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1.  Direct Autocrine Action of Insulin on β-Cells: Does It Make Physiological Sense? 
Diabetes  2013;62(7):2157-2163.
In recent years there has been a growing interest in the possibility of a direct autocrine effect of insulin on the pancreatic β-cell. Indeed, there have been numerous intriguing articles and several eloquent reviews written on the subject (1–3); however, the concept is still controversial. Although many in vitro experiments, a few transgenic mouse studies, and some human investigations would be supportive of the notion, there exist different insights, other studies, and circumstantial evidence that question the concept. Therefore, the idea of autocrine action of insulin remains a conundrum. Here we outline a series of thoughts, insights, and alternative interpretations of the available experimental evidence. We ask, how convincing are these, and what are the confusing issues? We agree that there is a clear contribution of certain downstream elements in the insulin signaling pathway for β-cell function and survival, but the question of whether insulin itself is actually the physiologically relevant ligand that triggers this signal transduction remains unsettled.
PMCID: PMC3712043  PMID: 23801714
2.  FoxO Feedback Control of Basal IRS-2 Expression in Pancreatic β-Cells Is Distinct From That in Hepatocytes 
Diabetes  2011;60(11):2883-2891.
Appropriate regulation of insulin receptor substrate 2 (IRS-2) expression in pancreatic β-cells is essential to adequately compensate for insulin resistance. In liver, basal IRS-2 expression is controlled via a temporal negative feedback of sterol regulatory element–binding protein 1 (SREBP-1) to antagonize transcription factors forkhead box class O (FoxO)1/FoxO3a at an insulin response element (IRE) on the IRS-2 promoter. The purpose of the study was to examine if a similar mechanism controlled IRS-2 expression in β-cells.
IRS-2 mRNA and protein expression, as well as IRS-2 gene promoter activity, were examined in isolated rat islets. Specific transcription factor association with the IRE on the IRS-2 promoter was examined by chromatin immunoprecipitation (ChIP) assay, and their nuclear translocation was examined by immunofluorescence. A direct in vivo effect of insulin on control of IRS-2 expression in liver and pancreatic islets was also investigated.
In IRS-2 promoter-reporter assays conducted in isolated islets, removal of the IRE decreased basal IRS-2 promoter activity in β-cells up to 80%. Activation of IRS signaling in isolated rat islets by insulin/IGF-I (used as an experimental in vitro tool) or downstream constitutive activation of protein kinase B (PKB) significantly decreased IRS-2 expression. In contrast, inhibition of phosphatidylinositol 3-kinase (PI3K) or PKB significantly increased IRS-2 levels in β-cells. ChIP assays indicated that transcription factors FoxO1 and FoxO3a associated with the IRE on the IRS-2 promoter in β-cells in a PI3K/PKB–dependent manner, whereas others, such as SREBP-1, the transcription factor binding to immunoglobulin heavy chain enhancer 3′, and the aryl hydrocarbon receptor nuclear translocator (ARNT), did not. However, only FoxO3a, not FoxO1, was capable of driving IRS-2 promoter activity via the IRE in β-cells. In vivo studies showed insulin was able to suppress IRS-2 expression via activation of SREBP-1 in the liver, but this mechanism was not apparent in pancreatic islets from the same animal.
The molecular mechanism for feedback control of IRS signaling to decrease IRS-2 expression in liver and β-cells is quite distinct, with a predominant role played by FoxO3a in β-cells.
PMCID: PMC3198101  PMID: 21933986
3.  Specific Glucose-Induced Control of Insulin Receptor Substrate-2 Expression Is Mediated via Ca2+-Dependent Calcineurin/NFAT Signaling in Primary Pancreatic Islet β-Cells 
Diabetes  2011;60(11):2892-2902.
Insulin receptor substrate-2 (IRS-2) plays an essential role in pancreatic islet β-cells by promoting growth and survival. IRS-2 turnover is rapid in primary β-cells, but its expression is highly regulated at the transcriptional level, especially by glucose. The aim was to investigate the molecular mechanism on how glucose regulates IRS-2 gene expression in β-cells.
Rat islets were exposed to inhibitors or subjected to adenoviral vector–mediated gene manipulations and then to glucose-induced IRS-2 expression analyzed by real-time PCR and immunoblotting. Transcription factor nuclear factor of activated T cells (NFAT) interaction with IRS-2 promoter was analyzed by chromatin immunoprecipitation assay and glucose-induced NFAT translocation by immunohistochemistry.
Glucose-induced IRS-2 expression occurred in pancreatic islet β-cells in vivo but not in liver. Modulating rat islet β-cell Ca2+ influx with nifedipine or depolarization demonstrated that glucose-induced IRS-2 gene expression was dependent on a rise in intracellular calcium concentration derived from extracellular sources. Calcineurin inhibitors (FK506, cyclosporin A, and a peptide calcineurin inhibitor [CAIN]) abolished glucose-induced IRS-2 mRNA and protein levels, whereas expression of a constitutively active calcineurin increased them. Specific inhibition of NFAT with the peptide inhibitor VIVIT prevented a glucose-induced IRS-2 transcription. NFATc1 translocation to the nucleus in response to glucose and association of NFATc1 to conserved NFAT binding sites in the IRS-2 promoter were demonstrated.
The mechanism behind glucose-induced transcriptional control of IRS-2 gene expression specific to the islet β-cell is mediated by the Ca2+/calcineurin/NFAT pathway. This insight into the IRS-2 regulation could provide novel therapeutic means in type 2 diabetes to maintain an adequate functional mass.
PMCID: PMC3198104  PMID: 21940781
4.  Islet Autoantigens: Structure, Function, Localization, and Regulation 
Islet autoantigens associated with autoimmune type 1 diabetes (T1D) are expressed in pancreatic β cells, although many show wider patterns of expression in the neuroendocrine system. Within pancreatic β cells, every T1D autoantigen is in one way or another linked to the secretory pathway. Together, these autoantigens play diverse roles in glucose regulation, metabolism of biogenic amines, as well as the regulation, formation, and packaging of secretory granules. The mechanism(s) by which immune tolerance to islet-cell antigens is lost during the development of T1D, remains unclear. Antigenic peptide creation for immune presentation may potentially link to the secretory biology of β cells in a number of ways, including proteasomal digestion of misfolded products, exocytosis and endocytosis of cell-surface products, or antigen release from dying β cells during normal or pathological turnover. In this context, we evaluate the biochemical nature and immunogenicity of the major autoantigens in T1D including (pro)insulin, GAD65, ZnT8, IA2, and ICA69.
In pancreatic β cells, every type 1 diabetes autoantigen including (pro)insulin, GAD65, ZnT8, IA2, and ICA69 is connected to the secretory pathway.
PMCID: PMC3405822  PMID: 22908193
5.  Islet Autoantigens: Structure, Function, Localization, and Regulation 
Cold Spring Harbor perspectives in medicine  2012;2(8):10.1101/cshperspect.a007658 a007658.
Islet autoantigens associated with autoimmune type 1 diabetes (T1D) are expressed in pancreatic β cells, although many show wider patterns of expression in the neuroendocrine system. Within pancreatic β cells, every T1D autoantigen is in one way or another linked to the secretory pathway. Together, these autoantigens play diverse roles in glucose regulation, metabolism of biogenic amines, as well as the regulation, formation, and packaging of secretory granules. The mechanism(s) by which immune tolerance to islet-cell antigens is lost during the development of T1D, remains unclear. Antigenic peptide creation for immune presentation may potentially link to the secretory biology of β cells in a number of ways, including proteasomal digestion of misfolded products, exocytosis, and endocytosis of cell-surface products, or antigen release from dying β cells during normal or pathological turnover. In this context, we evaluate the biochemical nature and immunogenicity of the major autoantigens in T1D including (pro)insulin, GAD65, ZnT8, IA2, and ICA69.
PMCID: PMC3405822  PMID: 22908193
6.  Supplementation of iron in pulmonary hypertension: Rationale and design of a phase II clinical trial in idiopathic pulmonary arterial hypertension 
Pulmonary Circulation  2013;3(1):100-107.
Our aim is to assess the safety and potential clinical benefit of intravenous iron (Ferinject) infusion in iron deficient patients with idiopathic pulmonary arterial hypertension (IPAH). Iron deficiency in the absence of anemia (1) is common in patients with IPAH; (2) is associated with inappropriately raised levels of hepcidin, the key regulator of iron homeostasis; and (3) correlates with disease severity and worse clinical outcomes. Oral iron absorption may be impeded by reduced absorption due to elevated hepcidin levels. The safety and benefits of parenteral iron replacement in IPAH are unknown. Supplementation of Iron in Pulmonary Hypertension (SIPHON) is a Phase II, multicenter, double-blind, randomized, placebo-controlled, crossover clinical trial of iron in IPAH. At least 60 patients will be randomized to intravenous ferric carboxymaltose (Ferinject) or saline placebo with a crossover point after 12 weeks of treatment. The primary outcome will be the change in resting pulmonary vascular resistance from baseline at 12 weeks, measured by cardiac catheterization. Secondary measures include resting and exercise hemodynamics and exercise performance from serial bicycle incremental and endurance cardiopulmonary exercise tests. Other secondary measurements include serum iron indices, 6-Minute Walk Distance, WHO functional class, quality of life score, N-terminal pro-brain natriuretic peptide (NT-proBNP), and cardiac anatomy and function from cardiac magnetic resonance. We propose that intravenous iron replacement will improve hemodynamics and clinical outcomes in IPAH. If the data supports a potentially useful therapeutic effect and suggest this drug is safe, the study will be used to power a Phase III study to address efficacy.
PMCID: PMC3641712  PMID: 23662181
exercise capacity; ferric carboxymaltose; iron; pulmonary arterial hypertension; pulmonary vascular resistance
7.  Increased Phagocyte-Like NADPH Oxidase and ROS Generation in Type 2 Diabetic ZDF Rat and Human Islets 
Diabetes  2011;60(11):2843-2852.
To determine the subunit expression and functional activation of phagocyte-like NADPH oxidase (Nox), reactive oxygen species (ROS) generation and caspase-3 activation in the Zucker diabetic fatty (ZDF) rat and diabetic human islets.
Expression of core components of Nox was quantitated by Western blotting and densitometry. ROS levels were quantitated by the 2′,7′-dichlorofluorescein diacetate method. Rac1 activation was quantitated using the gold-labeled immunosorbent assay kit.
Levels of phosphorylated p47phox, active Rac1, Nox activity, ROS generation, Jun NH2-terminal kinase (JNK) 1/2 phosphorylation, and caspase-3 activity were significantly higher in the ZDF islets than the lean control rat islets. Chronic exposure of INS 832/13 cells to glucolipotoxic conditions resulted in increased JNK1/2 phosphorylation and caspase-3 activity; such effects were largely reversed by SP600125, a selective inhibitor of JNK. Incubation of normal human islets with high glucose also increased the activation of Rac1 and Nox. Lastly, in a manner akin to the ZDF diabetic rat islets, Rac1 expression, JNK1/2, and caspase-3 activation were also significantly increased in diabetic human islets.
We provide the first in vitro and in vivo evidence in support of an accelerated Rac1–Nox–ROS–JNK1/2 signaling pathway in the islet β-cell leading to the onset of mitochondrial dysregulation in diabetes.
PMCID: PMC3198065  PMID: 21911753
8.  Leptin action via neurotensin neurons controls orexin, the mesolimbic dopamine system and energy balance 
Cell metabolism  2011;14(3):313-323.
Leptin acts on leptin receptor (LepRb)-expressing neurons throughout the brain, but the roles for many populations of LepRb neurons in modulating energy balance and behavior remain unclear. We found that the majority of LepRb neurons in the lateral hypothalamic area (LHA) contain neurotensin (Nts). To investigate the physiologic role for leptin action via these LepRbNts neurons, we generated mice null for LepRb specifically in Nts neurons (Nts-LepRbKO mice). Nts-LepRbKO mice demonstrate early-onset obesity, modestly increased feeding, and decreased locomotor activity. Furthermore, consistent with the connection of LepRbNts neurons with local OX neurons and the ventral tegmental area (VTA), Nts-LepRbKO mice exhibit altered regulation of OX neurons and the mesolimbic DA system. Thus, LHA LepRbNts neurons mediate physiologic leptin action on OX neurons and the mesolimbic DA system, and contribute importantly to the control of energy balance.
PMCID: PMC3183584  PMID: 21907138
leptin; neurotensin; obesity; orexin; dopamine; amphetamine
10.  Arf nucleotide binding site opener [ARNO] promotes sequential activation of Arf6, Cdc42 and Rac1 and insulin secretion in INS 832/13 β-cells and rat islets 
Biochemical pharmacology  2011;81(8):1016-1027.
Glucose-stimulated insulin secretion [GSIS] involves interplay between small G-proteins and their regulatory factors. Herein, we tested the hypothesis that Arf nucleotide binding site opener [ARNO], a guanine nucleotide exchange factor [GEF] for the small G-protein Arf6, mediates the functional activation of Arf6, and that ARNO/Arf6 signaling axis, in turn, controls the activation of Cdc42 and Rac1, which have been implicated in GSIS. Molecular biological [i.e., expression of inactive mutants or siRNA] and pharmacological approaches were employed to assess the roles for ARNO/Arf6 signaling pathway in insulin secretion in normal rat islets and INS 832/13 cells. Degrees of activation of Arf6 and Cdc42/Rac1 were quantitated by GST-GGA3 and PAK-1 kinase pull-down assays, respectively. ARNO is expressed in INS 832/13 cells, rat islets and human islets. Expression of inactive mutants of Arf6 [Arf6-T27N] or ARNO [ARNO-E156K] or siRNA-ARNO markedly reduced GSIS in isolated β-cells. secinH3, a selective inhibitor of ARNO/Arf6 signaling axis, also inhibited GSIS in INS 832/13 cells and rat islets. Stimulatory concentrations of glucose promoted Arf6 activation, which was inhibited by secinH3 or siRNA-ARNO, suggesting that ARNO/Arf6 signaling cascade is necessary for GSIS. secinH3 or siRNA-ARNO also inhibited glucose-induced activation of Cdc42 and Rac1 suggesting that ARNO/Arf6 might be upstream to Cdc42 and Rac1 activation steps, which are necessary for GSIS. Lastly, co-immunoprecipitation and confocal microscopic studies suggested increased association between Arf6 and ARNO in glucose-stimulated β-cells. These findings provide the first evidence to implicate ARNO in the sequential activation of Arf6, Cdc42 and Rac1 culminating in GSIS.
PMCID: PMC3073812  PMID: 21276423
Insulin secretion; pancreatic islet; ARNO; Arf6; Rac1; secinH3
11.  Modelling the Influence of Foot-and-Mouth Disease Vaccine Antigen Stability and Dose on the Bovine Immune Response 
PLoS ONE  2012;7(2):e30435.
Foot and mouth disease virus causes a livestock disease of significant global socio-economic importance. Advances in its control and eradication depend critically on improvements in vaccine efficacy, which can be best achieved by better understanding the complex within-host immunodynamic response to inoculation. We present a detailed and empirically parametrised dynamical mathematical model of the hypothesised immune response in cattle, and explore its behaviour with reference to a variety of experimental observations relating to foot and mouth immunology. The model system is able to qualitatively account for the observed responses during in-vivo experiments, and we use it to gain insight into the incompletely understood effect of single and repeat inoculations of differing dosage using vaccine formulations of different structural stability.
PMCID: PMC3281838  PMID: 22363437
12.  Glycogen synthase kinase 3β mediates high glucose-induced ubiquitination and proteasome degradation of insulin receptor substrate 1 
The Journal of endocrinology  2010;206(2):171-181.
High glucose (HG) has been shown to induce insulin resistance in both type 1 and type 2 diabetes. However, the molecular mechanism behind this phenomenon is unknown. Insulin receptor substrate (IRS) proteins are the key signaling molecules that mediate insulin’s intracellular actions. Genetic and biological studies have shown that reductions in IRS1 and/or IRS2 protein levels are associated with insulin resistance. In this study we have shown that proteasome degradation of IRS1, but not of IRS2, is involved in HG-induced insulin resistance in Chinese hamster ovary (CHO) cells as well as in primary hepatocytes. To further investigate the molecular mechanism by which HG induces insulin resistance, we examined various molecular candidates with respect to their involvement in the reduction in IRS1 protein levels. In contrast to the insulin-induced degradation of IRS1, HG-induced degradation of IRS1 did not require IR signaling or phosphatidylinositol 3-kinase/Akt activity. We have identified glycogen synthase kinase 3β (GSK3β or GSK3B as listed in the MGI Database) as a kinase required for HG-induced serine332 phosphorylation, ubiquitination, and degradation of IRS1. Overexpression of IRS1 with mutation of serine332 to alanine partially prevents HG-induced IRS1 degradation. Furthermore, overexpression of constitutively active GSK3β was sufficient to induce IRS1 degradation. Our data reveal the molecular mechanism of HG-induced insulin resistance, and support the notion that activation of GSK3β contributes to the induction of insulin resistance via phosphorylation of IRS1, triggering the ubiquitination and degradation of IRS1.
PMCID: PMC3072280  PMID: 20466847
13.  Glucose and Fatty Acids Synergize to Promote B-Cell Apoptosis through Activation of Glycogen Synthase Kinase 3β Independent of JNK Activation 
PLoS ONE  2011;6(4):e18146.
The combination of elevated glucose and free-fatty acids (FFA), prevalent in diabetes, has been suggested to be a major contributor to pancreatic β-cell death. This study examines the synergistic effects of glucose and FFA on β-cell apoptosis and the molecular mechanisms involved. Mouse insulinoma cells and primary islets were treated with palmitate at increasing glucose and effects on apoptosis, endoplasmic reticulum (ER) stress and insulin receptor substrate (IRS) signaling were examined.
Principal Findings
Increasing glucose (5–25 mM) with palmitate (400 µM) had synergistic effects on apoptosis. Jun NH2-terminal kinase (JNK) activation peaked at the lowest glucose concentration, in contrast to a progressive reduction in IRS2 protein and impairment of insulin receptor substrate signaling. A synergistic effect was observed on activation of ER stress markers, along with recruitment of SREBP1 to the nucleus. These findings were confirmed in primary islets. The above effects associated with an increase in glycogen synthase kinase 3β (Gsk3β) activity and were reversed along with apoptosis by an adenovirus expressing a kinase dead Gsk3β.
Glucose in the presence of FFA results in synergistic effects on ER stress, impaired insulin receptor substrate signaling and Gsk3β activation. The data support the importance of controlling both hyperglycemia and hyperlipidemia in the management of Type 2 diabetes, and identify pancreatic islet β-cell Gsk3β as a potential therapeutic target.
PMCID: PMC3082528  PMID: 21541314
14.  Protein Farnesylation–Dependent Raf/Extracellular Signal–Related Kinase Signaling Links to Cytoskeletal Remodeling to Facilitate Glucose-Induced Insulin Secretion in Pancreatic β-Cells 
Diabetes  2010;59(4):967-977.
Posttranslational prenylation (e.g., farnesylation) of small G-proteins is felt to be requisite for cytoskeletal remodeling and fusion of secretory vesicles with the plasma membrane. Here, we investigated roles of protein farnesylation in the signaling steps involved in Raf-1/extracellular signal–related kinase (ERK1/2) signaling pathway in glucose-induced Rac1 activation and insulin secretion in the pancreatic β-cell.
These studies were carried out in INS 832/13 cells and normal rat islets. Molecular biological (e.g., overexpression or small interfering RNA [siRNA]–mediated knockdown) and pharmacologic approaches were used to determine roles for farnesylation in glucose-mediated activation of ERK1/2, Rac1, and insulin secretion. Activation of ERK1/2 was determined by Western blotting. Rac1 activation (i.e., Rac1.GTP) was quantitated by p21-activated kinase pull-down assay. Insulin release was quantitated by enzyme-linked immunosorbent assay.
Coprovision of structure-specific inhibitors of farnesyl transferase (FTase; e.g., FTI-277 or FTI-2628) or siRNA-mediated knockdown of FTase β-subunit resulted in a significant inhibition of glucose-stimulated ERK1/2 and Rac1 activation and insulin secretion. Pharmacologic inhibition of Raf-1 kinase using GW-5074 markedly reduced the stimulatory effects of glucose on ERK1/2 phosphorylation, Rac1 activation, and insulin secretion, suggesting that Raf-1 kinase activation may be upstream to ERK1/2 and Rac1 activation leading to glucose-induced insulin release. Lastly, siRNA-mediated silencing of endogenous expression of ERK1/2 markedly attenuated glucose-induced Rac1 activation and insulin secretion.
Together, our findings provide the first evidence of a role for protein farnesylation in glucose-mediated regulation of the Raf/ERK signaling pathway culminating in the activation of Rac1, which has been shown to be necessary for cytoskeletal reorganization and exocytotic secretion of insulin.
PMCID: PMC2844844  PMID: 20071600
15.  Direct innervation and modulation of orexin neurons by lateral hypothalamic LepRb neurons 
Leptin, the adipose-derived hormonal signal of body energy stores, acts via the leptin receptor (LepRb) on neurons in multiple brain regions. We previously identified LepRb neurons in the lateral hypothalamic area (LHA), which are distinct from neighboring leptin-regulated melanin concentrating hormone (MCH)- or orexin (OX)-expressing cells. Neither the direct synaptic targets of LHA LepRb neurons nor their potential role in the regulation of other LHA neurons have been determined, however. We thus generated several adenoviral and transgenic systems in which cre recombinase promotes the expression of the tracer, wheat germ agglutinin (WGA), and utilized these in combination with LepRbcre mice to determine the neuronal targets of LHA LepRb neurons. This analysis revealed that, while some LHA LepRb neurons project to dopamine neurons in the ventral tegmental area (VTA), LHA LepRb neurons also densely innervate the LHA where they directly synapse with OX, but not MCH, neurons. Indeed, few other LepRb neurons in the brain project to the OX-containing region of the mouse LHA, and direct leptin action via LHA LepRb neurons regulates gene expression in OX neurons. These findings thus reveal a major role for LHA leptin action in the modulation of OX neurons, suggesting the importance of LHA LepRb neurons in the regulation of OX signaling that is crucial to leptin action and metabolic control.
PMCID: PMC2930259  PMID: 20739548
Leptin receptor; lateral hypothalamus; orexin; mesolimbic system; WGA; GFP
16.  Simvastatin as a Treatment for Pulmonary Hypertension Trial 
Rationale: In animal models of pulmonary hypertension, simvastatin has been shown to reduce pulmonary artery pressure and induce regression of associated right ventricular (RV) hypertrophy.
Objectives: To assess the therapeutic value of simvastatin in patients with pulmonary arterial hypertension (PAH).
Methods: Forty-two patients with PAH were randomized to receive either simvastatin (80 mg/d) or placebo in addition to current care for 6 months, and thereafter offered open-label simvastatin. The primary outcome was change in RV mass, assessed by cardiac magnetic resonance (CMR).
Measurements and Main Results: At 6 months, RV mass decreased by 5.2 ± 11 g in the statin group (P = 0.045) and increased 3.9 ± 14 g in the placebo group. The treatment effect was −9.1 g (P = 0.028). N-terminal pro–B-type natriuretic peptide (NT-proBNP) levels decreased significantly in the statin group (−75 ± 167 fmol/ml; P = 0.02) but not the placebo group (49 ± 224 fmol/ml; P = 0.43; overall treatment effect −124 fmol/ml; P = 0.041). There were no significant changes in other outcome measures (including 6-minute walk test, cardiac index, and circulating cytokines). From 6 to 12 months, both RV mass and NT-proBNP increased toward baseline values in 16 patients on active treatment who continued with simvastatin but remained stable in 18 patients who switched from placebo to simvastatin. Two patients required a reduction in dose but not cessation of simvastatin.
Conclusions: Simvastatin added to conventional therapy produces a small and transient early reduction in RV mass and NT-proBNP levels in patients with PAH, but this is not sustained over 12 months.
Clinical trial registered with (NCT00180713).
PMCID: PMC2874452  PMID: 20460548
hypertension, pulmonary; simvastatin; MRI; hypertrophy
17.  VTA Leptin Receptor Neurons Specifically Project to and Regulate CART Neurons of the Extended Central Amygdala 
Leptin acts via its receptor (LepRb) to regulate neural circuits in concert with body energy stores. In addition to acting on a number of hypothalamic structures, leptin modulates the mesolimbic dopamine (DA) system. To determine the sites at which LepRb neurons might directly influence the mesolimbic DA system, we examined the distribution of LepRb neurons and their projections within mesolimbic brain regions. Although the ventral tegmental area (VTA) contains DA LepRb neurons, LepRb neurons are absent from the amygdala and striatum. Also, LepRb-EGFPf mice (which label projections from LepRb neurons throughout the brain) reveal that few LepRb neurons project to the nucleus accumbens (NAc). In contrast, the central amygdala (CeA) and its rostral extension receive copious projections from LepRb neurons. Indeed, LepRb-specific anterograde tracing demonstrates (and retrograde tracing confirms) that VTA LepRb neurons project to the extended CeA (extCeA), but not the NAc. Consistently, leptin promotes CREB phosphorylation in the extCeA, but not NAc, of leptin-deficient animals. Furthermore, transgenic mice expressing the trans-synaptic tracer, wheat germ agglutinin, in LepRb neurons reveal the innervation of CeA cocaine and amphetamine regulated transcript (CART) neurons by LepRb neurons, and leptin suppresses the increased CeA CART expression of leptin-deficient animals. Thus, LepRb VTA neurons represent a subclass of VTA DA neurons that specifically innervate and control the extCeA; we hypothesize that these neurons primarily modulate CeA directed behaviors.
PMCID: PMC2864009  PMID: 20410123
midbrain; hypothalamus; obesity; striatum; dopamine
18.  Evolutionary Entropy Determines Invasion Success in Emergent Epidemics 
PLoS ONE  2010;5(9):e12951.
Standard epidemiological theory claims that in structured populations competition between multiple pathogen strains is a deterministic process which is mediated by the basic reproduction number () of the individual strains. A new theory based on analysis, simulation and empirical study challenges this predictor of success.
Principal Findings
We show that the quantity is a valid predictor in structured populations only when size is infinite. In this article we show that when population size is finite the dynamics of infection by multi-strain pathogens is a stochastic process whose outcome can be predicted by evolutionary entropy, S, an information theoretic measure which describes the uncertainty in the infectious age of an infected parent of a randomly chosen new infective. Evolutionary entropy characterises the demographic stability or robustness of the population of infectives. This statistical parameter determines the duration of infection and thus provides a quantitative index of the pathogenicity of a strain. Standard epidemiological theory based on as a measure of selective advantage is the limit as the population size tends to infinity of the entropic selection theory. The standard model is an approximation to the entropic selection theory whose validity increases with population size.
An epidemiological analysis based on entropy is shown to explain empirical observations regarding the emergence of less pathogenic strains of human influenza during the antigenic drift phase. Furthermore, we exploit the entropy perspective to discuss certain epidemiological patterns of the current H1N1 swine 'flu outbreak.
PMCID: PMC2944876  PMID: 20886082
19.  Involvement of Per-Arnt-Sim Kinase and Extracellular-Regulated Kinases-1/2 in Palmitate Inhibition of Insulin Gene Expression in Pancreatic β-Cells 
Diabetes  2009;58(9):2048-2058.
Prolonged exposure of pancreatic β-cells to simultaneously elevated levels of fatty acids and glucose (glucolipotoxicity) impairs insulin gene transcription. However, the intracellular signaling pathways mediating these effects are mostly unknown. This study aimed to ascertain the role of extracellular-regulated kinases (ERKs)1/2, protein kinase B (PKB), and Per-Arnt-Sim kinase (PASK) in palmitate inhibition of insulin gene expression in pancreatic β-cells.
MIN6 cells and isolated rat islets were cultured in the presence of elevated glucose, with or without palmitate or ceramide. ERK1/2 phosphorylation, PKB phosphorylation, and PASK expression were examined by immunoblotting and real-time PCR. The role of these kinases in insulin gene expression was assessed using pharmacological and molecular approaches.
Exposure of MIN6 cells and islets to elevated glucose induced ERK1/2 and PKB phosphorylation, which was further enhanced by palmitate. Inhibition of ERK1/2, but not of PKB, partially prevented the inhibition of insulin gene expression in the presence of palmitate or ceramide. Glucose-induced expression of PASK mRNA and protein levels was reduced in the presence of palmitate. Overexpression of wild-type PASK increased insulin and pancreatic duodenal homeobox-1 gene expression in MIN6 cells and rat islets incubated with glucose and palmitate, whereas overexpression of a kinase-dead PASK mutant in rat islets decreased expression of insulin and pancreatic duodenal homeobox-1 and increased C/EBPβ expression.
Both the PASK and ERK1/2 signaling pathways mediate palmitate inhibition of insulin gene expression. These findings identify PASK as a novel mediator of glucolipotoxicity on the insulin gene in pancreatic β-cells.
PMCID: PMC2731539  PMID: 19502418
20.  Leptin acts via leptin receptor-expressing lateral hypothalamic neurons to modulate the mesolimbic dopamine system and suppress feeding 
Cell metabolism  2009;10(2):89-98.
The lateral hypothalamic area (LHA) acts in concert with the ventral tegmental area (VTA) and other components of the mesolimbic dopamine (DA) system to control motivation, including the incentive to feed. The anorexigenic hormone, leptin, modulates the mesolimbic DA system, although the mechanisms underlying this control have remained incompletely understood. We show that leptin directly regulates a population of leptin receptor (LepRb)-expressing inhibitory neurons in the LHA, and that leptin action via these LHA LepRb neurons decreases feeding and body weight. Furthermore, these LHA LepRb neurons innervate the VTA, and leptin action on these neurons restores VTA expression of the rate-limiting enzyme in DA production along with mesolimbic DA content in leptin-deficient animals. Thus, these findings reveal that LHA LepRb neurons link anorexic leptin action to the mesolimbic DA system.
PMCID: PMC2723060  PMID: 19656487
obesity; tyrosine hydroxylase; VTA; nucleus accumbens
21.  Direct innervation of GnRH neurons by metabolic- & sexual odorant-sensing leptin receptor neurons in the hypothalamic ventral premammillary nucleus 
Leptin acts via its receptor (LepRb) on specific CNS neurons to signal the adequacy of long-term energy stores, thereby permitting the expenditure of resources on energy-intensive processes such as reproduction. The ventral premammillary nucleus of the hypothalamus (PMv), which has been implicated in the stimulation of gonadotropin release by olfactory cues, contains numerous LepRb neurons, suggesting a potential role for LepRb PMv neurons in transmitting both metabolic and odorant signals to the neuroendocrine reproductive system. Indeed, Fos-immunoreactivity (-IR) and electrophysiologic recordings revealed the direct activation of LepRb PMv neurons by leptin, and exposure to odors from mice of the opposite sex promoted Fos-IR in many LepRb PMv neurons. To determine the regions innervated by the LepRb PMv neurons, we utilized two novel cre-activated tract-tracing systems in Leprcre animals; data from these systems and from standard tracing techniques revealed that LepRb PMv neurons project to a subset of the regions, including the preoptic area (POA), that are innervated by the PMv as a whole. Furthermore, the retrograde accumulation in LepRb PMv neurons of a trans-synaptic tracer from GnRH neurons revealed the direct innervation of GnRH neurons by many LepRb PMv neurons. Thus, LepRb PMv neurons sense metabolic and sexual odorant cues and project to the rostral hypothalamus to directly innervate GnRH neurons. These results are consistent with a role for LepRb PMv neurons in regulating the reproductive axis in response to metabolic and odorant stimuli.
PMCID: PMC2675106  PMID: 19279251
Leptin receptor; hypothalamus; premammillary; reproduction; sexual odorants; energy homeostasis
22.  Complex interactions with females and rival males limit the evolution of sperm offence and defence 
Postcopulatory sexual selection favours males which are strong offensive and defensive sperm competitors. As a means of identifying component traits comprising each strategy, we used an experimental evolution approach. Separate populations of Drosophila melanogaster were selected for enhanced sperm offence and defence. Despite using a large outbred population and evidence of substantive genetic variation for each strategy, neither trait responded to selection in the two replicates of this experiment. Recent work with fixed chromosome lines of D. melanogaster suggests that complex genotypic interactions between females and competing males contribute to the maintenance of this variation. To determine whether such interactions could explain our lack of response to selection on sperm offence and defence, we quantified sperm precedence across multiple sperm competition bouts using an outbred D. melanogaster population exhibiting continuous genetic variation. Both offensive and defensive sperm competitive abilities were found to be significantly repeatable only across matings involving ejaculates of the same pair of males competing within the same female. These repeatabilities decreased when the rival male stayed the same but the female changed, and they disappeared when both the rival male and the female changed. Our results are discussed with a focus on the complex nature of sperm precedence and the maintenance of genetic variation in ejaculate characteristics.
PMCID: PMC2493577  PMID: 17507332
sperm competition; genetic interactions; artificial selection; virgin effects; Drosophila
23.  Dominant negative PKCɛ impairs apical actin remodeling in parallel with inhibition of carbachol-stimulated secretion in rabbit lacrimal acini 
Here we investigate the involvement of PKCɛ in apical actin remodeling in carbachol-stimulated exocytosis in reconstituted rabbit lacrimal acinar cells. Lacrimal acinar PKCɛ co-sedimented with actin filaments in an actin filament binding assay. Stimulation of acini with carbachol (100 μM, 2–15 min) significantly (p ≤ 0.05) increased PKCɛ recovery with actin filaments in two distinct biochemical assays, while confocal fluorescence microscopy showed a significant increase in PKCɛ association with apical actin in stimulated acini as evidenced by quantitative colocalization analysis. Overexpression of dominant negative (DN) PKCɛ in lacrimal acini using replication-defective adenovirus (Ad) resulted in profound alterations in apical and basolateral actin filaments while significantly inhibiting carbachol-stimulated secretion of bulk protein and β-hexosaminidase. The chemical inhibitor GF 109203X (10 μM, 3 hrs) which inhibits PKCα, β, δ and ɛ, also elicited more potent inhibition of carbachol-stimulated secretion relative to Gö 6976 (10 μM, 3 hrs) which inhibits only PKCα and β. Transduction of lacrimal acini with Ad encoding syncollin-GFP resulted in labeling of secretory vesicles that were discharged in response to carbachol stimulation while co-transduction of acini with Ad-DN-PKCɛ significantly inhibited carbachol-stimulated release of syncollin-GFP. Carbachol also increased the recovery of secretory component in culture medium while Ad-DN-PKCɛ transduction suppressed its carbachol-stimulated release. We propose that DN-PKCɛ alters lacrimal acinar apical actin remodeling, leading to inhibition of stimulated exocytosis and transcytosis.
PMCID: PMC1414898  PMID: 15930141
actin; PKCɛ; exocytosis; lacrimal gland; acinar epithelial cell
24.  Actin and non-muscle myosin II facilitate apical exocytosis of tear proteins in rabbit lacrimal acinar epithelial cells 
Journal of cell science  2005;118(Pt 20):4797-4812.
The acinar epithelial cells of the lacrimal gland exocytose the contents of mature secretory vesicles containing tear proteins at their apical membranes in response to secretagogues. Here we use time-lapse confocal fluorescence microscopy and fluorescence recovery after photobleaching to investigate the changes in actin filaments located beneath the apical membrane during exocytosis evoked by the muscarinic agonist, carbachol (100 μM). Time-lapse confocal fluorescence microscopy of apical actin filaments in reconstituted rabbit lacrimal acini transduced with replication-deficient adenovirus containing GFP-actin revealed a relatively quiescent apical actin array in resting acini. Carbachol markedly increased apical actin filament turnover and also promoted transient actin assembly around apparent fusion intermediates. Fluorescence recovery after photobleaching measurements revealed significant (p≤0.05) increases and decreases, respectively, in mobile fraction (Mf) and turnover times (t½) for apical actin filaments in carbachol-stimulated acini relative to untreated acini. The myosin inhibitors, 2,3-butanedione monoxime (BDM, 10 mM, 15 min) and ML-7 (40 μM, 15 min), significantly decreased carbachol-stimulated secretion of bulk protein and the exogenous secretory vesicle marker, syncollin-GFP; these agents also promoted accumulation of actin-coated structures which were enriched, in transduced acini, in syncollin-GFP, confirming their identity as fusion intermediates. Actin-coated fusion intermediates were sized consistent with incorporation of multiple rather than single secretory vesicles; moreover, BDM and ML-7 caused a shift towards formation of multiple secretory vesicle aggregates while significantly increasing the diameter of actin-coated fusion intermediates. Our findings suggest that the increased turnover of apical actin filaments and the interaction of actin with non-muscle myosin II assembled around aggregates of secretory vesicles facilitate exocytosis in lacrimal acinar epithelial cells.
PMCID: PMC1482462  PMID: 16219687
secretion; fluorescence recovery after photobleaching; confocal microscopy; actin; myosin
25.  Insulin-Like Growth Factor 1 Receptor Signaling Regulates Skin Development and Inhibits Skin Keratinocyte Differentiation 
Molecular and Cellular Biology  2006;26(7):2675-2687.
The insulin-like growth factor 1 receptor (IGF-1R) is a multifunctional receptor that mediates signals for cell proliferation, differentiation, and survival. Genetic experiments showed that IGF-1R inactivation in skin results in a disrupted epidermis. However, because IGF-1R-null mice die at birth, it is difficult to study the effects of IGF-1R on skin. By using a combined approach of conditional gene ablation and a three-dimensional organotypic model, we demonstrate that IGF-1R-deficient skin cocultures show abnormal maturation and differentiation patterns. Furthermore, IGF-1R-null keratinocytes exhibit accelerated differentiation and decreased proliferation. Investigating the signaling pathway downstream of IGF-1R reveals that insulin receptor substrate 2 (IRS-2) overexpression compensates for the lack of IGF-1R, whereas IRS-1 overexpression does not. We also demonstrate that phosphatidylinositol 3-kinase and extracellular signal-regulated kinase 1 and 2 are involved in the regulation of skin keratinocyte differentiation and take some part in mediating the inhibitory signal of IGF-1R on differentiation. In addition, we show that mammalian target of rapamycin plays a specific role in mediating IGF-1R impedance of action on keratinocyte differentiation. In conclusion, these results reveal that IGF-1R plays an inhibitory role in the regulation of skin development and differentiation.
PMCID: PMC1430337  PMID: 16537911

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