bluuebunny/crossref_metadata_2025 huggingface dataset:
Carotid Artery Intima-Media Thickness in Children With Type 1 Diabetes
Authors: Järvisalo Mikko J. , Putto-Laurila Anne , Jartti Laura , Lehtimäki Terho , Solakivi Tiina , Rönnemaa Tapani , Raitakari Olli T.
Published: March 2007

Postmortem studies have shown a relationship between diabetic state and atherosclerotic arterial lesions in adolescents. The aim of the present study was to determine the presence of increased subclinical atherosclerosis (measured as carotid intima-media thickness [IMT]) and its risk factors, including lipoprotein oxidation, in children with type 1 diabetes. We measured carotid IMT using high-resolution ultrasound in 85 children (mean age, 11 ± 2 years): 50 with type 1 diabetes (mean duration, 4.4 ± 3.0 years) and 35 healthy control subjects matched for age, sex, and body size. The susceptibility of LDL to oxidation was determined by measuring the formation of conjugated dienes induced by Cu2+ in 42 children (21 with diabetes and 21 control subjects). The mean carotid IMT was increased in children with diabetes (0.47 ± 0.04 vs. 0.42 ± 0.04 mm; P < 0.0001). Total cholesterol and LDL cholesterol concentrations were similar between the groups, but the children with diabetes had increased LDL diene formation rate (0.49 ± 0.06 vs. 0.45 ± 0.07 μmol/min; P < 0.05), suggesting increased in vitro LDL oxidizability. In a multivariate model for all subjects, the independent correlates for IMT were the diabetic state (P < 0.001), LDL cholesterol level (P < 0.001), and systolic blood pressure (P < 0.001). In children with diabetes but not in control subjects, LDL oxidizability correlated significantly with mean IMT (r = 0.47, P < 0.05), and this relationship remained significant after controlling for LDL cholesterol level. We conclude that type 1 diabetes is an independent risk factor for increased carotid IMT in children. These data also suggest that increased oxidative modification of LDL may be related to early structural atherosclerotic vascular changes in children with diabetes.
Is Reduced First-Phase Insulin Release the Earliest Detectable Abnormality in Individuals Destined to Develop Type 2 Diabetes?
Authors: Gerich John E.
Published: March 2007

Insulin is released from the pancreas in a biphasic manner in response to a square-wave increase in arterial glucose concentration. The first phase consists of a brief spike lasting ∼10 min followed by the second phase, which reaches a plateau at 2–3 h. It is widely thought that diminution of first-phase insulin release is the earliest detectable defect of β-cell function in individuals destined to develop type 2 diabetes and that this defect largely represents β-cell exhaustion after years of compensation for antecedent insulin resistance. In this article, the origins of these concepts are reviewed and recent evidence is presented suggesting that reductions in both phases of insulin release are equally early, that they precede insulin resistance other than that simply due to obesity, and that they therefore may represent the primary genetic risk factor predisposing individuals to type 2 diabetes.
Control Mechanisms of the Oscillations of Insulin Secretion In Vitro and In Vivo
Authors: Gilon Patrick , Ravier Magalie A. , Jonas Jean-Christophe , Henquin Jean-Claude
Published: March 2007

The mechanisms driving the pulsatility of insulin secretion in vivo and in vitro are still unclear. Because glucose metabolism and changes in cytosolic free Ca2+ ([Ca2+]c) in β-cells play a key role in the control of insulin secretion, and because oscillations of these two factors have been observed in single isolated islets and β-cells, pulsatile insulin secretion could theoretically result from [Ca2+]c or metabolism oscillations. We could not detect metabolic oscillations independent from [Ca2+]c changes in β-cells, and imposed metabolic oscillations were poorly effective in inducing oscillations of secretion when [Ca2+]c was kept stable, which suggests that metabolic oscillations are not the direct regulator of the oscillations of secretion. By contrast, tight temporal and quantitative correlations between the changes in [Ca2+]c and insulin release strongly suggest that [Ca2+]c oscillations are the direct drivers of insulin secretion oscillations. Metabolism may play a dual role, inducing [Ca2+]c oscillations (via changes in ATP-sensitive K+ channel activity and membrane potential) and amplifying the secretory response by increasing the efficiency of Ca2+ on exocytosis. The mechanisms underlying the oscillations of insulin secretion by the isolated pancreas and those observed in vivo remain elusive. It is not known how the functioning of distinct islets is synchronized, and the possible role of intrapancreatic ganglia in this synchronization requires confirmation. That pulsatile insulin secretion is beneficial in vivo, by preventing insulin resistance, is suggested by the greater hypoglycemic effect of exogenous insulin when it is infused in a pulsatile rather than continuous manner. The observation that type 2 diabetic patients have impaired pulsatile insulin secretion has prompted the suggestion that such dysregulation contributes to the disease and justifies the efforts toward understanding of the mechanism underlying the pulsatility of insulin secretion both in vitro and in vivo.
Glucose-Stimulated Oscillations in Free Cytosolic ATP Concentration Imaged in Single Islet β-Cells
Authors: Ainscow Edward K. , Rutter Guy A.
Published: March 2007

Normal glucose-stimulated insulin secretion is pulsatile, but the molecular mechanisms underlying this pulsatility are poorly understood. Oscillations in the intracellular free [ATP]/[ADP] ratio represent one possible mechanism because they would be expected to cause fluctuations in ATP-sensitive K+ channel activity and hence oscillatory Ca2+ influx. After imaging recombinant firefly luciferase, expressed via an adenoviral vector in single human or mouse islet β-cells, we report here that cytosolic free ATP concentrations oscillate and that these oscillations are affected by glucose. In human β-cells, oscillations were observed at both 3 and 15 mmol/l glucose, but the oscillations were of a longer wavelength at the higher glucose concentration (167 vs. 66 s). Mouse β-cells displayed oscillations in both cytosolic free [Ca2+] and [ATP] only at elevated glucose concentrations, both with a period of 120 s. To explore the causal relationship between [Ca2+] and [ATP] oscillations, the regulation of each was further investigated in populations of MIN6 β-cells. Incubation in Ca2+-free medium lowered cytosolic [Ca2+] but increased [ATP] in MIN6 cells at both 3 and 30 mmol/l glucose. Removal of external Ca2+ increased [ATP], possibly by decreasing ATP consumption by endoplasmic reticulum Ca2+-ATPases. These results allow a model to be constructed of the β-cell metabolic oscillator that drives nutrient-induced insulin secretion.
TRP Genes
Authors: Qian Feng , Huang Ping , Ma Li , Kuznetsov Andrey , Tamarina Natalia , Philipson Louis H.
Published: March 2007

Nonselective cation channels may play a role in insulin secretion by regulating pancreatic β-cell plasma membrane potential, Ca2+ 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 β-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 Ca2+ entry in β-cells and may be interesting targets for manipulating β-cell function.
Accurate Assessment of β-Cell Function
Authors: Bergman Richard N. , Ader Marilyn , Huecking Katrin , Van Citters Gregg
Published: March 2007

Only in the last decade did modeling studies predict, and knockout experiments confirm, that type 2 diabetes is a “2-hit” disease in which insulin resistance is necessarily accompanied by β-cell defect(s) preventing the compensatory upregulation of insulin secretion. This long- delayed insight was associated with the development of a constant, the “disposition index,” describing the β-cell sensitivity-secretion relationship as a rectangular hyperbola. Shifts in insulin sensitivity are accompanied by compensatory alterations in β-cell sensitivity to glucose. Insulin-sensitive subjects do not require a massive insulin response to exogenous glucose to maintain a normal blood glucose. But if their insulin sensitivity decreases by 80%, as in late pregnancy, they need a fivefold greater insulin response to achieve an identical disposition index. Women with gestational diabetes have an insulin response similar to that of normal volunteers; at first glance, this suggests similar islet function, but the utility of the disposition index is to normalize this response to the amplitude of third trimester insulin resistance, revealing severe β-cell deficiency. The index is a quantitative, convenient, and accurate tool in analyzing epidemiologic data and identifying incipient impaired glucose tolerance. Separate major issues remain, however: the causes of insulin resistance, the causes of the failure of adequate β-cell compensation in type 2 diabetes, and the nature of the signal(s) from insulin-resistant tissues that fail to elicit the appropriate β-cell increment in sensitivity to glucose and other stimuli. The disposition index is likely to remain the most accurate physiologic measure for testing hypotheses and solutions to these challenges in whole organisms.
Assessing Insulin Secretion by Modeling in Multiple-Meal Tests
Authors: Mari Andrea , Tura Andrea , Gastaldelli Amalia , Ferrannini Ele
Published: March 2007

We developed a mathematical model of the glucose control of insulin secretion capable of quantifying β-cell function from a physiological meal test. The model includes a static control, i.e., a secretion component that is a function of plasma glucose concentration (the dose-response function), and a dynamic control, i.e., a secretion component that is proportional to the positive values of the glucose concentration derivative. Furthermore, the dose-response function is assumed to be modulated by a time-varying potentiation factor. To test the model, nine nondiabetic control subjects and nine type 2 diabetic patients received three standardized mixed meals over a period of 14–15 h. Blood samples were drawn for the measurement of glucose, insulin, and C-peptide concentration. The dose-response function, the parameter of the dynamic control, and the potentiation factor were determined by fitting the model to glucose and C-peptide concentrations. In diabetic patients, the dose-response function was shifted to the right (glucose concentration at a reference insulin secretion of 300 pmol · min−1 · m−2 was 11.7 ± 1.1 vs. 7.2 ± 0.7 mmol/l; P < 0.05), and decreased in slope (53 ± 15 vs. 148 ± 38 pmol · min−1 · m−2 · mmol−1 · l; P < 0.05) and the parameter of the dynamic control was decreased (220 ± 67 vs. 908 ± 276 pmol · m−2 · mmol−1 · l; P < 0.05) compared with the nondiabetic control subjects. Furthermore, potentiation was markedly blunted and delayed: maximum potentiation was observed at the first meal in normal subjects and at the second meal (about 4 h later) in diabetic subjects; the mean time for the potentiation factor was higher (7.1 ± 0.2 vs. 5.9 ± 0.2 h; P < 0.01), and the size of potentiation was reduced (2.6 ± 0.5 vs. 7.2 ± 1.5 fold increase; P < 0.005). In conclusion, our model of insulin secretion extracts multiple indexes of β-cell function from a physiological meal test. Use of the model in patients with type 2 diabetes retrieves known defects in insulin secretion but also uncovers new facets of β-cell dysfunction.
Rationale and Methods for the Estimation of Insulin Secretion in a Given Patient
Authors: Vague Philippe , Nguyen Loan
Published: March 2007

Although the plasma insulin assay is now 40 years old, it is not widely used in clinical practice. However, simple methods (such as the various indexes relating fasting insulin to fasting glucose), the increase in plasma insulin at the 30th minute of an oral glucose tolerance test, and the increase in insulin or C-peptide after stimulation by glucagon are relatively reliable compared with more sophisticated approaches to assess β-cell sensitivity to glucose, kinetics of insulin secretion, residual insulin secretion, or insulin sensitivity. But these measures are of no decisive help in distinguishing between the various forms of impaired fasting glucose or non–insulin-dependent diabetes, such as type 2 diabetes, slow type 1 diabetes, the various forms of maturity-onset diabetes of the young, or the mitochondrial genome defects. No data are available to show that the measurement of plasma insulin may be of help to adapt the treatment of a diabetic patient, except for the need of insulin therapy. There are some suggestions that fasting plasma insulin and, more precisely, the homeostasis model assessment indexes may help to predict the progression toward diabetes or the progressive deterioration of β-cell function in diabetic patients.
Challenges and Strategies for Investigating the Genetic Complexity of Common Human Diseases
Authors: Rich Stephen S. , Concannon Patrick
Published: March 2007

There is substantial interest in the identification of genes underlying susceptibility to complex human diseases because of the potential utility of such genes in disease prediction or therapy. Type 1 diabetes is an example of one such disorder and is presumed to arise from the effect of multiple genes and environmental factors. One identified locus has a major effect on type 1 diabetes susceptibility (IDDM1), whereas other loci have significant, yet small, individual effects (IDDM2, IDDM15). It is unclear whether susceptibility for type 1 diabetes arises because of the effects of loci acting independently or whether there are important interactions between loci. Although genetic tools are continuing to be developed to enable examination of candidate regions, the means to identify and narrow “true” susceptibility regions continues to be limited by the lack of statistical power resulting from inadequately sized collections of families. This report provides an evaluation of the approaches for identification of regions harboring type 1 diabetes genes, methods to identify the gene regions that interact to define the risk for type 1 diabetes, and efforts to fine-map the variants responsible.
Triggering of Insulin Release by a Combination of cAMP Signal and Nutrients
Authors: Komatsu Mitsuhisa , Sato Yoshihiko , Yamada Satoko , Yamauchi Keishi , Hashizume Kiyoshi , Aizawa Toru
Published: March 2007

Nutrient augmentation of Ca2+-triggered insulin release occurs in an ATP-sensitive K+ (KATP) channel–independent manner. Here, using rat islets, we explored the possibility of the KATP channel-independent nutrient triggering of insulin release. In the presence of 250 μmol/l diazoxide, simultaneous application of forskolin and 16.7 mmol/l glucose strongly stimulated insulin release: fourfold and eightfold increases with 1 and 30 μmol/l forskolin, respectively. α-Ketoisocaproate (KIC) and 3-isobutylmethylxanthine (IBMX) could be used in place of glucose and forskolin, respectively, to trigger insulin release in the presence of diazoxide. Triggering of insulin release by a combination of nutrients and forskolin was not attenuated by 10 μmol/l nifedipine (a blocker of voltage-dependent Ca2+ channels) and 2 μmol/l thapsigargin (an inhibitor of intracellular Ca2+-ATPase), ascertaining independence of this phenomenon from Ca2+ entry and from intracellular Ca2+ liberation. As anticipated, the action of glucose and KIC was greatly (>80%) suppressed by inhibition of mitochondrial metabolism by 2 mmol/l sodium azide (NaN3). A combination of palmitate and dimethyl glutamate (a cell-permeable glutamate donor), but not either one alone, weakly but unequivocally triggered insulin release when applied simultaneously with forskolin. In this case, however, mitochondrial poisoning by azide was without effect. The finding suggests that a combination of induced palmitoylation and cytosolic glutamate accumulation partially reconstituted signaling beyond mitochondrial metabolism in the β-cell upon glucose stimulation. In conclusion, a combination of cAMP signal and nutrients potently triggers insulin release under full activation of the KATP channel, indicating the multiplicity of driving force for insulin exocytosis.
Regulation of pdx-1 Gene Expression
Authors: Melloul Danielle , Marshak Sonya , Cerasi Erol
Published: March 2007

The homeodomain-containing transcription factor pancreatic duodenal homeobox 1 (PDX-1) plays a key role in pancreas development and in β-cell function. Upstream sequences of the gene up to about −6 kb show islet-specific activity in transgenic mice. Attempts to identify functional regulatory elements involved in the controlled expression of the pdx-1 gene led to the identification of distinct distal β-cell-specific enhancers in human and rat genes. Three additional sequences, conserved between the mouse and the human 5′-flanking regions, two of which are also found in the chicken gene, conferred β-cell-specific expression on a reporter gene, albeit to different extents. A number of transcription factors binding to and modulating the transcriptional activity of the regulatory elements were identified, such as hepatocyte nuclear factor (HNF)-3β, HNF-1α, SP1/3, and, interestingly, PDX-1 itself. A fourth conserved region was localized to the proximal promoter around an E-box motif and was found to bind members of the upstream stimulatory factor (USF) family of transcription factors. We postulate that disruption of pdx-1 cis-acting regulatory sequences and/or mutations or functional impairment of transcription factors controlling the expression of the gene can lead to diabetes.
Glucose-Regulated Gene Expression Maintaining the Glucose-Responsive State of β-Cells
Authors: Schuit Frans , Flamez Daisy , De Vos Anick , Pipeleers Daniel
Published: March 2007

The mammalian β-cell has particular properties that synthesize, store, and secrete insulin in quantities that are matched to the physiological demands of the organism. To achieve this task, β-cells are regulated both acutely and chronically by the extracellular glucose concentration. Several in vivo and in vitro studies indicate that preservation of the glucose-responsive state of β-cells is lost when the extracellular glucose concentration chronically deviates from the normal physiological condition. Experiments with the protein synthesis inhibitor cycloheximide suggest that the maintenance of the functional state of β-cells depends on protein(s) with rapid turnover. Analysis of newly synthesized proteins via two-dimensional gel electrophoresis and high-density gene expression microarrays demonstrates that the glucose-dependent preservation of β-cell function is correlated with glucose regulation of a large number of β-cell genes. Two different microarray analyses of glucose regulation of the mRNA profile in β-cells show that the sugar influences expression of multiple genes involved in energy metabolism, the regulated insulin biosynthetic/secretory pathway, membrane transport, intracellular signaling, gene transcription, and protein synthesis/degradation. Functional analysis of some of these regulated gene clusters has provided new evidence for the concept that cataplerosis, the conversion of mitochondrial metabolites into lipid intermediates, is a major metabolic pathway that allows β-cell activation independently of closure of ATP-sensitive potassium channels.
Regulation of Intracellular Ca2+ Stores by Multiple Ca2+-Releasing Messengers
Authors: Cancela Jose M. , Petersen Ole H.
Published: March 2007

Although glucose-elicited insulin secretion depends on Ca2+ entry through voltage-gated Ca2+ channels in the surface cell membrane of the pancreatic β-cell, there is also ample evidence for an important role of intracellular Ca2+ stores, particularly in relation to hormone- or neurotransmitter-induced insulin secretion. There is now direct evidence for Ca2+ entry-induced release of Ca2+ from the endoplasmic reticulum in neurons, but with regard to glucose stimulation of β-cells, there is conflicting evidence about the operation of such a process. This finding suggests that the sensitivity of the Ca2+ release channels in the endoplasmic reticulum membrane varies under different conditions and therefore is regulated. Recent evidence from studies of pancreatic acinar cells has revealed combinatorial roles of multiple messengers in setting the sensitivity of the endoplasmic reticulum for Ca2+ release. Here we focus on the possible combinatorial roles of inositol 1,4,5-trisphosphate, cyclic ADP-ribose, and nicotinic acid adenine dinucleotide phosphate in β-cell function.
Intracellular Targeting of Protein Kinases and Phosphatases
Authors: Alto Neal , Carlisle Michel Jennifer J. , Dodge Kimberly L. , Langeberg Lorene K. , Scott John D.
Published: March 2007

Compartmentalization of kinases and phosphatases is a key determinant in the specificity of second messenger-mediated signaling events. Localization of the cAMP-dependent protein kinase (PKA) and other signaling enzymes is mediated by interaction with A-kinase anchoring proteins (AKAPs). This study focused on recent advances that further our understanding of AKAPs, with particular emphasis on the bidirectional regulation of signaling events by AKAP signaling complexes and their contribution to the control of actin reorganization events.
Regulation of Pancreatic β-Cell Glucokinase
Authors: Matschinsky Franz M.
Published: March 2007

Glucokinase (GK) serves as glucose sensor in pancreatic β-cells and in other glucose sensor cells in the body. Biochemical genetic studies have characterized many activating and inactivating GK mutants that have been discovered in patients with hyperinsulinemic hypoglycemia or diabetes, all inherited as autosomal dominant traits. Mathematical modeling of the kinetic data of recombinant human wild-type and mutant GK accurately predicts the effects of GK mutations on the threshold of glucose-stimulated insulin release and glucose homeostasis. Structure/function studies of the enzyme suggest the existence of a hitherto unknown allosteric activator site of the enzyme that has significant implications for the physiological chemistry of GK-containing cells, particularly the pancreatic β-cells. Glucose is the preeminent positive regulator of β-cell GK expression and involves molecular mechanisms that are still to be elucidated in detail, but seem to have a specific requirement for increased glucose metabolism. Pharmaceutical chemists, motivated by the clear tenets of the GK glucose-sensor paradigm, have searched for and have discovered a novel class of GK activator molecules. The therapeutic application of this basic discovery offers a new principle for drug therapy of diabetes.
Insulin Receptor Signaling and Sarco/Endoplasmic Reticulum Calcium ATPase in β-Cells
Authors: Borge Prabakhar D. , Moibi Jacob , Greene Scott R. , Trucco Matteo , Young Robert A. , Gao Zhiyong , Wolf Bryan A.
Published: March 2007

Glucose is the main physiological secretagogue for insulin secretion by pancreatic β-cells, and the major biochemical mechanisms involved have been elucidated. In particular, an increase in intracellular calcium is important for insulin exocytosis. More recently, it has become apparent that the β-cell also has many of the elements of the insulin receptor signal transduction pathway, including the insulin receptor and insulin receptor substrate (IRS) proteins 1 and 2. Studies with transgenic models have shown that the β-cell-selective insulin receptor knockout and the IRS-1 knockout lead to reduced glucose-induced insulin secretion. Overexpression of the insulin receptor and IRS-1 in β-cells results in increased insulin secretion and increased cytosolic Ca2+. We have thus postulated the existence of a novel autocrine-positive feedback loop of insulin on its own secretion involving interaction with the insulin receptor signal transduction pathway and regulation of intracellular calcium homeostasis. Our current working hypothesis is that this glucose-dependent interaction occurs at the level of IRS-1 and the sarco(endo)plasmic reticulum calcium ATPase, the calcium pump of the endoplasmic reticulum.
Effect of the Insulin Mimetic L-783,281 on Intracellular [Ca2+] and Insulin Secretion From Pancreatic β-Cells
Authors: Roper Michael G. , Qian Wei-jun , Zhang Bei B. , Kulkarni Rohit N. , Kahn C. Ronald , Kennedy Robert T.
Published: March 2007

L-783,281, an antidiabetic fungal metabolite that has previously been shown to activate insulin signaling in CHO cells, was tested for its effect on intracellular Ca2+ ([Ca2+]i) and insulin secretion in single mouse pancreatic β-cells. Application of 10 μmol/l L-783,281 for 40 s to isolated β-cells in the presence of 3 mmol/l glucose increased [Ca2+]i to 178 ± 10% of basal levels (n = 18) as measured by fluo-4 fluorescence. L-767,827, an inactive structural analog of the insulin mimetic, had no effect on β-cell [Ca2+]i. The L-783,281-evoked [Ca2+]i increase was reduced by 82 ± 4% (n = 6, P < 0.001) in cells incubated with 1 μmol/l of the SERCA (sarco/endoplasmic reticulum calcium ATPase) pump inhibitor thapsigargin and reduced by 33 ± 6% (n = 6, P < 0.05) in cells incubated with 20 μmol/l of the l-type Ca2+-channel blocker nifedipine. L-783,281–stimulated [Ca2+]i increases were reduced to 31 ± 3% (n = 9, P < 0.05) and 48 ± 10% (n = 6, P < 0.05) of control values by the phosphatidylinositol 3-kinase (PI3-K) inhibitors LY294002 (25 μmol/l) and wortmannin (100 nmol/l), respectively. In β-cells from IRS-1–/– mice, 10 μmol/l L-783,281 had no significant effect on [Ca2+]i (n = 5). L-783,281 also resulted in insulin secretion at single β-cells. Application of 10 μmol/l L-783,281 for 40 s resulted in 12.2 ± 2.1 (n = 14) exocytotic events as measured by amperometry, whereas the inactive structural analog had no stimulatory effect on secretion. Virtually no secretion was evoked by L-783,281 in IRS-1–/– β-cells. LY294002 (25 μmol/l) significantly reduced the effect of the insulin mimetic on β-cell exocytosis. It is concluded that L-783,281 evokes [Ca2+]i increases and exocytosis in β-cells via an IRS-1/PI3-K–dependent pathway and that the [Ca2+]i increase involves release of Ca2+ from intracellular stores.
Two Generations of Insulinotropic Imidazoline Compounds
Authors: Efendic Suad , Efanov Alexander M. , Berggren Per-Olof , Zaitsev Sergei V.
Published: March 2007

The imidazoline RX871024 increased basal- and glucose-stimulated insulin release in vitro and in vivo. The compound inhibited activity of ATP-sensitive K+ channels as well as voltage-gated K+ channels, which led to membrane depolarization, an increase in the cytosolic Ca2+ concentration ([Ca2+]i), and insulin release. Importantly, RX871024 also enhanced the insulinotropic effect of glucose in cells with clamped [Ca2+]i but in the presence of high ATP and Ca2+concentration inside the cell. We believe that the latter effect on insulin exocytosis was at least in part mediated by a rise in diacylglycerol, which then activated protein kinase C (PKC) and increased the generation of arachidonic acid (AA) metabolites. Activation of both the PKC and AA pathways resulted in potentiation of glucose effects on insulin secretion. Unlike RX871024, the novel imidazoline BL11282 did not block ATP-dependent K+ channels, but similarly to RX871024, it stimulated insulin secretion in depolarized or permeabilized islets. Accordingly, BL11282 did not influence glucose and insulin levels under basal conditions either in vitro or in vivo, but it markedly enhanced the insulinotropic effects of glucose. BL11282 restored the impaired insulin response to glucose in islets from spontaneously diabetic GK rats. We conclude that BL11282 belongs to a new class of insulinotropic compounds that demonstrate a strong glucose-dependent effect on insulin exocytosis.
Recent Advances in the Okamoto Model
Authors: Okamoto Hiroshi , Takasawa Shin
Published: March 2007

Twenty years ago, we first proposed our hypothesis on β-cell damage and its prevention (the Okamoto model), according to which poly(ADP-ribose) synthetase/polymerase (PARP) activation is critically involved in the consumption of NAD+, leading to energy depletion and cell death by necrosis. Recently, the model was reconfirmed by results using PARP knockout mice and has been recognized as providing the basis for necrotic death of various cells and tissues. Based on the model, we proposed two signal systems in β-cells: one is the CD38-cyclic ADP-ribose (cADPR) signal system for insulin secretion, and the other is the regenerating gene protein (Reg)-Reg receptor system for β-cell regeneration. The physiological and pathological significance of the two signal systems in a variety of cells and tissues as well as in pancreatic β-cells has recently been recognized. Here, we describe the Okamoto model and its descendents, the CD38-cADPR signal system and the Reg-Reg receptor system, focusing on recent advances and how their significance came to light. Because PARP is involved in Reg gene transcription to induce β-cell regeneration, and the PARP activation reduces the cellular NAD+ to decrease the formation of cADPR (a second messenger for insulin secretion) and further to cause necrotic β-cell death, PARP and its inhibitors have key roles in the induction of β-cell regeneration, the maintenance of insulin secretion, and the prevention of β-cell death.
Production and Characterization of Reg Knockout Mice
Authors: Unno Michiaki , Nata Koji , Noguchi Naoya , Narushima Yoichi , Akiyama Takako , Ikeda Takayuki , Nakagawa Kei , Takasawa Shin , Okamoto Hiroshi
Published: March 2007

Reg (regenerating gene) was isolated as a gene specifically expressed in regenerating islets. We have demonstrated in vitro and in vivo that the exogenous addition of rat and human Reg gene products, Reg/REG proteins, induced β-cell replication via the Reg receptor and thereby ameliorated experimental diabetes. In the present study, we produced Reg knockout mice by homologous recombination. The Reg gene disruption resulted in a null mutation. Knockout mice developed normally. Islets from the Reg knockout mice appeared morphologically indistinguishable from those of normal controls. However, [3H]thymidine incorporation in isolated islets from Reg knockout mice was decreased. When hyperplastic islets were induced by the injection of goldthioglucose, the average islet size in Reg knockout mice was significantly smaller than that of control Reg+/+ mice. We then produced transgenic mice carrying the Reg gene under the control of the rat insulin II promoter (Ins-Reg) to express Reg in β-cells. Isolated islets from the Ins-Reg transgenic mice showed increased [3H]thymidine incorporation. By intercrossing, we produced NOD mice carrying the Ins-Reg transgene and found that development of diabetes in the resultant Ins-Reg transgenic NOD mice was significantly retarded, coinciding with an increase in the pancreatic β-cell mass. These results indicate that Reg plays an important role in β-cell growth/regeneration.
Activation of Nuclear Factor-κB by Depolarization and Ca2+ Influx in MIN6 Insulinoma Cells
Authors: Bernal-Mizrachi Ernesto , Wen Wu , Shornick Michael , Permutt M. Alan
Published: March 2007

The purpose of the current study was to determine whether nuclear factor-κB (NF-κB) activation is a component of the depolarization/Ca2+-dependent signaling in β-cells. MIN6 cells were transfected with a plasmid containing five tandem repeats of NF-κB binding sites linked to a luciferase reporter. The results of these experiments showed that KCl induced depolarization-activated NF-κB-dependent transcription (3.8-fold at 45 mmol/l, P < 0.01) in a concentration-dependent manner. Tumor necrosis factor-α (TNF-α), a known inducer of NF-κB signaling, activated this construct by 3.4-fold (P < 0.01). The response of NF-κB to depolarization was inhibited by the Ca2+-channel blocker verapamil and by the mitogen-activated protein kinase kinase (MEK) inhibitor PD98059 (70 and 62%, respectively). TNF-α, glucose, and KCl treatment resulted in inhibitory κBα degradation by Western blot analysis. TNF-α treatment and depolarization activation of NF-κB differed significantly in that TNF-α activation was not blocked by PD98059. Transfection with PKA, MEK, and MEK kinase induced NF-κB-dependent transcription by 20-, 90-, and 300-fold, respectively, suggesting that these pathways contribute to the activation in the depolarization response. These findings demonstrate that depolarization/Ca2+ influx, as well as TNF-α treatment, can activate NF-κB-dependent transcription in pancreatic β-cells, but by different signaling pathways. The current studies show that Ca2+ signals in pancreatic β-cells can activate transcription factors involved in the regulation of cell cycle and apoptosis. These findings now add NF-κB to the list of depolarization-induced transcription factors in pancreatic β-cells.
Proinsulin 2 Knockout NOD Mice
Authors: Dubois-Lafforgue Danièle , Mogenet Laurent , Thébault Karine , Jami Jacques , Krief Patricia , Boitard Christian
Published: March 2007

Insulin is a major disease determinant in type 1 diabetes, type 2 diabetes, and related disorders. The role of variations in the expression of the insulin gene has been proposed in genetic susceptibility to the three pathological conditions in humans. In contrast to humans, rodents express two proinsulin isoforms. One isoform, proinsulin 1, is expressed exclusively in islets. The second, proinsulin 2, is expressed in islets and in other tissues, especially the thymus. We took advantage of the expression of these two isoforms to introduce a null proinsulin 2 allele in NOD mice and to evaluate the consequence of a variation of proinsulin 2 gene expression on the development of type 1 diabetes on the NOD genetic background. Heterozygote NOD mutant mice carrying a null proinsulin 2 mutation showed an increased incidence of type 1 diabetes at successive backcross generations. Plasma glucose and insulin levels were identical in prediabetic mutant and in wild-type mice at 4 weeks of age. Variation in insulin gene expression is hypothesized to interfere with diabetes development at both the islet and the thymus level.
Glucose-Dependent Promotion of Insulin Release From Mouse Pancreatic Islets by the Insulin-Mimetic Compound L-783,281
Authors: Westerlund Johanna , Wolf Bryan A. , Bergsten Peter
Published: March 2007

An insulin-mimetic compound (L-783,281) was used in an attempt to determine the role of the β-cell insulin receptor (IR) on insulin release. Islets were isolated from C57Bl/6j mice and cultured for 1 to 4 days. Insulin release from individual islets perifused in the presence of 3 mmol/l glucose was 10.5 ± 1.4 pg/min. Addition of 10 μmol/l L-783,281 had no effect on the rate of insulin secretion. When L-783,281 was added to perifusion medium containing 11 mmol/l glucose, the insulin-mimetic compound significantly increased insulin release. Insulin release from the isolated islet is pulsatile. In the presence of 3 mmol/l glucose, addition of L-783,281 significantly decreased the frequency of the oscillations from 0.35 ± 0.03 to 0.22 ± 0.04 oscillations/min. Addition of L-783,281 to perifusion medium containing 11 mmol/l glucose had no effect on the frequency of the insulin pulses (0.30 ± 0.05 oscillations/min). These results indicate that activation of the β-cell IR by L-783,281 augments insulin release in the presence of a stimulatory glucose concentration. At nonstimulatory glucose concentrations, activation of the β-cell IR may affect mechanisms related to the frequency of the insulin pulses.
Modeling Phasic Insulin Release
Authors: Nesher Rafael , Cerasi Erol
Published: March 2007

The cellular and molecular mechanisms of insulin secretion are being intensively investigated, yet most researchers are seemingly unaware of the complexity of the dynamic regulation of the secretion. In this article, we summarize studies of the physiology of insulin secretion performed over several decades. The insulin response of perifused islets of rats, perfused rat pancreas, or that of a human, to a square-wave glucose stimulus is biphasic, a transient first-phase response of 4- to 10-min duration followed by a gradual rise in secretion rates (second-phase response). Several hypotheses have been proposed to account for the phasic nature of insulin secretion; they are briefly discussed in this review. We have favored the hypothesis that nutrient stimulators such as glucose, in addition to a primary and almost immediate secretory signal, with time induce both stimulatory and inhibitory messages in the β-cell, and those messages modulate the primary insulinogenic signal. Indeed, studies in the rat pancreas and in humans have demonstrated that short stimulations with glucose generate a state of refractoriness of the insulin secretion, which we have termed time-dependent inhibition (TDI). Nonnutrient secretagogues such as arginine induce strong TDI independent of the duration of stimulation. Once the agent is removed, TDI persists for a considerable period. In contrast, prolonged stimulations with glucose (and other nutrients) lead to the amplification of the insulin response to subsequent stimuli; this can be demonstrated in the perfused rat pancreas, in perifused islets from several rodents, and in humans. We have termed this stimulatory signal time-dependent potentiation (TDP). The generation of TDP requires higher glucose concentrations and prolonged stimulation; the effect is retained for some time after cessation of the stimulus. Of major interest is the observation that, while the acute insulin response to glucose is severely reduced in glucose-intolerant animals and humans, TDP seems to be intact. The cellular mechanisms of TDI and TDP are poorly understood, but data reviewed here suggest that they are distinct from those that lead to the acute insulin response to stimuli. A model is proposed whereby the magnitude and kinetics of the insulin response to a given stimulus reflect the balance between TDP and TDI. Researchers studying the cellular and molecular mechanisms of insulin release are urged to take into consideration these complex and opposing factors which regulate insulin secretion.
β-Cell Protein Kinases and the Dynamics of the Insulin Response to Glucose
Authors: Nesher Rafael , Anteby Eyal , Yedovizky Michael , Warwar Nasim , Kaiser Nurit , Cerasi Erol
Published: March 2007

A full biphasic insulin response is the most sensitive index for well-coupled β-cell signal transduction. While first-phase insulin response is extremely sensitive to potentiating and inhibiting modulations, full expression of second-phase response requires near maximally activated β-cell fuel metabolism. In the isolated rat pancreas, accelerated calcium entry or activation of protein kinase (PK)-A or PKC result in no insulin response in the absence of fuel metabolism. At submaximal levels of β-cell fuel secretagogue, arginine (which promotes calcium entry) or glucagon (which activates PKA) produces a small first-phase insulin response but minimal or no second-phase response; carbachol (which activates PKC and promotes calcium entry) generates biphasic insulin response in the presence of minimal fuel (3.3 mmol/l glucose). Glucagon produces full biphasic response in the presence of 10.0 mmol/l glucose, whereas arginine requires near-maximal stimulatory glucose (16.7 mmol) to produce full biphasic insulin response. Thus, PKA and PKC signal pathways potentiate primary signals generated by fuel secretagogues to induce full biphasic insulin response, while calcium recruitment alone is insufficient to potentiate primary signals generated at low levels of fuel secretagogue. We suggest that three families of PKs (calmodulin-dependent PK [CaMK], PKA, and PKC) function as distal amplifiers for stimulus–secretion coupling signals originating from fuel metabolism, as well as from incretins acting through membrane receptors, adenylate cyclase, and phospholipase C. Several isoenzymes of PKA and PKC are present in pancreatic β-cells, but the specific function of most is still undefined. Each PK isoenzyme is activated and subsequently phosphorylates its specific effector protein by binding to a highly specific anchoring protein. Some diabetes-related β-cell derangements may be linked to abnormal function of one or more PK isoenzymes. Identification and characterization of the specific function of the individual PK isoenzymes may provide the tool to improve the insulin response of the diabetic patient.
The Effects of Cerulenin, an Inhibitor of Protein Acylation, on the Two Phases of Glucose-Stimulated Insulin Secretion
Authors: Straub Susanne G. , Yajima Hiroki , Komatsu Mitsuhisa , Aizawa Toru , Sharp Geoffrey W.G.
Published: March 2007

The potential role of protein acylation in the control of biphasic insulin secretion has been studied in isolated rat pancreatic islets. The protein acylation inhibitor cerulenin inhibited both phases of glucose-stimulated insulin secretion. However, it did not affect the secretory response to a depolarizing concentration of KCl in either the absence or presence of diazoxide. Therefore, cerulenin has no deleterious effect on the l-type Ca2+ channels or subsequent events in Ca2+ stimulus–secretion coupling. Advantage was taken of this to study the effect of cerulenin on the KATP channel–independent pathway of glucose signaling. In the presence of KCl and diazoxide, cerulenin powerfully inhibited the augmentation of insulin release by glucose and palmitate. Similar inhibition of the augmentation of release by glucose and palmitate was seen under Ca2+-free conditions in the presence of 12-O-tetradecanoylphorbol-13-acetate and forskolin. As neither glucose oxidation nor the effect of glucose to inhibit fatty acid oxidation is affected by cerulenin, these data suggest that protein acylation is involved in the KATP channel–independent pathway of glucose signaling.
Molecular Detection of Circulating β-Cells After Islet Transplantation
Authors: Ritz-Laser Beate , Oberholzer José , Toso Christian , Brulhart Marie-Claude , Zakrzewska Katerina , Ris Frédéric , Bucher Pascal , Morel Philippe , Philippe Jacques
Published: March 2007

Islet transplantation is a promising treatment for type 1 diabetes. However, islet grafts are submitted to multiple injuries, including immunosuppressive drug toxicity, hyperglycemia, hypoxia, unspecific inflammatory reactions, as well as allo- and autoimmune destruction. Therapeutic approaches to these damage mechanisms require early detection of islet injury, which is currently not feasible because of the lack of efficient markers. Based on the hypothesis of islet dissociation and release of islet cells into the circulation during islet injury, we designed a highly sensitive and specific molecular assay, able to detect two β-cells per milliliter of venous blood by RT-PCR of insulin mRNA. We report that circulating β-cells can be demonstrated up to 10 weeks after intraportal islet transplantation, as assessed after six islet grafts in four type 1 diabetic patients. Furthermore, our results suggest that the time during which circulating islet cells can be detected may depend on the graft environment and the immunosuppressive regimen. This test may allow better estimation of islet cell loss and identification of factors involved in islet graft injury.
Elevation of Cytotoxic Lymphocyte Gene Expression Is Predictive of Islet Allograft Rejection in Nonhuman Primates
Authors: Han Dongmei , Xu Xiumin , Pastori Ricardo L. , Ricordi Camillo , Kenyon Norma S.
Published: March 2007

Hyperglycemia and increased insulin requirements are indicators of ongoing islet allograft rejection, but there are no methods to predict or confirm rejection. Elevation of cytotoxic lymphocyte (CL) gene expression in peripheral blood (PB) has been correlated with renal allograft rejection in humans, but no published study has assessed the utility of monitoring these markers as predictors of rejection before the onset of clinical symptoms. We have established quantitative real-time PCR methods to determine the levels of mRNA transcripts for the CL genes granzyme B (GB), perforin, and fas ligand in blood samples from rhesus and cynomolgus monkeys. Four rhesus monkeys with long-term islet allograft function were studied. Antirejection (anti-CD154) therapy was discontinued, and weekly PB samples were obtained to determine whether the levels of mRNA transcripts for CL genes correlated with and/or were predictive of islet allograft rejection, defined as a loss of C-peptide production. For all monkeys, elevation of CL gene expression preceded rejection by 83–197 days, with GB as the best predictor. Elevated mRNA levels were sustained for 2–2.5 months in three of four animals and 1 month in the other, thus suggesting that the testing of these parameters may have practical applications in clinical islet cell transplantation.
Human Skeletal Muscle Expresses a Glycogen-Targeting Subunit of PP1 That Is Identical to the Insulin-Sensitive Glycogen-Targeting Subunit GL of Liver
Authors: Munro Shonagh , Cuthbertson Daniel J.R. , Cunningham Joan , Sales Mark , Cohen Patricia T.W.
Published: March 2007

Insulin has been previously shown to regulate the expression of the hepatic glycogen-targeting subunit, GL, of protein phosphatase 1 (PP1) and is believed to control the activity of the PP1-GL complex by modulation of the level of phosphorylase a, which allosterically inhibits the activity of PP1-GL. These mechanisms contribute to the ability of insulin to increase hepatic glycogen synthesis. Human GL shows >88% amino acid identity to its rat and mouse homologs, with complete conservation of the phosphorylase a binding site. GL is highly expressed in the liver and present at appreciable levels in heart tissue of all three species. Surprisingly, GL is highly expressed in human skeletal muscle while only being detected at very low levels in rat, mouse, and rabbit skeletal muscle. The amino acid sequence of GL predicted from the cDNA is identical in human liver and skeletal muscle and encoded by a gene on chromosome 8 at p23.1. The species-specific difference in the level of expression of GL mRNA and protein in skeletal muscle has important implications for understanding the mechanisms by which insulin regulates glycogen synthesis in human skeletal muscle and for questions regarding whether rodents are appropriate models for this purpose.