, and gaba transmit?

Although neurotransmitters are present in pancreatic islets of Langerhans and can be shown to alter hormone secretion, their precise physiological roles in islet function and their cellular mechanisms of action are unclear. Recent research has identified specific neurotransmitter receptor isoforms in islets that may be important physiologically, because selective receptor agonists activate islet ion channels, modify intracellular [Ca2+], and affect secretion. This article focuses on the putative roles of acetylcholine, glutamate, and GABA in islet function. It has been hypothesized that acetylcholine potentiates insulin secretion by either promoting Ca release from cellular stores, activating a store depletion-activated channel, or activating a novel Na channel. GABA and glutamate, in contrast, have been proposed to mediate a novel paracrine signaling pathway whereby α- and β-cells communicate within the islet. The evidence supporting these hypotheses will be critically evaluated.

Gap junction coupling and calcium waves in the pancreatic islet.

The pancreatic islet is a highly coupled, multicellular system that exhibits complex spatiotemporal electrical activity in response to elevated glucose levels. The emergent properties of islets, which differ from those arising in isolated islet cells, are believed to arise in part by gap junctional coupling, but the mechanisms through which this coupling occurs are poorly understood. To uncover these mechanisms, we have used both high-speed imaging and theoretical modeling of the electrical activity in pancreatic islets under a reduction in the gap junction mediated electrical coupling. Utilizing islets from a gap junction protein connexin 36 knockout mouse model together with chemical inhibitors, we can modulate the electrical coupling in the islet in a precise manner and quantify this modulation by electrophysiology measurements. We find that after a reduction in electrical coupling, calcium waves are slowed as well as disrupted, and the number of cells showing synchronous calcium oscillations is reduced. This behavior can be reproduced by computational modeling of a heterogeneous population of beta-cells with heterogeneous levels of electrical coupling. The resulting quantitative agreement between the data and analytical models of islet connectivity, using only a single free parameter, reveals the mechanistic underpinnings of the multicellular behavior of the islet.

Modulation of the Bursting Properties of Single Mouse Pancreatic β-Cells by Artificial Conductances

Glucose triggers bursting activity in pancreatic islets, which mediates the Ca2+ uptake that triggers insulin secretion. Aside from the channel mechanism responsible for bursting, which remains unsettled, it is not clear whether bursting is an endogenous property of individual beta-cells or requires an electrically coupled islet. While many workers report stochastic firing or quasibursting in single cells, a few reports describe single-cell bursts much longer (minutes) than those of islets (15-60 s). We studied the behavior of single cells systematically to help resolve this issue. Perforated patch recordings were made from single mouse beta-cells or hamster insulinoma tumor cells in current clamp at 30-35 degrees C, using standard K+-rich pipette solution and external solutions containing 11.1 mM glucose. Dynamic clamp was used to apply artificial KATP and Ca2+ channel conductances to cells in current clamp to assess the role of Ca2+ and KATP channels in single cell firing. The electrical activity we observed in mouse beta-cells was heterogeneous, with three basic patterns encountered: 1) repetitive fast spiking; 2) fast spikes superimposed on brief (<5 s) plateaus; or 3) periodic plateaus of longer duration (10-20 s) with small spikes. Pattern 2 was most similar to islet bursting but was significantly faster. Burst plateaus lasting on the order of minutes were only observed when recordings were made from cell clusters. Adding gCa to cells increased the depolarizing drive of bursting and lengthened the plateaus, whereas adding gKATP hyperpolarized the cells and lengthened the silent phases. Adding gCa and gKATP together did not cancel out their individual effects but could induce robust bursts that resembled those of islets, and with increased period. These added currents had no slow components, indicating that the mechanisms of physiological bursting are likely to be endogenous to single beta-cells. It is unlikely that the fast bursting (class 2) was due to oscillations in gKATP because it persisted in 100 microM tolbutamide. The ability of small exogenous currents to modify beta-cell firing patterns supports the hypothesis that single cells contain the necessary mechanisms for bursting but often fail to exhibit this behavior because of heterogeneity of cell parameters.

An ATP-Sensitive Cl − Channel Current That Is Activated by Cell Swelling, cAMP, and Glyburide in Insulin-Secreting Cells

Although chloride ions are known to modulate insulin release and islet electrical activity, themechanism or mechanisms mediating these effects are unclear. However, numerous studies of islet Cl− fluxes have suggested that Cl− movements and glucose and sulfonylurea sensitive and are blocked by stilbene-derivative Cl− channel blockers. We now show for the first time that insulin-secreting cells have a Cl− channel current, which we term ICl,islet· The current is activated by hypotonic conditions, 1–10 μmol/l glyburide and 0.5 mmol/l 8-bromoadenosine 3′:5′-cyclic monophosphate sodium. ICl,islet is mediated by Cl− channels, since replacing [Cl−]o with less permeant aspartate reduces current amplitude and depolarizes its reversal potential. In addition, 100 μmol/l 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) or glyburide, which blocks the Cl− channels of other cell types, block ICl,islet· Reducing [ATP]i reduces the amplitude of the current, suggesting thatit may be under metabolic control. The current is time-independent and shows strong outward-rectification beyond ∼0 mV. At potentials associated with the silent phase of islet electrical activity (approximately −65 mV), ICl,islet mediates a large inward current, which would be expected to depolarize islet membrane potential. Thus, activation of this novel current byincreased intracellular cAMP, sulfonylureas, or ATP may contribute to the well-known depolarizing effects of these agents.

Connexin-36 Gap Junctions Regulate In Vivo First- and Second-Phase Insulin Secretion Dynamics and Glucose Tolerance in the Conscious Mouse

Insulin is secreted from the islets of Langerhans in coordinated pulses. These pulses are thought to lead to plasma insulin oscillations, which are putatively more effective in lowering blood glucose than continuous levels of insulin. Gap-junction coupling of β-cells by connexin-36 coordinates intracellular free calcium oscillations and pulsatile insulin release in isolated islets, however a role in vivo has not been shown. We test whether loss of gap-junction coupling disrupts plasma insulin oscillations and whether this impacts glucose tolerance. We characterized the connexin-36 knockout (Cx36−/−) mouse phenotype and performed hyperglycemic clamps with rapid sampling of insulin in Cx36−/− and control mice. Our results show that Cx36−/− mice are glucose intolerant, despite normal plasma insulin levels and insulin sensitivity. However, Cx36−/− mice exhibit reduced insulin pulse amplitudes and a reduction in first-phase insulin secretion. These changes are similarly found in isolated Cx36−/− islets. We conclude that Cx36 gap junctions regulate the in vivo dynamics of insulin secretion, which in turn is important for glucose homeostasis. Coordinated pulsatility of individual islets enhances the first-phase elevation and second-phase pulses of insulin. Because these dynamics are disrupted in the early stages of type 2 diabetes, dysregulation of gap-junction coupling could be an important factor in the development of this disease.