TRP Genes: Candidates for Nonselective Cation Channels and Store-operated Channels in Insulin-secreting Cells

Diabetes. Feb, 2002  |  Pubmed ID: 11815479

Nonselective cation channels may play a role in insulin secretion by regulating pancreatic beta-cell plasma membrane potential, Ca(2+) homeostasis, and thereby glucose signaling. Transient receptor potential channel (TRPC)-related genes encode nonselective cation channels, some of which are similar to those described for beta-cells. Some TRPC-like channels are activated via G-protein--coupled mechanisms, some have been reported to be calcium-store-operated channels (SOC), and others are activated by novel signaling molecules or are sensitive to pressure and osmotic strength. Here we report the cloning and expression of mSTRPC4 from a mouse insulinoma cDNA library. mSTRPC4 encoded a protein of 97 kd, expressed in both endocrine cells and the brain. Stable cell lines expressing mSTRPC4 showed abundant mSTRPC4 protein, but no reproducible currents could be detected. mSTRPC4 therefore probably functions as a heteromultimer. We also report that LTRPC2, a G-protein and adenosine 5'-diphosphoribose (ADPR)-activated nonselective cation channel, is also expressed in human islets. TRPC-like channels may provide a pathway for depolarization or Ca(2+) entry in beta-cells and may be interesting targets for manipulating beta-cell function.

Insulin Secretory Deficiency and Glucose Intolerance in Rab3A Null Mice

The Journal of Biological Chemistry. Mar, 2003  |  Pubmed ID: 12510060

Insulin secretory dysfunction of the pancreatic beta-cell in type-2 diabetes is thought to be due to defective nutrient sensing and/or deficiencies in the mechanism of insulin exocytosis. Previous studies have indicated that the GTP-binding protein, Rab3A, plays a mechanistic role in insulin exocytosis. Here, we report that Rab3A(-/-) mice develop fasting hyperglycemia and upon a glucose challenge show significant glucose intolerance coupled to ablated first-phase insulin release and consequential insufficient insulin secretion in vivo, without insulin resistance. The in vivo insulin secretory response to arginine was similar in Rab3A(-/-) mice as Rab3A(+/+) control animals, indicating a phenotype reminiscent of insulin secretory dysfunction found in type-2 diabetes. However, when a second arginine dose was given 10 min after, there was a negligible insulin secretory response in Rab3A(-/-) mice, compared with that in Rab3A(+/+) animals, that was markedly increased above that to the first arginine stimulus. There was no difference in beta-cell mass or insulin production between Rab3A(-/-) and Rab3A(+/+) mice. However, in isolated islets, secretagogue-induced insulin release (by glucose, GLP-1, glyburide, or fatty acid) was approximately 60-70% lower in Rab3A(-/-) islets compared with Rab3A(+/+) controls. Nonetheless, there was a similar rate of glucose oxidation and glucose-induced rise in cytosolic [Ca(2+)](i) flux between Rab3A(-/-) and Rab3A(+/+) islet beta-cells, indicating the mechanistic role of Rab3A lies downstream of generating secondary signals that trigger insulin release, at the level of secretory granule transport and/or exocytosis. Thus, Rab3A plays an important in vivo role facilitating the efficiency of insulin exocytosis, most likely at the level of replenishing the ready releasable pool of beta-granules. Also, this study indicates, for the first time, that the in vivo insulin secretory dysfunction found in type-2 diabetes can lie solely at the level of defective insulin exocytosis.

Visualizing Superoxide Production in Normal and Diabetic Rat Islets of Langerhans

The Journal of Biological Chemistry. Mar, 2003  |  Pubmed ID: 12514170

Oxygen free radicals have been implicated in beta-cell dysfunction and apoptosis associated with type 1 and type 2 diabetes mellitus. The roles of free radicals in diabetes have thus far been defined indirectly by monitoring oxidative tissue damage and the effects of antioxidants, free radical scavengers, and overexpression of superoxide dismutase. We employed the superoxide-mediated oxidation of hydroethidine to ethidium to dynamically and directly assess the relative rates of mitochondrial superoxide anion generation in isolated islets in response to glucose stimulation. Superoxide content of isolated islets increased in response to glucose stimulation. We next compared the oxyradical levels in Zucker lean control and Zucker diabetic fatty rat islets by digital imaging microfluorometry. The superoxide content of Zucker diabetic fatty islets was significantly higher than Zucker lean control islets under resting conditions, relatively insensitive to elevated glucose concentrations, and correlated temporally with a decrease in glucose-induced hyperpolarization of the mitochondrial membrane. Importantly, superoxide levels were elevated in islets from young, pre-diabetic Zucker diabetic fatty animals. Overproduction of superoxide was associated with perturbed mitochondrial morphology and may contribute to abnormal glucose signaling found in the Zucker diabetic fatty model of type 2 diabetes mellitus.

Modeling of Ca2+ Flux in Pancreatic Beta-cells: Role of the Plasma Membrane and Intracellular Stores

American Journal of Physiology. Endocrinology and Metabolism. Jul, 2003  |  Pubmed ID: 12644446

We have developed a detailed mathematical model of ionic flux in beta-cells that includes the most essential channels and pumps in the plasma membrane. This model is coupled to equations describing Ca2+, inositol 1,4,5-trisphosphate (IP3), ATP, and Na+ homeostasis, including the uptake and release of Ca2+ by the endoplasmic reticulum (ER). In our model, metabolically derived ATP activates inward Ca2+ flux by regulation of ATP-sensitive K+ channels and depolarization of the plasma membrane. Results from the simulations support the hypothesis that intracellular Na+ and Ca2+ in the ER can be the main variables driving both fast (2-7 osc/min) and slow intracellular Ca2+ concentration oscillations (0.3-0.9 osc/min) and that the effect of IP3 on Ca2+ leak from the ER contributes to the pattern of slow calcium oscillations. Simulations also show that filling the ER Ca2+ stores leads to faster electrical bursting and Ca2+ oscillations. Specific Ca2+ oscillations in isolated beta-cell lines can also be simulated.

Small-conductance Calcium-activated K+ Channels Are Expressed in Pancreatic Islets and Regulate Glucose Responses

Diabetes. Aug, 2003  |  Pubmed ID: 12882916

Glucose-stimulated insulin secretion is associated with transients of intracellular Ca(2+) concentration [Ca(2+)](i) in the pancreatic beta-cell. We identified the expression and function of specific small-conductance Ca(2+)-activated K(+) (SK) channel genes in insulin-secreting cells. The presence of mRNA for SK1, -2, -3, and -4 (intermediate-conductance Ca(2+)-activated K(+) 1 [IK1]) channels was demonstrated by RT-PCR in rodent islets and insulinoma cells. SK2 and -3 proteins in mouse islets were detected by immunoblot and immunocytochemistry. In the tTA-SK3 tet-off mouse, a normal amount of SK3 protein was present in islets, but it became undetectable after exposure to doxycycline (DOX), which inhibits the transcription of the tTA-SK3 gene. The SK/IK channel-blockers apamin, dequalinium, and charybdotoxin caused increases in average [Ca(2+)](i) levels and in frequency of [Ca(2+)](i) oscillations in wild-type mouse islets. In SK3-tTA tet-off mice, the addition of apamin with glucose and tetraethylammonium (TEA) caused a similar elevation in [Ca(2+)](i), which was greatly diminished after DOX suppression of SK3 expression. We conclude that SK1, -2, -3, and IK1 (SK4) are expressed in islet cells and insulin-secreting cells and are able to influence glucose-induced calcium responses, thereby regulating insulin secretion.

Direct Imaging Shows That Insulin Granule Exocytosis Occurs by Complete Vesicle Fusion

Proceedings of the National Academy of Sciences of the United States of America. Jun, 2004  |  Pubmed ID: 15197259

Confocal imaging of GFP-tagged secretory granules combined with the use of impermeant extracellular dyes permits direct observation of insulin packaged in secretory granules, trafficking of these granules to the plasma membrane, exocytotic fusion of granules with the plasma membrane, and eventually the retrieval of membranes by endocytosis. Most such studies have been done in tumor cell lines, using either confocal methods or total internal reflectance microscopy. Here we compared these methods by using GFP-syncollin or PC3-GFP plus rhodamine dextrans to study insulin granule dynamics in insulinoma cells, normal mouse islets, and primary pancreatic beta cells. We found that most apparently docked granules did not fuse with the plasma membrane after stimulation. Granules that did fuse typically fused completely, but a few dextran-filled granules lingered at the membrane. Direct recycling of granules occurred only rarely. Similar results were obtained with both confocal and total internal reflection microscopy, although each technique had advantages for particular aspects of the granule life cycle. We conclude that insulin exocytosis involves a prolonged interaction of secretory granules with the plasma membrane, and that the majority of exocytotic events occur by full, not partial, fusion.

Does the Glucose-dependent Insulin Secretion Mechanism Itself Cause Oxidative Stress in Pancreatic Beta-cells?

Diabetes. Aug, 2004  |  Pubmed ID: 15277370

Glucose-dependent insulin secretion (GDIS), reactive oxygen species (ROS) production, and oxidative stress in pancreatic beta-cells may be tightly linked processes. Here we suggest that the same pathways used in the activation of GDIS (increased glycolytic flux, ATP-to-ADP ratio, and intracellular Ca2+ concentration) can dramatically enhance ROS production and manifestations of oxidative stress and, possibly, apoptosis. The increase in ROS production and oxidative stress produced by GDIS activation itself suggests a dual role for metabolic insulin secretagogues, as an initial sharp increase in insulin secretion rate can be accompanied by progressive beta-cell injury. We propose that therapeutic strategies targeting enhancement of GDIS should be carefully considered in light of possible loss of beta-cell function and mass.

FRET-based Voltage Probes for Confocal Imaging: Membrane Potential Oscillations Throughout Pancreatic Islets

American Journal of Physiology. Cell Physiology. Jul, 2005  |  Pubmed ID: 15758044

Insulin secretion is dependent on coordinated pancreatic islet physiology. In the present study, we found a way to overcome the limitations of cellular electrophysiology to optically determine cell membrane potential (V(m)) throughout an islet by using a fast voltage optical dye pair. Using laser scanning confocal microscopy (LSCM), we observed fluorescence (Förster) resonance energy transfer (FRET) with the fluorescent donor N-(6-chloro-7-hydroxycoumarin-3-carbonyl)-dimyristoylphosphatidyl-ethanolamine and the acceptor bis-(1,3-diethylthiobarbiturate) trimethine oxonol in the plasma membrane of essentially every cell within an islet. The FRET signal was approximately linear from V(m) -70 to +50 mV with a 2.5-fold change in amplitude. We evaluated the responses of islet cells to glucose and tetraethylammonium. Essentially, every responding cell in a mouse islet displayed similar time-dependent changes in V(m). When V(m) was measured simultaneously with intracellular Ca2+, all active cells showed tight coupling of V(m) to islet cell Ca2+ changes. Our findings indicate that FRET-based, voltage-sensitive dyes used in conjunction with LSCM imaging could be extremely useful in studies of excitation-secretion coupling in intact islets of Langerhans.

5-amino-imidazole Carboxamide Riboside Acutely Potentiates Glucose-stimulated Insulin Secretion from Mouse Pancreatic Islets by KATP Channel-dependent and -independent Pathways

Biochemical and Biophysical Research Communications. May, 2005  |  Pubmed ID: 15823553

AMP-activated protein kinase (AMPK) is an important signaling effector that couples cellular metabolism and function. The effects of AMPK activation on pancreatic beta-cell function remain unresolved. We used 5-amino-imidazole carboxamide riboside (AICAR), an activator of AMPK, to define the signaling mechanisms linking the activation of AMPK with insulin secretion. Application of 300 microM AICAR to mouse islets incubated in 5-14 mM glucose significantly increased AMPK activity and potentiated insulin secretion. AICAR inhibited ATP-sensitive K(+) (K(ATP)) channels and increased the frequency of glucose-induced calcium oscillations in islets incubated in 8-14 mM glucose. At lower glucose concentration (5mM) AICAR did not affect K(ATP) activity or intracellular ([Ca(2+)](i)). AICAR also did not inhibit (86)Rb(+) efflux from islets isolated from Sur1(-/-) mice that lack K(ATP) channels yet significantly potentiated glucose stimulated insulin secretion. Our data suggest that AICAR stimulates insulin secretion by both K(ATP) channel-dependent and -independent pathways.

Adenine Nucleotide Regulation in Pancreatic Beta-cells: Modeling of ATP/ADP-Ca2+ Interactions

American Journal of Physiology. Endocrinology and Metabolism. Nov, 2005  |  Pubmed ID: 15985450

Glucose metabolism stimulates insulin secretion in pancreatic beta-cells. A consequence of metabolism is an increase in the ratio of ATP to ADP ([ATP]/[ADP]) that contributes to depolarization of the plasma membrane via inhibition of ATP-sensitive K+ (K(ATP)) channels. The subsequent activation of calcium channels and increased intracellular calcium leads to insulin exocytosis. Here we evaluate new data and review the literature on nucleotide pool regulation to determine the utility and predictive value of a new mathematical model of ion and metabolic flux regulation in beta-cells. The model relates glucose consumption, nucleotide pool concentration, respiration, Ca2+ flux, and K(ATP) channel activity. The results support the hypothesis that beta-cells maintain a relatively high [ATP]/[ADP] value even in low glucose and that dramatically decreased free ADP with only modestly increased ATP follows from glucose metabolism. We suggest that the mechanism in beta-cells that leads to this result can simply involve keeping the total adenine nucleotide concentration unchanged during a glucose elevation if a high [ATP]/[ADP] ratio exits even at low glucose levels. Furthermore, modeling shows that independent glucose-induced oscillations of intracellular calcium can lead to slow oscillations in nucleotide concentrations, further predicting an influence of calcium flux on other metabolic oscillations. The results demonstrate the utility of comprehensive mathematical modeling in understanding the ramifications of potential defects in beta-cell function in diabetes.

Delayed-rectifier (KV2.1) Regulation of Pancreatic Beta-cell Calcium Responses to Glucose: Inhibitor Specificity and Modeling

American Journal of Physiology. Endocrinology and Metabolism. Oct, 2005  |  Pubmed ID: 16014354

The delayed-rectifier (voltage-activated) K(+) conductance (K(V)) in pancreatic islet beta-cells has been proposed to regulate plasma membrane repolarization during responses to glucose, thereby determining bursting and Ca(2+) oscillations. Here, we verified the expression of K(V)2.1 channel protein in mouse and human islets of Langerhans. We then probed the function of K(V)2.1 channels in islet glucose responses by comparing the effect of hanatoxin (HaTx), a specific blocker of K(V)2.1 channels, with a nonspecific K(+) channel blocker, tetraethylammonium (TEA). Application of HaTx (1 microM) blocked delayed-rectifier currents in mouse beta-cells, resulting in a 40-mV rightward shift in threshold of activation of the voltage-dependent outward current. In the presence of HaTx, there was negligible voltage-activated outward current below 0 mV, suggesting that K(V)2.1 channels form the predominant part of this current in the physiologically relevant range. We then employed HaTx to study the role of K(V)2.1 in the beta-cell Ca(2+) responses to elevated glucose in comparison with TEA. Only HaTx was able to induce slow intracellular Ca(2+) concentration ([Ca(2+)](i)) oscillations in cells stimulated with 20 mM glucose, whereas TEA induced an immediate rise in [Ca(2+)](i) followed by rapid oscillations. In human islets, HaTx acted in a similar fashion. The data were analyzed using a detailed mathematical model of ionic flux and Ca(2+) regulation in beta-cells. The results can be explained by a specific HaTx effect on the K(V) current, whereas TEA affects multiple K(+) conductances. The results underscore the importance of K(V)2.1 channel in repolarization of the pancreatic beta-cell plasma membrane and its role in regulating insulin secretion.

Inositol (1,4,5)-trisphosphate Dynamics and Intracellular Calcium Oscillations in Pancreatic Beta-cells

Diabetes. Nov, 2005  |  Pubmed ID: 16249428

Glucose-stimulated insulin secretion is associated with transients of intracellular calcium concentration ([Ca2+]i) in the pancreatic beta-cell. We tested the hypothesis that inositol (1,4,5)-trisphosphate [Ins(1,4,5)P3] [Ca2+]i release is incorporated in glucose-induced [Ca2+]i oscillations in mouse islets and MIN6 cells. We found that depletion of intracellular Ca2+ stores with thapsigargin increased the oscillation frequency by twofold and inhibited the slow recovery phase of [Ca2+]i oscillations. We employed a pleckstrin homology domain-containing fluorescent biosensor, phospholipase C partial differential pleckstrin homology domain-enhanced green fluorescent protein, to visualize Ins(1,4,5)P3 dynamics in insulin-secreting MIN6 cells and mouse islets in real time using a video-rate confocal system. In both types of cells, stimulation with carbamoylcholine (CCh) and depolarization with KCl results in an increase in Ins(1,4,5)P3 accumulation in the cytoplasm. When stimulated with glucose, the Ins(1,4,5)P3 concentration in the cytoplasm oscillates in parallel with oscillations of [Ca2+]i. Maximal accumulation of Ins(1,4,5)P3 in these oscillations coincides with the peak of [Ca2+]i and tracks changes in frequencies induced by the voltage-gated K+ channel blockade. We show that Ins(1,4,5)P3 release in insulin-secreting cells can be stimulated by depolarization-induced Ca2+ flux. We conclude that Ins(1,4,5)P3 concentration oscillates in parallel with [Ca2+]i in response to glucose stimulation, but it is not the driving force for [Ca2+]i oscillations.

Oxidative Reactive Species in Cell Injury: Mechanisms in Diabetes Mellitus and Therapeutic Approaches

Annals of the New York Academy of Sciences. Dec, 2005  |  Pubmed ID: 16533924

Mammalian cells are continuously subject to insult from reactive species. Most of the pathogenic mechanisms that have been considered to date reflect overproduction of reactive oxygen species (ROS) or a peculiar failure in intracellular defenses against ROS. We have attempted to consider briefly the most important mechanisms of ROS production, defense, and reactive species-induced cell damage and approaches to therapy, focusing on the example of diabetes mellitus. An improved understanding of these mechanisms should facilitate development of antioxidant intervention strategies leading to reduction in diseases associated with oxidative stress.

Reversal of Diabetes in Non-obese Diabetic Mice Without Spleen Cell-derived Beta Cell Regeneration

Science (New York, N.Y.). Mar, 2006  |  Pubmed ID: 16556844

Autoimmune destruction of beta cells is the predominant cause of type 1 diabetes mellitus (T1DM) in humans and is modeled in non-obese diabetic (NOD) mice. Many therapeutic interventions prevent the development of T1DM in NOD mice, but few can induce its reversal once established. Intervention with Freund's complete adjuvant, semi-allogeneic splenocytes, and temporary islet transplantation has been reported to cure NOD mice of established T1DM. Using the same approach, we report here that this treatment cured 32% of NOD mice of established diabetes (>340 milligrams per deciliter blood glucose), although beta cells in these mice were not derived from donor splenocytes.

Downstream Regulatory Element Antagonistic Modulator Regulates Islet Prodynorphin Expression

American Journal of Physiology. Endocrinology and Metabolism. Sep, 2006  |  Pubmed ID: 16621893

Calcium-binding proteins regulate transcription and secretion of pancreatic islet hormones. Here, we demonstrate neuroendocrine expression of the calcium-binding downstream regulatory element antagonistic modulator (DREAM) and its role in glucose-dependent regulation of prodynorphin (PDN) expression. DREAM is distributed throughout beta- and alpha-cells in both the nucleus and cytoplasm. As DREAM regulates neuronal dynorphin expression, we determined whether this pathway is affected in DREAM(-/-) islets. Under low glucose conditions, with intracellular calcium concentrations of <100 nM, DREAM(-/-) islets had an 80% increase in PDN message compared with controls. Accordingly, DREAM interacts with the PDN promoter downstream regulatory element (DRE) under low calcium (<100 nM) conditions, inhibiting PDN transcription in beta-cells. Furthermore, beta-cells treated with high glucose (20 mM) show increased cytoplasmic calcium (approximately 200 nM), which eliminates DREAM's interaction with the DRE, causing increased PDN promoter activity. As PDN is cleaved into dynorphin peptides, which stimulate kappa-opioid receptors expressed predominantly in alpha-cells of the islet, we determined the role of dynorphin A-(1-17) in glucagon secretion from the alpha-cell. Stimulation with dynorphin A-(1-17) caused alpha-cell calcium fluctuations and a significant increase in glucagon release. DREAM(-/-) islets also show elevated glucagon secretion in low glucose compared with controls. These results demonstrate that PDN transcription is regulated by DREAM in a calcium-dependent manner and suggest a role for dynorphin regulation of alpha-cell glucagon secretion. The data provide a molecular basis for opiate stimulation of glucagon secretion first observed over 25 years ago.

Functional MR Microimaging of Pancreatic Beta-cell Activation

Cell Transplantation. 2006  |  Pubmed ID: 16719054

The increasing incidence of diabetes and the need to further understand its cellular basis has resulted in the development of new diagnostic and therapeutic techniques. Nonetheless, the quest to noninvasively ascertain beta-cell mass and function has not been achieved. Manganese (Mn)-enhanced MRI is presented here as a tool to image beta-cell functionality in cell culture and isolated islets. Similar to calcium, extracellular Mn was taken up by glucose-activated beta-cells resulting in 200% increase in MRI contrast enhancement, versus nonactivated cells. Similarly, glucose-activated islets showed an increase in MRI contrast up to 45%. Although glucose-stimulated Ca influx was depressed in the presence of 100 microM Mn, no significant effect was seen at lower Mn concentrations. Moreover, islets exposed to Mn showed normal glucose sensitivity and insulin secretion. These results demonstrate a link between image contrast enhancement and beta-cell activation in vitro, and provide the basis for future noninvasive in vivo imaging of islet functionality and beta-cell mass.

Visualizing Calcium Signaling in Cells by Digitized Wide-field and Confocal Fluorescent Microscopy

Methods in Molecular Biology (Clifton, N.J.). 2006  |  Pubmed ID: 16719350

Calcium (Ca2+) is a fundamentally important component of cellular signal transduction. Dynamic changes in the concentration of Ca2+ ([Ca2+]) in the cytoplasm and within organelles are tightly controlled and regulate a diverse array of biological activities, including fertilization, cell division, gene expression, cellular metabolism, protein biosynthesis, secretion, muscle contraction, intercellular communication, and cell death. Measurement of intracellular [Ca2+] is essential to understanding the role of Ca2+ and for defining the underlying regulatory mechanisms in any cellular process. A broad range of synthetic and biosynthetic fluorescent Ca2+ sensors are available that enable the visualization and quantification of subcellular spatio-temporal [Ca2+] gradients. This chapter describes the application of wide-field digitized video fluorescence microfluorometry and confocal microscopy to quantitatively image Ca2+ in cells with high temporal and spatial resolution.

Cold Climate Genes and the Prevalence of Type 2 Diabetes Mellitus

Medical Hypotheses. 2006  |  Pubmed ID: 16797871

Type 2 diabetes mellitus (T2D) is approaching epidemic proportions globally. However, some human populations, such as Western-Europeans, have a lower prevalence compared with urban or westernized groups with origins in warmer climates. To explain this conspicuous trend we have developed a hypothesis suggesting that pressure for survival on ancestral Western-Europeans (or on other human populations) in extremely cold climates could lead to selection for a combination of specific genes or alleles, which we have named cold climate genes, promoting adaptation to these condition. The possible molecular basis for the effects of these genes could lead to decreasing susceptibility to T2D. The possible candidates for cold climate genes have been evaluated from three areas: the uncoupling proteins, maternally-transmitted mitochondrial genes, and mitochondrial biogenesis. Conclusions/significance: The possible existence of cold climate genes can lead to both increased thermogenesis and decreased prevalence of T2D. This may help explain the variations in prevalence of T2D in different ethnic groups. This consideration suggests testable experimental approaches towards prevention and therapies for T2D.

Recovery of Islet Beta-cell Function in Streptozotocin- Induced Diabetic Mice: an Indirect Role for the Spleen

Diabetes. Dec, 2006  |  Pubmed ID: 17130468

Limitations in islet beta-cell transplantation as a therapeutic option for type 1 diabetes have prompted renewed interest in islet regeneration as a source of new islets. In this study we tested whether severely diabetic adult C57BL/6 mice can regenerate beta-cells. Diabetes was induced in C57BL/6 mice with high-dose streptozotocin (160-170 mg/kg). In the absence of islet transplantation, all diabetic mice remained diabetic (blood glucose >400 mg/dl), and no spontaneous reversal of diabetes was observed. When syngeneic islets (200/mouse) were transplanted into these diabetic mice under a single kidney capsule, stable restoration of euglycemia for >/=120 days was achieved. Removal of the kidney bearing the transplanted islets at 120 days posttransplantation revealed significant restoration of endogenous beta-cell function. This restoration of islet function was associated with increased beta-cell mass, as well as beta-cell hypertrophy and proliferation. The restoration of islet cell function was facilitated by the presence of a spleen; however, the facilitation was not due to the direct differentiation of spleen-derived cells into beta-cells. This study supports the possibility of restoring beta-cell function in diabetic individuals and points to a role for the spleen in facilitating this process.

Reactive Species, Cellular Repair and Risk Factors in the Onset of Type 2 Diabetes Mellitus: Review and Hypothesis

Current Diabetes Reviews. May, 2006  |  Pubmed ID: 18220630

Insulin resistance (IRe) and a failure of insulin secretion are the major features of the early pathophysiology of type-2 diabetes mellitus (T2D) but the etiology is still not well understood. We suggest that: 1. The cellular mechanisms that protect against oxidative stress per se are capable of creating a reactive species-dependent IRe. 2. Reactive species-induced mitochondrial dysfunction can lead to disruption of lipid metabolism, increased intracellular lipid content, and can also contribute to lipid-dependent IRe in myocytes and adipocytes. 3. Metabolic secretagogues that stimulate insulin secretion by the activation of initial steps in the glucose-stimulated insulin secretion pathway can also lead to increased reactive species production and cellular destruction contributing to beta-cell damage and apoptosis. These events that underlie the repair mechanisms in beta-cells, muscle and adipocytes, are important factors in the early etiology of T2D, leading to both IRe and decreased insulin secretion. This hypothesis is supported by data from multiple disciplines and includes aging, obesity and genetic factors in promoting multiple failures in this system leading to the onset of T2D. On the basis of this hypothesis therapeutic strategies should be directed towards increasing insulin secretion and reducing IRe without increasing reactive species production or concentration. Pharmacological or other approaches that result in the activation of mitochondrial biogenesis could be beneficial for both IRe and T2D.

Modulation of the Pancreatic Islet Beta-cell-delayed Rectifier Potassium Channel Kv2.1 by the Polyunsaturated Fatty Acid Arachidonate

The Journal of Biological Chemistry. Mar, 2007  |  Pubmed ID: 17197450

Glucose stimulates both insulin secretion and hydrolysis of arachidonic acid (AA) esterified in membrane phospholipids of pancreatic islet beta-cells, and these processes are amplified by muscarinic agonists. Here we demonstrate that nonesterified AA regulates the biophysical activity of the pancreatic islet beta-cell-delayed rectifier channel, Kv2.1. Recordings of Kv2.1 currents from INS-1 insulinoma cells incubated with AA (5 mum) and subjected to graded degrees of depolarization exhibit a significantly shorter time-to-peak current interval than do control cells. AA causes a rapid decay and reduced peak conductance of delayed rectifier currents from INS-1 cells and from primary beta-cells isolated from mouse, rat, and human pancreatic islets. Stimulating mouse islets with AA results in a significant increase in the frequency of glucose-induced [Ca(2+)] oscillations, which is an expected effect of Kv2.1 channel blockade. Stimulation with concentrations of glucose and carbachol that accelerate hydrolysis of endogenous AA from islet phosphoplipids also results in accelerated Kv2.1 inactivation and a shorter time-to-peak current interval. Group VIA phospholipase A(2) (iPLA(2)beta) hydrolyzes beta-cell membrane phospholipids to release nonesterified fatty acids, including AA, and inhibiting iPLA(2)beta prevents the muscarinic agonist-induced accelerated Kv2.1 inactivation. Furthermore, glucose and carbachol do not significantly affect Kv2.1 inactivation in beta-cells from iPLA(2)beta(-/-) mice. Stably transfected INS-1 cells that overexpress iPLA(2)beta hydrolyze phospholipids more rapidly than control INS-1 cells and also exhibit an increase in the inactivation rate of the delayed rectifier currents. These results suggest that Kv2.1 currents could be dynamically modulated in the pancreatic islet beta-cell by phospholipase-catalyzed hydrolysis of membrane phospholipids to yield non-esterified fatty acids, such as AA, that facilitate Ca(2+) entry and insulin secretion.

Protective Role for Nitric Oxide During the Endoplasmic Reticulum Stress Response in Pancreatic Beta-cells

American Journal of Physiology. Endocrinology and Metabolism. Jun, 2007  |  Pubmed ID: 17264231

Higher requirements for disulfide bond formation in professional secretory cells may affect intracellular redox homeostasis, particularly during an endoplasmic reticulum (ER) stress response. To assess this hypothesis, we investigated the effects of the ER stress response on the major redox couple (GSH/GSSG), endogenous ROS production, expression of genes involved in ER oxidative protein folding, general antioxidant defense, and thiol metabolism by use of the well-validated MIN6 beta-cell as a model and mouse islets. The data revealed that glucose concentration-dependent decreases in the GSH/GSSG ratio were further decreased significantly by ER-derived oxidative stress induced by inhibiting ER-associated degradation with the specific proteasome inhibitor lactacystin (10 microM) in mouse islets. Notably, minimal cell death was observed during 12-h treatments. This was likely attributed to the upregulation of genes encoding the rate limiting enzyme for glutathione synthesis (gamma-glutamylcysteine ligase), as well as genes involved in antioxidant defense (glutathione peroxidase, peroxiredoxin-1) and ER protein folding (Grp78/BiP, PDI, Ero1). Gene expression and reporter assays with a NO synthase inhibitor (Nomega-nitro-L-arginine methyl ester, 1-10 mM) indicated that endogenous NO production was essential for the upregulation of several ER stress-responsive genes. Specifically, gel shift analyses demonstrate NO-independent binding of the transcription factor NF-E2-related factor to the antioxidant response element Gclc-ARE4 in MIN6 cells. However, endogenous NO production was necessary for activation of Gclc-ARE4-driven reporter gene expression. Together, these data reveal a distinct protective role for NO during the ER stress response, which helps to dissipate ROS and promote beta-cell survival.

Ethics of Hematopoietic Stem Cell Transplantation in Type 1 Diabetes Mellitus

JAMA : the Journal of the American Medical Association. Jul, 2007  |  Pubmed ID: 17635886

Kv2.1 Ablation Alters Glucose-induced Islet Electrical Activity, Enhancing Insulin Secretion

Cell Metabolism. Sep, 2007  |  Pubmed ID: 17767909

Voltage-gated potassium currents (Kv), primarily due to Kv2.1 channels, are activated by glucose-stimulated pancreatic beta cell depolarization, but the exact role (or roles) of this channel in regulating insulin secretion remains uncertain. Here we report that, compared with controls, Kv2.1 null mice have reduced fasting blood glucose levels and elevated serum insulin levels. Glucose tolerance is improved and insulin secretion is enhanced compared to control animals, with similar results in isolated islets in vitro. Isolated Kv2.1(-/-) beta cells have residual Kv currents, which are decreased by 83% at +50 mV compared with control cells. The glucose-induced action potential (AP) duration is increased while the firing frequency is diminished, similar to the effect of specific toxins on control cells but substantially different from the effect of the less specific blocker tetraethylammonium. These results reveal the specific role of Kv2.1 in modulating glucose-stimulated APs of beta cells, exposing additional important currents involved in regulating physiological insulin secretion.

Insulin Gene Mutations As a Cause of Permanent Neonatal Diabetes

Proceedings of the National Academy of Sciences of the United States of America. Sep, 2007  |  Pubmed ID: 17855560

We report 10 heterozygous mutations in the human insulin gene in 16 probands with neonatal diabetes. A combination of linkage and a candidate gene approach in a family with four diabetic members led to the identification of the initial INS gene mutation. The mutations are inherited in an autosomal dominant manner in this and two other small families whereas the mutations in the other 13 patients are de novo. Diabetes presented in probands at a median age of 9 weeks, usually with diabetic ketoacidosis or marked hyperglycemia, was not associated with beta cell autoantibodies, and was treated from diagnosis with insulin. The mutations are in critical regions of the preproinsulin molecule, and we predict that they prevent normal folding and progression of proinsulin in the insulin secretory pathway. The abnormally folded proinsulin molecule may induce the unfolded protein response and undergo degradation in the endoplasmic reticulum, leading to severe endoplasmic reticulum stress and potentially beta cell death by apoptosis. This process has been described in both the Akita and Munich mouse models that have dominant-acting missense mutations in the Ins2 gene, leading to loss of beta cell function and mass. One of the human mutations we report here is identical to that in the Akita mouse. The identification of insulin mutations as a cause of neonatal diabetes will facilitate the diagnosis and possibly, in time, treatment of this disorder.

Regulation of CAMP Dynamics by Ca2+ and G Protein-coupled Receptors in the Pancreatic Beta-cell: a Computational Approach

American Journal of Physiology. Cell Physiology. Dec, 2007  |  Pubmed ID: 17928534

In this report we describe a mathematical model for the regulation of cAMP dynamics in pancreatic beta-cells. Incretin hormones such as glucagon-like peptide 1 (GLP-1) increase cAMP and augment insulin secretion in pancreatic beta-cells. Imaging experiments performed in MIN6 insulinoma cells expressing a genetically encoded cAMP biosensor and loaded with fura-2, a calcium indicator, showed that cAMP oscillations are differentially regulated by periodic changes in membrane potential and GLP-1. We modeled the interplay of intracellular calcium (Ca(2+)) and its interaction with calmodulin, G protein-coupled receptor activation, adenylyl cyclases (AC), and phosphodiesterases (PDE). Simulations with the model demonstrate that cAMP oscillations are coupled to cytoplasmic Ca(2+) oscillations in the beta-cell. Slow Ca(2+) oscillations (<1 min(-1)) produce low-frequency cAMP oscillations, and faster Ca(2+) oscillations (>3-4 min(-1)) entrain high-frequency, low-amplitude cAMP oscillations. The model predicts that GLP-1 receptor agonists induce cAMP oscillations in phase with cytoplasmic Ca(2+) oscillations. In contrast, observed antiphasic Ca(2+) and cAMP oscillations can be simulated following combined glucose and tetraethylammonium-induced changes in membrane potential. The model provides additional evidence for a pivotal role for Ca(2+)-dependent AC and PDE activation in coupling of Ca(2+) and cAMP signals. Our results reveal important differences in the effects of glucose/TEA and GLP-1 on cAMP dynamics in MIN6 beta-cells.

Insulin Mutation Screening in 1,044 Patients with Diabetes: Mutations in the INS Gene Are a Common Cause of Neonatal Diabetes but a Rare Cause of Diabetes Diagnosed in Childhood or Adulthood

Diabetes. Apr, 2008  |  Pubmed ID: 18162506

Insulin gene (INS) mutations have recently been described as a cause of permanent neonatal diabetes (PND). We aimed to determine the prevalence, genetics, and clinical phenotype of INS mutations in large cohorts of patients with neonatal diabetes and permanent diabetes diagnosed in infancy, childhood, or adulthood.

Mutations in the Insulin Gene Can Cause MODY and Autoantibody-negative Type 1 Diabetes

Diabetes. Apr, 2008  |  Pubmed ID: 18192540

Mutations in the insulin (INS) gene can cause neonatal diabetes. We hypothesized that mutations in INS could also cause maturity-onset diabetes of the young (MODY) and autoantibody-negative type 1 diabetes.

Glucose Homeostasis, Insulin Secretion, and Islet Phospholipids in Mice That Overexpress IPLA2beta in Pancreatic Beta-cells and in IPLA2beta-null Mice

American Journal of Physiology. Endocrinology and Metabolism. Feb, 2008  |  Pubmed ID: 17895289

Studies with genetically modified insulinoma cells suggest that group VIA phospholipase A(2) (iPLA(2)beta) participates in amplifying glucose-induced insulin secretion. INS-1 insulinoma cells that overexpress iPLA(2)beta, for example, exhibit amplified insulin-secretory responses to glucose and cAMP-elevating agents. To determine whether similar effects occur in whole animals, we prepared transgenic (TG) mice in which the rat insulin 1 promoter (RIP) drives iPLA(2)beta overexpression, and two characterized TG mouse lines exhibit similar phenotypes. Their pancreatic islet iPLA(2)beta expression is increased severalfold, as reflected by quantitative PCR of iPLA(2)beta mRNA, immunoblotting of iPLA(2)beta protein, and iPLA(2)beta enzymatic activity. Immunofluorescence microscopic studies of pancreatic sections confirm iPLA(2)beta overexpression in RIP-iPLA(2)beta-TG islet beta-cells without obviously perturbed islet morphology. Male RIP-iPLA(2)beta-TG mice exhibit lower blood glucose and higher plasma insulin concentrations than wild-type (WT) mice when fasting and develop lower blood glucose levels in glucose tolerance tests, but WT and TG blood glucose levels do not differ in insulin tolerance tests. Islets from male RIP-iPLA(2)beta-TG mice exhibit greater amplification of glucose-induced insulin secretion by a cAMP-elevating agent than WT islets. In contrast, islets from male iPLA(2)beta-null mice exhibit blunted insulin secretion, and those mice have impaired glucose tolerance. Arachidonate incorporation into and the phospholipid composition of RIP-iPLA(2)beta-TG islets are normal, but they exhibit reduced Kv2.1 delayed rectifier current and prolonged glucose-induced action potentials and elevations of cytosolic Ca(2+) concentration that suggest a molecular mechanism for the physiological role of iPLA(2)beta to amplify insulin secretion.

When BAD is Good for Beta Cells

Cell Metabolism. Apr, 2008  |  Pubmed ID: 18396130

BAD, a proapoptotic member of the Bcl-2 family of proteins, is regulated by phosphorylation. A recent study (Danial et al., 2008) suggests a phosphorylation-state-dependent bifunctional role of BAD in the regulation of glucose-stimulated insulin secretion and beta cell mass.

Reversible Translocation of EYFP-tagged STIM1 is Coupled to Calcium Influx in Insulin Secreting Beta-cells

Cell Calcium. Dec, 2008  |  Pubmed ID: 18452988

Calcium (Ca(2+)) signaling regulates insulin secretion in pancreatic beta-cells. STIM1 has been proposed to function as an endoplasmic reticulum (ER) Ca(2+) sensor regulating store-operated Ca(2+) entry (SOCE). Here we studied the translocation of EYFP-STIM1 in response to ER calcium depletion in mouse insulinoma MIN6 cells by fluorescent microscopy. While in resting cells EYFP-STIM1 is co-localized with an ER marker, in thapsigargin (Tg)-stimulated cells it occupied highly defined areas of the peri-PM space in punctae adjacent to, but not entirely coincident with the ER. Co-staining with fluorescent phalloidin revealed that EYFP-STIM1 punctae was located in actin-poor areas. Use of the SOCE blocker in MIN6 cells, 2-aminoethoxy diphenylborate (2-APB), prevented store depletion-dependent translocation of EYFP-STIM1 to the PM in a concentration-dependent (3.75-100muM) and reversible manner. TIRF microscopy revealed that 2-APB treatment led to the reversible disappearance of peri-PM EYFP-STIM1 punctae, while the ER structure in this compartment remained grossly unaffected. We conclude from this data that in these cells EYFP-STIM1 is delivered to a peri-PM location from the ER upon store depletion and this trafficking is reversibly blocked by 2-APB.

Diagnosis and Treatment of Neonatal Diabetes: a United States Experience

Pediatric Diabetes. Oct, 2008  |  Pubmed ID: 18662362

Mutations in KCNJ11, ABCC8, or INS are the cause of permanent neonatal diabetes mellitus in about 50% of patients diagnosed with diabetes before 6 months of age and in a small fraction of those diagnosed between 6 and 12 months. The aim of this study was to identify the genetic cause of diabetes in 77 consecutive patients referred to the University of Chicago with diabetes diagnosed before 1 yr of age.

The Chemistrode: a Droplet-based Microfluidic Device for Stimulation and Recording with High Temporal, Spatial, and Chemical Resolution

Proceedings of the National Academy of Sciences of the United States of America. Nov, 2008  |  Pubmed ID: 18974218

Microelectrodes enable localized electrical stimulation and recording, and they have revolutionized our understanding of the spatiotemporal dynamics of systems that generate or respond to electrical signals. However, such comprehensive understanding of systems that rely on molecular signals-e.g., chemical communication in multicellular neural, developmental, or immune systems-remains elusive because of the inability to deliver, capture, and interpret complex chemical information. To overcome this challenge, we developed the "chemistrode," a plug-based microfluidic device that enables stimulation, recording, and analysis of molecular signals with high spatial and temporal resolution. Stimulation with and recording of pulses as short as 50 ms was demonstrated. A pair of chemistrodes fabricated by multilayer soft lithography recorded independent signals from 2 locations separated by 15 mum. Like an electrode, the chemistrode does not need to be built into an experimental system-it is simply brought into contact with a chemical or biological substrate, and, instead of electrical signals, molecular signals are exchanged. Recorded molecular signals can be injected with additional reagents and analyzed off-line by multiple, independent techniques in parallel (e.g., fluorescence correlation spectroscopy, MALDI-MS, and fluorescence microscopy). When recombined, these analyses provide a time-resolved chemical record of a system's response to stimulation. Insulin secretion from a single murine islet of Langerhans was measured at a frequency of 0.67 Hz by using the chemistrode. This article characterizes and tests the physical principles that govern the operation of the chemistrode to enable its application to probing local dynamics of chemically responsive matter in chemistry and biology.

The Alpha-cell Conundrum: ATP-sensitive K+ Channels and Glucose Sensing

Diabetes. Feb, 2009  |  Pubmed ID: 19171747

Leptin Deficiency and Beta-cell Dysfunction Underlie Type 2 Diabetes in Compound Akt Knockout Mice

Molecular and Cellular Biology. Jun, 2009  |  Pubmed ID: 19289493

Phenotypic analyses of mice null for the individual Akt isoforms suggested that they are functionally distinct and that only Akt2 plays a role in diabetes. We show here that Akt isoforms play compensatory and complementary roles in glucose homeostasis and diabetes. Insulin resistance in Akt2(-/-) mice was inhibited by haplodeficiency of Pten, suggesting that other Akt isoforms can compensate for Akt2 function. Haplodeficiency of Akt1 in Akt2(-/-) mice, however, converts prediabetes to overt type 2 diabetes, which is also reversed by haplodeficiency of Pten. Akt3 does not appear to contribute significantly to diabetes. Overt type 2 diabetes in Akt1(+/-) Akt2(-/-) mice is manifested by hyperglycemia due to beta-cell dysfunction combined with impaired glucose homeostasis due to markedly decreased leptin levels. Restoring leptin levels was sufficient to restore normal blood glucose and insulin levels in Akt1(+/-) Akt2(-/-) and Akt2(-/-) mice, suggesting that leptin-deficiency is the predominant cause of diabetes in these mice. These results uncover a new mechanism linking Akt to diabetes, provide a therapeutic strategy, and show that diabetes induced as a consequence of cancer therapy, via Akt inhibition, could be reversed by leptin therapy.

Tooth Discoloration in Patients with Neonatal Diabetes After Transfer Onto Glibenclamide: a Previously Unreported Side Effect

Diabetes Care. Aug, 2009  |  Pubmed ID: 19435956

OBJECTIVE To assess if tooth discoloration is a novel side effect of sulfonylurea therapy in patients with permanent neonatal diabetes due to mutations in KCNJ11. RESEARCH DESIGN AND METHODS A total of 67 patients with a known KCNJ11 mutation who had been successfully transferred from insulin injections onto oral sulfonylureas were contacted and asked about the development of tooth discoloration after transfer. RESULTS Altered tooth appearance was identified in 5 of the 67 patients. This was variable in severity, ranging from mild discoloration/staining (n = 4) to loss of enamel (n = 1) and was only seen in patients taking glibenclamide (glyburide). CONCLUSIONS These previously unreported side effects may relate to the developing tooth and/or to the high local concentrations in the children who frequently chewed glibenclamide tablets or took it as a concentrated solution. Given the multiple benefits of sulfonylurea treatment for patients with activating KCNJ11 mutations, this association warrants further investigation but should not preclude such treatment.

A Nanoporous, Transparent Microcontainer for Encapsulated Islet Therapy

Journal of Diabetes Science and Technology. Mar, 2009  |  Pubmed ID: 19746206

Present-day islet encapsulation techniques such as polymer microcapsules and microelectromechanical system (MEMS)-based biocapsules have shown promise in insulin replacement therapy, but they each have limitations-the permeability characteristics of existing polymeric capsules cannot be strictly controlled because of tortuosity and the large size of present-day MEMS biocapsules leads to necrotic regions within the encapsulation volume. We report on a new microcontainer to encapsulate and immunoprotect islets/beta cells that may be used for allo- or xenotransplantation in cell-based therapy. The microcontainers have membranes containing nanoslots to permit the bidirectional transport of nutrients, secretagogues, and cellular products while immunoprotecting the encapsulated cells. The 300-microm microcontainers were fabricated from an epoxy-based polymer, SU-8, with 50-microm-thick walls. Arrays of 25-nm wide slots were created in the SU-8 microcontainer lid. Isolated mouse islets were encapsulated in the microcontainer, and their physiological response to glucose was studied with fluorescence and two-photon imaging over 48 hours. The physiological response of the encapsulated islets was indistinguishable from controls. An agarose-filled microcontainer was imaged with magnetic resonance imaging to demonstrate the feasibility of future noninvasive, in vivo imaging. The SU-8 microcontainers maintained mechanical integrity upon islet loading and mechanical manipulation. Islet encapsulation, as well as the ability to visualize islet function within these transparent microcontainers, was demonstrated.

The Granular Chloride Channel ClC-3 is Permissive for Insulin Secretion

Cell Metabolism. Oct, 2009  |  Pubmed ID: 19808024

Insulin secretion from pancreatic beta cells is dependent on maturation and acidification of the secretory granule, processes necessary for prohormone convertase cleavage of proinsulin. Previous studies in isolated beta cells revealed that acidification may be dependent on the granule membrane chloride channel ClC-3, in a step permissive for a regulated secretory response. In this study, immuno-EM of beta cells revealed colocalization of ClC-3 and insulin on secretory granules. Clcn3(-/-) mice as well as isolated islets demonstrate impaired insulin secretion; Clcn3(-/-) beta cells are defective in regulated insulin exocytosis and granular acidification. Increased amounts of proinsulin were found in the majority of secretory granules in the Clcn3(-/-) mice, while in Clcn3(+/+) cells, proinsulin was confined to the immature secretory granules. These results demonstrate that in pancreatic beta cells, chloride channels, specifically ClC-3, are localized on insulin granules and play a role in insulin processing as well as insulin secretion through regulation of granular acidification.

In Vitro Processing and Secretion of Mutant Insulin Proteins That Cause Permanent Neonatal Diabetes

American Journal of Physiology. Endocrinology and Metabolism. Mar, 2010  |  Pubmed ID: 19952343

Permanent neonatal diabetes mellitus is a rare form of insulin-requiring diabetes presenting within the first few weeks or months of life. Mutations in the insulin gene are the second most common cause of this form of diabetes. These mutations are located in critical regions of preproinsulin and are likely to prevent normal processing or folding of the preproinsulin/proinsulin molecule. To characterize these mutations, we transiently expressed proinsulin-GFP fusion proteins in MIN6 mouse insulinoma cells. Our study revealed three groups of mutant proteins: 1) mutations that result in retention of proinsulin in the endoplasmic reticulum (ER) and attenuation of secretion of cotransfected wild-type insulin: C43G, F48C, and C96Y; 2) mutations with partial ER retention, partial recruitment to granules, and attenuation of secretion of wild-type insulin: G32R, G32S, G47V, G90C, and Y108C; and 3) similar to (2) but with no significant attenuation of wild-type insulin secretion: A24D and R89C. The mutant insulin proteins do not prevent targeting of wild-type insulin to secretory granules, but most appear to lead to decreased secretion of wild-type insulin. Each of the mutants triggers the expression of the proapoptotic gene Chop, indicating the presence of ER stress.

Update in Neonatal Diabetes

Current Opinion in Endocrinology, Diabetes, and Obesity. Feb, 2010  |  Pubmed ID: 19952737

Here we give context to new data on neonatal diabetes mellitus, a rare group of insulin-requiring monogenic forms of diabetes presenting at birth or shortly thereafter. Genetic studies are critical in the diagnosis and treatment of these patients. The most common causes of neonatal diabetes are activating mutations in the two protein subunits of the ATP-sensitive potassium channel. These are responsible for about half of all cases of permanent neonatal diabetes and some cases of transient neonatal diabetes. Identification of these mutations allows patients treated with insulin to be transferred to sulfonylureas, but associated conditions and other causes must be considered.

Neonatal Diabetes Mellitus: a Model for Personalized Medicine

Trends in Endocrinology and Metabolism: TEM. Aug, 2010  |  Pubmed ID: 20434356

Neonatal diabetes mellitus occurs in approximately 1 out of every 100,000 live births. It can be either permanent or transient, and recent studies indicate that is likely to have an underlying genetic cause, particularly when diagnosed before 6 months of age. Permanent neonatal diabetes is most commonly due to activating mutations in either of the genes encoding the two subunits of the ATP-sensitive potassium channel. In most of these patients, switching from insulin to oral sulfonylurea therapy leads to improved metabolic control, as well as possible amelioration of occasional associated neurodevelopmental disabilities. It remains to be determined what is the most appropriate treatment of other causes. The diagnosis and treatment of neonatal diabetes, therefore, represents a model for personalized medicine.

Glucose Sensing in the Pancreatic Beta Cell: a Computational Systems Analysis

Theoretical Biology & Medical Modelling. 2010  |  Pubmed ID: 20497556

Pancreatic beta-cells respond to rising blood glucose by increasing oxidative metabolism, leading to an increased ATP/ADP ratio in the cytoplasm. This leads to a closure of KATP channels, depolarization of the plasma membrane, influx of calcium and the eventual secretion of insulin. Such mechanism suggests that beta-cell metabolism should have a functional regulation specific to secretion, as opposed to coupling to contraction. The goal of this work is to uncover contributions of the cytoplasmic and mitochondrial processes in this secretory coupling mechanism using mathematical modeling in a systems biology approach.

Blood Sugar Measurement in Zebrafish Reveals Dynamics of Glucose Homeostasis

Zebrafish. Jun, 2010  |  Pubmed ID: 20515318

The adult zebrafish has the potential to become an important model for diabetes-related research. To realize this potential, small-scale methods for analyzing pancreas function are required. The measurement of blood glucose level is a commonly used method for assessing beta-cell function, but the small size of the zebrafish presents challenges both for collecting blood samples and for measuring glucose. We have developed methods for collecting microsamples of whole blood and plasma for the measurement of hematocrit and blood glucose. We demonstrate that two hand-held glucose meters designed for use by human diabetics return valid results with zebrafish blood. Additionally, we present methods for fasting and for performing postprandial glucose and intraperitoneal glucose tolerance tests. We find that the dynamics of zebrafish blood glucose homeostasis are consistent with patterns reported for other omnivorous teleost fish.

Disruption of the Clock Components CLOCK and BMAL1 Leads to Hypoinsulinaemia and Diabetes

Nature. Jul, 2010  |  Pubmed ID: 20562852

The molecular clock maintains energy constancy by producing circadian oscillations of rate-limiting enzymes involved in tissue metabolism across the day and night. During periods of feeding, pancreatic islets secrete insulin to maintain glucose homeostasis, and although rhythmic control of insulin release is recognized to be dysregulated in humans with diabetes, it is not known how the circadian clock may affect this process. Here we show that pancreatic islets possess self-sustained circadian gene and protein oscillations of the transcription factors CLOCK and BMAL1. The phase of oscillation of islet genes involved in growth, glucose metabolism and insulin signalling is delayed in circadian mutant mice, and both Clock and Bmal1 (also called Arntl) mutants show impaired glucose tolerance, reduced insulin secretion and defects in size and proliferation of pancreatic islets that worsen with age. Clock disruption leads to transcriptome-wide alterations in the expression of islet genes involved in growth, survival and synaptic vesicle assembly. Notably, conditional ablation of the pancreatic clock causes diabetes mellitus due to defective beta-cell function at the very latest stage of stimulus-secretion coupling. These results demonstrate a role for the beta-cell clock in coordinating insulin secretion with the sleep-wake cycle, and reveal that ablation of the pancreatic clock can trigger the onset of diabetes mellitus.

Calcium-activated and Voltage-gated Potassium Channels of the Pancreatic Islet Impart Distinct and Complementary Roles During Secretagogue Induced Electrical Responses

The Journal of Physiology. Sep, 2010  |  Pubmed ID: 20643768

Glucose-induced β-cell action potential (AP) repolarization is regulated by potassium efflux through voltage gated (Kv) and calcium activated (K(Ca)) potassium channels. Thus, ablation of the primary Kv channel of the β-cell, Kv2.1, causes increased AP duration. However, Kv2.1(-/-) islet electrical activity still remains sensitive to the potassium channel inhibitor tetraethylammonium. Therefore, we utilized Kv2.1(-/-) islets to characterize Kv and K(Ca) channels and their respective roles in modulating the β-cell AP. The remaining Kv current present in Kv2.1(-/-) β-cells is inhibited with 5 μM CP 339818. Inhibition of the remaining Kv current in Kv2.1(-/-) mouse β-cells increased AP firing frequency by 39.6% but did not significantly enhance glucose stimulated insulin secretion (GSIS). The modest regulation of islet AP frequency by CP 339818 implicates other K(+) channels, possibly K(Ca) channels, in regulating AP repolarization. Blockade of the K(Ca) channel BK with slotoxin increased β-cell AP amplitude by 28.2%, whereas activation of BK channels with isopimaric acid decreased β-cell AP amplitude by 30.6%. Interestingly, the K(Ca) channel SK significantly contributes to Kv2.1(-/-) mouse islet AP repolarization. Inhibition of SK channels decreased AP firing frequency by 66% and increased AP duration by 67% only when Kv2.1 is ablated or inhibited and enhanced GSIS by 2.7-fold. Human islets also express SK3 channels and their β-cell AP frequency is significantly accelerated by 4.8-fold with apamin. These results uncover important repolarizing roles for both Kv and K(Ca) channels and identify distinct roles for SK channel activity in regulating calcium- versus sodium-dependent AP firing.

Glucose-induced ERM Protein Activation and Translocation Regulates Insulin Secretion

American Journal of Physiology. Endocrinology and Metabolism. Nov, 2010  |  Pubmed ID: 20739507

A key step in regulating insulin secretion is insulin granule trafficking to the plasma membrane. Using live-cell time-lapse confocal microscopy, we observed a dynamic association of insulin granules with filamentous actin and PIP2-enriched structures. We found that the scaffolding protein family ERM, comprising ezrin, radixin, and moesin, are expressed in β-cells and target both F-actin and PIP2. Furthermore, ERM proteins are activated via phosphorylation in a glucose- and calcium-dependent manner. This activation leads to a translocation of the ERM proteins to sites on the cell periphery enriched in insulin granules, the exocyst complex docking protein Exo70, and lipid rafts. ERM scaffolding proteins also participate in insulin granule trafficking and docking to the plasma membrane. Overexpression of a truncated dominant-negative ezrin construct that lacks the ERM F-actin binding domain leads to a reduction in insulin granules near the plasma membrane and impaired secretion. Conversely, overexpression of a constitutively active ezrin results in more granules near the cell periphery and an enhancement of insulin secretion. Diabetic mouse islets contain less active ERM, suggestive of a novel mechanism whereby impairment of insulin granule trafficking to the membrane through a complex containing F-actin, PIP2, Exo70, and ERM proteins contributes to defective insulin secretion.

Conditional Gene Targeting in Mouse Pancreatic ß-Cells: Analysis of Ectopic Cre Transgene Expression in the Brain

Diabetes. Dec, 2010  |  Pubmed ID: 20802254

Conditional gene targeting has been extensively used for in vivo analysis of gene function in β-cell biology. The objective of this study was to examine whether mouse transgenic Cre lines, used to mediate β-cell- or pancreas-specific recombination, also drive Cre expression in the brain.

Clinical and Molecular Genetics of Neonatal Diabetes Due to Mutations in the Insulin Gene

Reviews in Endocrine & Metabolic Disorders. Sep, 2010  |  Pubmed ID: 20938745

Over the last decade our insight into the causes of neonatal diabetes has greatly expanded. Neonatal diabetes was once considered a variant of type 1 diabetes that presented early in life. Recent advances in our understanding of this disorder have established that neonatal diabetes is not an autoimmune disease, but rather is a monogenic form of diabetes resulting from mutations in a number of different genes encoding proteins that play a key role in the normal function of the pancreatic beta-cell. Moreover, a correct genetic diagnosis can affect treatment and clinical outcome. This is especially true for patients with mutations in the genes KCNJ11 or ABCC8 that encode the two protein subunits (Kir6.2 and SUR1, respectively) of the ATP-sensitive potassium channel. These patients can be treated with oral sulfonylurea drugs with better glycemic control and quality of life. Recently, mutations in the insulin gene (INS) itself have been identified as another cause of neonatal diabetes. In this article, we review the role of INS mutations in the pathophysiology of neonatal diabetes.

The Cost-effectiveness of Personalized Genetic Medicine: the Case of Genetic Testing in Neonatal Diabetes

Diabetes Care. Mar, 2011  |  Pubmed ID: 21273495

Neonatal diabetes mellitus is a rare form of diabetes diagnosed in infancy. Nearly half of patients with permanent neonatal diabetes have mutations in the genes for the ATP-sensitive potassium channel (KCNJ11 and ABCC8) that allow switching from insulin to sulfonylurea therapy. Although treatment conversion has dramatic benefits, the cost-effectiveness of routine genetic testing is unknown.

Erratum To: Clinical and Molecular Genetics of Neonatal Diabetes Due to Mutations in the Insulin Gene

Reviews in Endocrine & Metabolic Disorders. Feb, 2011  |  Pubmed ID: 21286820

Who Should Have Genetic Testing for Maturity-onset Diabetes of the Young?

Clinical Endocrinology. Oct, 2011  |  Pubmed ID: 21521318

Maturity-onset diabetes of the young (MODY) is a clinically heterogeneous group of monogenic disorders characterized by autosomal dominant inheritance of young-onset, non-insulin-dependent diabetes. The genes involved are important in beta cell development, function and regulation and lead to disorders in glucose sensing and insulin secretion. Heterozygous GCK mutations cause impaired glucokinase activity resulting in stable, mild hyperglycaemia that rarely requires treatment. HNF1A mutations cause a progressive insulin secretory defect that is sensitive to sulphonylureas, most often resulting in improved glycaemic control compared with other diabetes treatment. MODY owing to mutations in the HNF4A gene results in a similar phenotype, including sensitivity to sulphonylurea treatment. HNF1B mutations most frequently cause developmental renal disease (particularly renal cysts) but may also cause MODY in isolation or may cause the renal cysts and diabetes syndrome (RCAD syndrome). Mutations in NEUROD1, PDX1 (IPF1), CEL and INS are rare causes of MODY. MODY is often misdiagnosed as type 1 or type 2 diabetes. However, a correct genetic diagnosis impacts treatment and identifies at-risk family members. Thus, it is important to consider a diagnosis of MODY in appropriate individuals and to pursue genetic testing to establish a molecular diagnosis.

Creation of the Web-based University of Chicago Monogenic Diabetes Registry: Using Technology to Facilitate Longitudinal Study of Rare Subtypes of Diabetes

Journal of Diabetes Science and Technology. Jul, 2011  |  Pubmed ID: 21880229

Monogenic diabetes is a group of disorders caused by mutations in any one of a number of genes. Although a monogenic diagnosis--estimated to represent as much as 2% of all diabetes patients--can have a transformational impact on treatment, the majority of monogenic cases remain unidentified and little is known about their natural history. We thus created the first United States Monogenic Diabetes Registry (http://www.kovlerdiabetescenter.org/registry/) for individuals with either neonatal diabetes diagnosed before 1 year of age or with a phenotype suggestive of maturity-onset diabetes of the young.

Coupling of Metabolic, Second Messenger Pathways and Insulin Granule Dynamics in Pancreatic Beta-cells: a Computational Analysis

Progress in Biophysics and Molecular Biology. Nov, 2011  |  Pubmed ID: 21920379

Insulin secretory responses to nutrient stimuli and hormonal modulators in pancreatic beta-cells are controlled by a variety of secondary messengers. We have analyzed numerous mechanisms responsible for regulated exocytosis in these cells and present an integrated mathematical model of cytosolic Ca²⁺, cAMP and granule dynamics. The insulin-containing granules in the beta-cell were divided into four classes: a large "reserve" granule pool, a smaller pool of the morphologically docked granules that is chemically 'primed' for release or the "readily releasable pool", and a pool of "restless newcomer granules" that undergoes preferential exocytosis. The model incorporates glucose and other aspects of metabolism, the cAMP amplifying pathway, insulin granule dynamics and the exocyst concept for granule binding. The values of most of the model parameters were inferred from available experimental data. The model can generate both the fast first phase and slow biphasic insulin secretion found experimentally in response to a step increase of membrane potential or of glucose. The numerical simulations have also reproduced a variety of experimental conditions, such as periodic stimulation by high K⁺ and the potentiation induced in islets by pre-incubation with cAMP pathway activators. The explicit incorporation of Ca²⁺ channels, Ca²⁺ and cAMP dynamics allows the model to be further connected to current models for calcium and metabolic dynamics and provides an interpretation of the roles of the triggering and amplifying pathways of glucose-stimulated insulin secretion. The model may be important in the identification of pharmacological targets for improving insulin secretion in type 2 diabetes.

Neonatal Diabetes: an Expanding List of Genes Allows for Improved Diagnosis and Treatment

Current Diabetes Reports. Dec, 2011  |  Pubmed ID: 21993633

There has been major progress in recent years uncovering the genetic causes of diabetes presenting in the first year of life. Twenty genes have been identified to date. The most common causes accounting for the majority of cases are mutations in the genes encoding the two subunits of the ATP-sensitive potassium channel (K(ATP)), KCNJ11 and ABCC8, and the insulin gene (INS), as well as abnormalities in chromosome 6q24. Patients with activating mutations in KCNJ11 and ABCC8 can be treated with oral sulfonylureas in lieu of insulin injections. This compelling example of personalized genetic medicine leading to improved glucose regulation and quality of life may-with continued research-be repeated for other forms of neonatal diabetes in the future.

SUMO Down-regulates GLP-1 Stimulated CAMP Generation and Insulin Secretion

American Journal of Physiology. Endocrinology and Metabolism. Jan, 2012  |  Pubmed ID: 22234371

GLP-1 based incretin therapy is becoming central to the treatment of Type-2 diabetes. Activation of incretin hormone receptors results in rapid elevation of cAMP followed by enhanced insulin secretion. However, the incretin effect may be significantly impaired in diabetes. The objective of this study is to investigate down-regulation of GLP-1 signaling by Small Ubiquitin related MOdifier protein, (SUMO). Mouse islets exposed to high glucose showed increased expression of endogenous SUMO transcripts and its conjugating enzyme Ubc-9. Over-expression of SUMO-1 in MIN6 cells and primary mouse beta cells resulted in reduced static and real-time estimates of intracellular cAMP upon receptor stimulation with exendin-4, a GLP-1R agonist. GLP1-R was covalently modified by SUMO. Over-expression of SUMO-1 attenuated cell surface trafficking of GLP-1R which resulted in significantly reduced insulin secretion when stimulated by exendin-4, a GLP-1R agonist. Partial knock-down of SUMO conjugating enzyme Ubc-9 resulted in enhanced exendin-4 stimulated insulin secretion in mouse islets exposed to high glucose. Thus, SUMO modification of the GLP-1R could be a contributing factor to reduced incretin responsiveness. Elucidating mechanisms of GLP-1R regulation by sumoylation will help improve our understanding of incretin biology and of GLP-1 based treatment of Type-2 diabetes.