Department of Physiology

Permanent URI for this collectionhttps://hdl.handle.net/1807/16980

Physiology is an integrative study of how living organisms function at all levels including (and not exclusive to) genomic, proteomic, molecular, etc. With the inclusion of sophisticated experimental equipments made available, the department strives to understand the fundamental physiological process in both healthy and disease human and translate these understandings to applications of clinical care.

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Serotonin Regulation of the Prefrontal Cortex: Cognitive Relevance and the Impact of Developmental Perturbation
(2019-07-17) Sargin, Derya; Jeoung, Ha-Seul; Goodfellow, Nathalie M; Lambe, Evelyn K
The prefrontal cortex is essential for both executive function and emotional regulation. The interrelationships among these behavioral domains are increasingly recognized, as well as their sensitivity to serotonin (5-hydroxytryptamine, 5-HT). Prefrontal cortex receives serotonergic inputs from the dorsal and median raphe nuclei and is modulated by multiple subtypes of 5-HT receptor across its layers and cell types. Extremes of serotonergic modulation alter mood regulation in vulnerable individuals, yet the impact of serotonin under more typical physiological parameters remains unclear. In this regard, new tools are permitting a closer examination of the behavioral impact of the serotonin system. Optogenetic and chemogenetic manipulations of dorsal raphe 5-HT neurons reveal that serotonin has a greater impact on executive function than previously appreciated. Domains that appear sensitive to fluctuations in 5-HT neuronal excitability include patience and cognitive flexibility. This work is broadly consistent with ex vivo research investigating how 5-HT regulates prefrontal cortex and its output projections. A growing literature suggests 5-HT modulation of these prefrontal circuits is unexpectedly flexible to alteration during development by genetic, behavioral, environmental or pharmacological manipulations, with lasting repercussions for cognition and emotional regulation. Here, we review the cellular and circuit mechanisms of prefrontal serotonergic modulation, investigate recent research into the cognitive consequences of the serotonergic system, and probe the lasting consequences of developmental perturbations. Understanding both the complexity of the prefrontal serotonin system and its sensitivity during development are essential to learn more about the vulnerabilities of this system in mood and anxiety disorders and the underappreciated cognitive consequences of these disorders and their treatment.
Endogenous Acetylcholine and Its Modulation of Cortical Microcircuits to Enhance Cognition
(Springer, 2020) Venkatesan, Sridevi; Jeoung, Ha-Seul; Chen, Tianhui; Power, Saige K.; Liu, Yupeng; Lambe, Evelyn K.
Acetylcholine regulates the cerebral cortex to sharpen sensory perception and enhance attentional focus. The cellular and circuit mechanisms of this cholinergic modulation are under active investigation in sensory and prefrontal cortex, but the universality of these mechanisms across the cerebral cortex is not clear. Anatomical maps suggest that the sensory and prefrontal cortices receive distinct cholinergic projections and have subtle differences in the expression of cholinergic receptors and the metabolic enzyme acetylcholinesterase. First, we briefly review this anatomical literature and the recent progress in the field. Next, we discuss in detail the electrophysiological effects of cholinergic receptor subtypes and the cell and circuit consequences of their stimulation by endogenous acetylcholine as established by recent optogenetic work. Finally, we explore the behavioral ramifications of in vivo manipulations of endogenous acetylcholine. We find broader similarities than we expected between the cholinergic regulation of sensory and prefrontal cortex, but there are some differences and some gaps in knowledge. In visual, auditory, and somatosensory cortex, the cell and circuit mechanisms of cholinergic sharpening of sensory perception have been probed in vivo with calcium imaging and optogenetic experiments to simultaneously test mechanism and measure the consequences of manipulation. By contrast, ascertaining the links between attentional performance and cholinergic modulation of specific prefrontal microcircuits is more complicated due to the nature of the required tasks. However, ex vivo optogenetic manipulations point to differences in the cholinergic modulation of sensory and prefrontal cortex. Understanding how and where acetylcholine acts within the cerebral cortex to shape cognition is essential to pinpoint novel treatment targets for the perceptual and attention deficits found in multiple psychiatric and neurological disorders.
Complementary and antagonistic effects of combined glucagon-like peptide-2 and glucagon-like peptide-1 receptor agonist administration on parameters relevant to short bowel syndrome
(Wiley, 2021-11-26) Srikrishnaraj, Arjuna; Jeong, Hyerin; Brubaker, Patricia L
Short Bowel Syndrome (SBS) is characterized by debilitating malabsorption requiring parenteral nutrition. The intestinotrophic glucagon-like peptide-2 receptor agonist, h[Gly2]GLP2, is currently used to treat patients with SBS. Recent evidence suggests that GLP-1 receptor agonists such as Exendin-4 (Ex4) may also be beneficial in SBS given their ability to increase intestinal growth and delay gastric emptying (GE).
Circadian GLP-1 secretion in mice is dependent on the intestinal microbiome for maintenance of diurnal metabolic homeostasis
(2020) Martchenko, Sarah E.; Martchenko, Alexandre; Cox, Brian J.; Naismith, Kendra; Waller, Alison; Gurges, Patrick; Sweeney, Maegan E.; Philpott, Dana J.; Brubaker, Patricia L.
Essential role of Munc18-1 in the regulation of glucagon-like peptide-1 secretion
(Oxford Academic, 2019) Campbell, Jhenielle R.; Martchenko, Alexandre; Sweeney, Maegan E.; Michael F., Maalouf; Psichas, Arianna; Gribble, Fiona M.; Reimann, Frank; Brubaker, Patricia L.
A Single Course of Synthetic Glucocorticoids in Pregnant Guinea Pigs Programs Behaviour and Stress Response in Two Generations of Offspring
(Oxford University Press, 2018-10-31) Moisiadis, Vasilis G; Mouratidis, Alexandros; Kostaki, Alisa; Matthews, Stephen G
Treatment with a single course of synthetic glucocorticoids (sGC) is the standard of care for pregnant women who are at risk for preterm delivery. Animal studies have demonstrated that multiple course sGC can program altered hypothalamic-pituitary-adrenal (HPA) axis response to stress in first- (F1) and second-generation (F2) offspring. This study sought to determine whether HPA axis activity and stress-associated behaviours (locomotor activity, attention) are altered following a single course of sGC in F1 and F2 female and male offspring. Pregnant guinea pigs (F0) received sGC (1 mg/kg) or saline on gestational days 50&51. HPA function and behaviour were assessed in juvenile and adult F1-F2 offspring of both sexes following maternal transmission. In F1, sGC increased the HPA stress response in females, but decreased responsiveness in males (P<0.05). sGC exposure (F0) produced the opposite effects in F2 (P<0.05). Reduced HPA responsiveness in F2 females was associated with reduced expression of proopiomelanocortin mRNA and increased expression of glucocorticoid receptor in the anterior pituitary (P<0.05). Locomotor activity and prepulse inhibition were reduced by sGC in adult F1 offspring. No behavioural changes were observed in F2 animals. These data indicate that effects of antenatal treatment with a single course of sGC are present in F2 following maternal transmission. However, there are fewer effects on HPA activity and behaviour in F1 and F2 offspring compared to treatment with multiple courses of sGC.
Astrocyte-mediated regulation of multidrug resistance p-glycoprotein in fetal and neonatal brain endothelial cells: age-dependent effects
(Wiley Open Access, 2016-08-22) Baello, Stephanie; Iqbal, Majid; Gibb, William; Matthews, Stephen G
Brain endothelial cells (BECs) form a major component of the blood-brain barrier (BBB). In late gestation, these cells express high levels of the multidrug transporter p-glycoprotein (P-gp; encoded by Abcb1), which prevents the passage of an array of endogenous factors and xenobiotics into the fetal brain. P-gp levels in the BECs increase dramatically in late gestation, coincident with astrocyte differentiation. However, the role of astrocytes in modulating P-gp in the developing BBB is unknown. We hypothesized that factors produced by astrocytes positively regulate P-gp in BECs. Astrocytes and BECs were isolated from fetal and postnatal guinea pigs. Levels of Abcb1 mRNA and P-gp were increased in BECs co-cultured with astrocytes compared to BECs in monoculture. Moreover, postnatal astrocytes enhanced P-gp function in fetal BECs but fetal astrocytes had no effect on postnatal BECs. These effects were dependent on secreted proteins with a molecular weight in the range of 3-100 kDa. LC/MS-MS revealed significant differences in proteins secreted by fetal and postnatal astrocytes. We propose that astrocytes are critical modulators of P-gp at the developing BBB. As such, aberrations in astrocyte maturation, observed in neurodevelopmental disorders, will likely decrease P-gp at the BBB. This would allow increased transfer of P-gp endogenous and exogenous substrates into the brain, many of which have neurodevelopmental consequences.
Glucocorticoids modify effects of TGF-β1 on multidrug resistance in the fetal blood-brain barrier
(Taylor and Francis, 2016-04-28) Baello, Stephanie; Iqbal, Majid; Kearney, Samantha; Kuthiala, Shikah; Bloise, Enrrico; Gibb, William; Matthews, Stephen G
Transforming growth factor-β1 (TGF-β1) increases P-glycoprotein (P-gp; encoded by Abcb1) activity in fetal brain endothelial cells (BECs). P-gp is important for fetal brain protection against xenobiotics including synthetic glucocorticoids (sGC). We hypothesized that antenatal sGC would modify P-gp responsiveness to TGF-β1 in fetal BECs. Pregnant guinea pigs were treated with dexamethasone or vehicle (N = 5/group) on gestational day (GD) 48-49 and BECs derived on GD50. In BECs from control fetuses, TGF-β1 increased Abcb1 mRNA and P-gp function, by approximately 5-fold and 55% respectively, as well as tight junction function. In contrast, TGF-β1 had no effect on these parameters in BECs from sGC-exposed fetuses. Moreover, levels of TGF-β1 responsive gene, Smad7, were increased 3-fold in BECs from control fetuses after TGF-β1 but not in sGC-exposed fetuses. In conclusion, antenatal sGC alters responsiveness to TGF-β1 in fetal BECs. This study has identified novel mechanisms by which TGF-β1 and sGC modulate fetal brain protection against xenobiotics and other P-gp substrates.
The DNA methylation landscape of enhancers in the guinea pig hippocampus
(Future Medicine, 2018-04-04) Boureau, Lisa; Constantinof, Andrea; Moisiadis, Vasilis G; Matthews, Stephen G; Szyf, Moshe
Aims: To determine the state of methylation of DNA molecules in the guinea pig hippocampus that are associated with either poised or active enhancers. Methods: We used sequential ChIP-bisulfite-sequencing with an antibody to H3K4me1 to map the state of methylation of DNA that is found within enhancers. Actively transcribing transcription start sites (TSS) were mapped by ChIP-sequencing with an antibody to RNApolII-PS5. Total DNA methylation was mapped using reduced representation bisulfite sequencing (RRBS). Results: DNA that overlaps with H3K4me1 binding regions in the genome is heavily methylated. However, DNA molecules that are found in H3K4me1 chromatin are hypomethylated, while DNA found in enhancers that are associated with active transcription is further demethylated. Differential: methylation in enhancers is spotted in single CGs, bimodal and corresponds to transcription factor binding sites. Conclusions: Our study delineates the DNA methylation status of H3K4 me1 bound regions in the hippocampus in active and inactive genes.
Insulin-like growth factor binding protein-4 inhibits epithelial growth and proliferation in the rodent intestine
(American Physiological Society, 2018-04-06) Austin, Kaori; Tsang, Derek; Chalmers, Jennifer A.; Maalouf, Michael F.; Brubaker, Patricia L.
Insulin-like growth factor binding protein-4 (IGFBP-4) is a binding protein that modulates the action of IGF-1, a growth factor whose presence is required for the intestinotrophic effects of glucagon-like peptide-2 (GLP-2). GLP-2 is a gut hormone that utilizes both IGF-1 and epidermal growth factor (EGF) as intermediary factors to promote intestinal growth. Thus, to elucidate the mechanism through which IGFBP-4 regulates IGF-1 activity in the intestine, proliferation assays were conducted using rat intestinal epithelial (IEC-6) cells. IGF-1 and EGF synergistically enhanced proliferation, an effect that was dose-dependently decreased by IGFBP-4 (p<0.05-0.001) in an IGF-1 receptor(R)- and MEK1/2- but not PI3K-dependent manner (p>0.05 for IGFBP-4 effects with IGF-1R- and MEK1/2-inhibitors). Intestinal organoids derived from IGFBP-4 knockout mice demonstrated significantly greater Ki67 expression and an enhanced surface area increase in response to IGF-1 treatment, compared to organoids from control mice (p<0.05-0.01). GLP-2 is also known to increase the mucosal expression of IGFBP-4 mRNA. To investigate whether this occurs through the actions of its intermediaries, IGF-1 and EGF, inducible intestinal epithelial-IGF-1R knockout and control mice were treated for 10d with and without the pan-ErB inhibitor, CI-1033. However, no differences in mucosal IGFBP-4 mRNA expression were found for any of the treatment groups (p>0.05). Consistently, IEC-6 cells treated with IGF-1 and/or EGF displayed no alteration in IGFBP-4 mRNA or in cellular and secreted IGFBP-4 protein (p>0.05). Overall, this study establishes that endogenous IGFBP-4 plays an important role in inhibiting IGF-1-induced intestinal epithelial proliferation, and that mucosal IGFBP-4 expression is independent of IGF-1 and EGF.
Impact of bacterial and viral challenge on multidrug resistance in first- and third-trimester human placenta
(Elsevier, 2015-05-08) Lye, Phetcharawan; Bloise, Enrrico; Javam, Mohsen; Gibb, William; Lye, Stephen J; Matthews, Stephen G
The ABC transporters P-glycoprotein (P-gp, official gene symbol ABCB1) and breast cancer resistance protein (BCRP, official gene symbol ABCG2) protect the conceptus from exposure to toxins and xenobiotics present in the maternal circulation. Viral or bacterial challenges alter expression of placental multidrug transporters in rodents. We hypothesized that exposure to lipopolysaccharide (LPS, bacterial antigen) and polyinosinic-polycytidylic acid (poly(I:C), viral antigen) would decrease P-gp and BCRP in the human placenta. Placental explants from first and third trimesters were challenged with 0.1 to 10 μg/mL LPS or 1 to 50 μg/mL poly(I:C) for 4 or 24 hours; mRNA levels, protein expression, and localization were assessed by quantitative real-time PCR, Western blot analysis, and immunohistochemistry, respectively. Toll-like receptor (TLR)-3 and TLR-4 mRNA expression increased from the first to third trimester (P < 0.01), and the receptors localized to cytotrophoblasts in the first trimester and to syncytiotrophoblasts in the third trimester. LPS exposure in first-trimester explants decreased (P < 0.001) ABCB1 and ABCG2 mRNA and protein levels. In contrast, poly(I:C) decreased (P < 0.05) ABCB1, TLR-3, and TLR-4 mRNA levels in the third trimester but not first trimester. LPS and poly(I:C) treatments increased (P < 0.01) IL-8 and chemokine ligand 2. Results suggest that bacterial infections likely alter exposure of the conceptus to toxins and drugs during early pregnancy, whereas viral infections may disrupt fetal protection in later stages of pregnancy.
Effect of oxygen on multidrug resistance in the first trimester human placenta
(Elsevier, 2013-05-27) Lye, P; Bloise, E; Dunk, C; Javam, M; Gibb, W; Lye, S J; Matthews, S G
Introduction: The multidrug resistance proteins, P-glycoprotein (P-gp, encoded by the ABCB1 gene) and breast cancer resistance protein (BCRP, encoded by ABCG2) are highly expressed in the first trimester placenta. These transporters protect the fetus from exposure to maternally derived toxins and xenobiotics. Since oxygen is a regulator of multidrug resistance in various tissues, we hypothesized that changes in oxygen tension alter placental ABCB1/P-gp and ABCG2/BCRP expression in the first trimester. Methods: Placental specimens were collected from first (n=7), second (n=5) and term pregnancies (n=5). First trimester placental villous explants were incubated (24 or 48h) in different oxygen tension (3-20%). ABCB1, ABCG2 and VEGFA mRNA expression levels were assessed by RT-PCR and protein was localized by IHC. Results: ABCB1 is expressed most highly in the first trimester placenta (p<0.05), whereas ABCG2 expression does not change significantly over pregnancy. P-gp and BCRP staining is present in the syncytiotrophoblast and in cytotrophoblasts. ABCG2 mRNA is increased in hyperoxic (20%) conditions after 48h (p<0.05). In contrast, hypoxia (3%) did not change ABCB1 mRNA expression but significantly increased VEGFA mRNA (p<0.05). Hypoxia resulted in increased BCRP staining in cytotrophoblasts and in the microvillous membrane of the syncytium. Whereas, hypoxia resulted in increased P-gp staining in proliferating cytotrophoblasts. Conclusion: We conclude that placental multidrug resistance expression, specifically ABCG2, is regulated by oxygen tension in the first trimester. It is possible that changes in placental oxygen supply are capable of altering fetal drug exposure especially during early pregnancy.
The ontogeny of P-glycoprotein in the developing human blood-brain barrier: implication for opioid toxicity in neonates
(Nature Publishing Group, 2015-02-18) Lam, Jessica; Baello, Stephanie; Iqbal, Majid; Kelly, Lauren E; Shannon, Patrick T; Chitayat, David; Matthews, Stephen G; Koren, Gideon
Neonates have been shown to have a heightened sensitivity to the central depressive effects of opioids compared to older infants and adults. The limited development of P-glycoprotein (P-gp) may limit the ability of the neonate to efflux morphine from the brain back to the systemic circulation. The objective of the study was to determine the ontogeny of P-gp in the human brain.
TGF-β1 regulation of multidrug resistance P-glycoprotein in the developing male blood-brain barrier
(Oxford University Press, 2014-02-01) Baello, Stephanie; Iqbal, Majid; Bloise, Enrrico; Javam, Mohsen; Gibb, William; Matthews, Stephen G
P-glycoprotein (P-gp), an efflux transporter encoded by the abcb1 gene, protects the developing fetal brain. Levels of P-gp in endothelial cells of the blood-brain barrier (BBB) increase dramatically during the period of peak brain growth. This is coincident with increased release of TGF-β1 by astrocytes and neurons. Although TGF-β1 has been shown to modulate P-gp activity in a number of cell types, little is known about how TGF-β1 regulates brain protection. In the present study, we hypothesized that TGF-β1 increases abcb1 expression and P-gp activity in fetal and postnatal BBB in an age-dependent manner. We found TGF-β1 to potently regulate abcb1 mRNA and P-gp function. TGF-β1 increased P-gp function in brain endothelial cells (BECs) derived from fetal and postnatal male guinea pigs. These effects were more pronounced earlier in gestation when compared with BECs derived postnatally. To investigate the signaling pathways involved, BECs derived at gestational day 50 and postnatal day 14 were exposed to ALK1 and ALK5 inhibitors and agonists. Through inhibition of ALK5, we demonstrated that ALK5 is required for the TGF-β1 effects on P-gp function. Activation of ALK1, by the agonist BMP-9, produced similar results to TGF-β1 on P-gp function. However, TGF-β1 signaling through the ALK1 pathway is age-dependent as dorsomorphin, an ALK1 inhibitor, attenuated TGF-β1-mediated effects in BECs derived at postnatal day 14 but not in those derived at gestational day 50. In conclusion, TGF-β1 regulates P-gp at the fetal and neonatal BBB and both ALK5 and ALK1 pathways are implicated in the regulation of P-gp function. Aberrations in TGF-β1 levels at the developing BBB may lead to substantial changes in fetal brain exposure to P-gp substrates, triggering consequences for brain development.
Acute Effects of Viral Exposure on P-Glycoprotein Function in the Mouse Fetal Blood-Brain Barrier
(Karger, 2017-02-22) Bloise, Enrrico; Petropoulos, Sophie; Iqbal, Majid; Kostaki, Alisa; Ortiga-Carvalho, Tania Maria; Gibb, William; Matthews, Stephen G
Background/Aims: Viral infection during pregnancy is known to affect the fetal brain. The toll-like receptor (TLR)-3 is a pattern recognition receptor activated by viruses known to elicit adverse fetal neurological outcomes. The P-glycoprotein (P-gp) efflux transporter protects the developing fetus by limiting the transfer of substrates across both the placenta and the fetal blood-brain barrier (BBB). As such, inhibition of P-gp at these blood-barrier sites may result in increased exposure of the developing fetus to environmental toxins and xenobiotics present in the maternal circulation. We hypothesized that viral exposure during pregnancy would impair P-gp function in the placenta and in the developing BBB. Here we investigated whether the TLR-3 ligand, polyinosinic:polycytidylic acid (PolyI:C), increased accumulation of one P-gp substrate in the fetus and in the developing fetal brain. Methods: Pregnant C57BL/6 mice (GD15.5) were injected (i.p.) with PolyI:C (5 mg/kg or 10 mg/kg) or vehicle (saline). [3 H]digoxin (P-gp substrate) was injected (i.v.) 3 or 23h post-treatment and animals were euthanized 1h later. Maternal plasma, ‘fetal-units’ (fetal membranes, amniotic fluid and whole fetus), and fetal brains were collected. Results: PolyI:C exposure (4h) significantly elevated maternal plasma IL-6 (P<0.001) and increased [3H]digoxin accumulation in the fetal brain (P<0.05). In contrast, 24h after PolyI:C exposure, no effect on IL-6 or fetal brain accumulation of P-gp substrate was observed. Conclusion: Viral infection modeled by PolyI:C causes acute increases in fetal brain accumulation of P-gp substrates and by doing so, may increase fetal brain exposure to xenobiotics and environmental toxins present in the maternal circulation.
Suppression of circadian secretion of glucagon-like peptide-1 by the saturated fatty acid, palmitate
(Wiley, 2017-12-18) Martchenko, A.; Oh, R.H.; Wheeler, S.E.; Gurges, P.; Chalmers, J.A.; Brubaker, P.L.
Aim: Glucagon-like peptide-1 is an incretin hormone secreted by the intestinal L-cell with a circadian rhythm that parallels expression of the core clock gene, Bmal1. Although feeding rats a high-fat/high-sucrose Western diet impairs rhythmic glucagon-like peptide-1 release, the mechanisms underlying this effect remain unclear. Therefore, the aim of this study was to determine the pathway(s) by which the saturated fat, palmitate, a major component of the Western diet, impairs circadian glucagon-like peptide-1 secretion. Methods: Murine mGLUTag L-cells were synchronized and the effects of palmitate pretreatment on gene expression and glucagon-like peptide-1 secretion were determined, in addition to metabolite quantification, mitochondrial function analysis, and enzyme inhibition and activation assays. Glucagon-like peptide-1 secretion was also analyzed in ileal crypt cultures from control and Bmal1 knockout mice. Results: Pre-treatment with palmitate dampened Bmal1 mRNA and protein expression and glucagon-like peptide-1 secretion at 8 but not 20 hours after cell synchronization (p<0.05-0.001). Glucagon-like peptide-1 release was also impaired in Bmal1 knockout cultures as compared to wild-type controls (p<0.001). Palmitate pre-treatment reduced expression of the Bmal1 downstream target, nicotinamide phosphoribosyltransferase, the rate-limiting enzyme in the synthesis of NAD+. This was paralleled by dampening of total NAD+ levels, as well as impaired mitochondrial function and ATP production (p<0.05-0.001). Whereas direct inhibition of 3 nicotinamide phosphoribosyltransferase also decreased glucagon-like peptide-1 release, activation of this enzyme restored glucagon-like peptide-1 secretion in the presence of palmitate. Conclusion: Palmitate impairs L-cell clock function at the peak of Bmal1 gene expression, thereby impairing mitochondrial function and ultimately rhythmic glucagon-like peptide-1 secretion.
IKKβ inhibition prevents fat-induced beta cell dysfunction in vitro and in vivo in rodents
(Springer Berlin Heidelberg, 2017-07-20) Ivovic, Aleksandar; Oprescu, Andrei I; Koulajian, Khajag; Mori, Yusaku; Eversley, Judith A; Zhang, Liling; Nino-Fong, Rodolfo; Lewis, Gary F; Donath, Marc Y; Karin, Michael; Wheeler, Michael B; Ehses, Jan; Volchuk, Allen; Chan, Catherine B; Giacca, Adria
Aims/Hypothesis: We have previously shown that oxidative stress plays a causal role in beta cell dysfunction induced by fat. Here, we address whether the proinflammatory kinase inhibitor of (nuclear factor) κB kinase β (IKKβ), which is activated by oxidative stress, is also implicated. Methods: Fat (oleate or olive oil) was infused intravenously in Wistar rats for 48 h with or without the IKKβ inhibitor salicylate. Thereafter, beta cell function was evaluated in vivo using hyperglycaemic clamps or ex vivo in islets isolated from fat-treated rats. We also exposed rat islets to oleate in culture, with or without salicylate and 4(2′-aminoethyl)amino-1,8-dimethylimidazo(1,2-a)quinoxaline; BMS-345541 (BMS, another inhibitor of IKKβ) and evaluated beta cell function in vitro. Furthermore, oleate was infused in mice treated with BMS and in beta cell-specific Ikkb-null mice. Results: 48 h infusion of fat impaired beta-cell function in vivo, assessed using the disposition index (DI), in rats (saline: 1.41 ± 0.13; oleate: 0.95 ± 0.11; olive oil [OLO]: 0.87 ± 0.15; p < 0.01 for both fats vs saline) and in mice (saline: 2.51 ± 0.39; oleate: 1.20 ± 0.19; p < 0.01 vs saline) and ex vivo (i.e., insulin secretion, units are pmol insulin islet−1 h−1) in rat islets (saline: 1.51 ± 0.13; oleate: 1.03 ± 0.10; OLO: 0.91 ± 0.13; p < 0.001 for both fats vs saline) and the dysfunction was prevented by co-infusion of salicylate in rats (oleate + salicylate: 1.30 ± 0.09; OLO + salicylate: 1.33 ± 0.23) or BMS in mice (oleate + BMS: 2.25 ± 0.42) in vivo and by salicylate in rat islets ex vivo (oleate + salicylate: 1.74 ± 0.31; OLO + salicylate: 1.54 ± 0.29). In cultured islets, 48 h exposure to oleate impaired beta-cell function ([in pmol insulin islet−1 h−1] control: 0.66 ± 0.12; oleate: 0.23 ± 0.03; p < 0.01 vs saline), an effect prevented by both inhibitors (oleate + salicylate: 0.98 ± 0.08; oleate + BMS: 0.50 ± 0.02). Genetic inhibition of IKKβ also prevented fat-induced beta-cell dysfunction ex vivo ([in pmol insulin islet−1 h−1] control saline: 0.16 ± 0.02; control oleate: 0.10 ± 0.02; knockout oleate: 0.17 ± 0.04; p < 0.05 control saline vs. control oleate) and in vivo (DI: control saline: 3.86 ± 0.40; control oleate: 1.95 ± 0.29; knockout oleate: 2.96 ± 0.24; p < 0.01 control saline vs control oleate). Conclusions/interpretation: Our results demonstrate a causal role for IKKβ in fat-induced beta cell dysfunction in vitro, ex vivo and in vivo.
Quantitative Proteomics of Intestinal Mucosa From Male Mice Lacking Intestinal Epithelial Insulin Receptors
(2017-06-07) Jensen, Stina Rikke; Schoof, Erwin M; Wheeler, Sarah E; Hvid, Henning; Ahnfelt-Rønne, Jonas; Hansen, Bo Falck; Nishimura, Erica; Olsen, Grith Skytte; Kislinger, Thomas; Brubaker, Patricia L.
The goal of the present study was to determine whether loss of the insulin receptor alters the molecular landscape of the intestinal mucosa, using intestinal-epithelial insulin receptor knockout (IE-irKO) mice and both genetic (IRfl/fl and Villin-cre) controls. Quantitative proteomic analysis by liquid chromatography mass spectrometry was applied to jejunal and colonic mucosa from mice fed a normal chow diet and mice fed a Western diet (WD). Jejunal mucosa from IE-irKO mice demonstrated alterations in all intestinal cell lineages: Paneth, goblet, absorptive, and enteroendocrine cells. Only goblet and absorptive cells were affected in the colon. Also, a marked effect of WD consumption was found on the gut proteome. A substantial reduction was detected in Paneth cell proteins with antimicrobial activity, including lysozyme C-1, angiogenin-4, cryptdin-related sequence 1C-3 and -2, α-defensin 17, and intelectin-1a. The key protein expressed by goblet cells, mucin-2, was also reduced in the IE-irKO mice. Proteins involved in lipid metabolism, including aldose reductase-related protein 1, 15-hydroxyprostaglandin dehydrogenase, apolipoprotein A-II, and pyruvate dehydrogenase kinase isozyme 4, were increased in the mucosa of WD-fed IE-irKO mice compared with controls. In contrast, expression of the nutrient-responsive gut hormones, glucose-dependent insulinotropic polypeptide and neurotensin, was reduced in the jejunal mucosa of IE-irKO mice, and the expression of proteins of the P-type adenosine triphosphatases and the solute carrier-transporter family was reduced in the colon of WD-fed IE-irKO mice. In conclusion, IE-irKO mice display a distinct molecular phenotype, suggesting a biological role of insulin and its receptor in determining differentiated cell specificity in the intestinal epithelium.
Elucidating the Biological Roles of Insulin and Its Receptor in Murine Intestinal Growth and Function
(Oxford University Press, 2017-08-01) Jensen, Stina Rikke; Wheeler, Sarah E; Hvid, Henning; Ahnfelt-Rønne, Jonas; Hansen, Bo Falck; Nishimura, Erica; Olsen, Grith Skytte; Brubaker, Patricia L.
The role of the intestinal insulin receptor (IR) is not well understood. We therefore explored the effect of insulin (300 nmol/kg per day for 12 days) on the intestine in sex-matched C57Bl/6J mice. The intestinal and metabolic profiles were also characterized in male and female intestinal-epithelial IR knockout (IE-irKO) mice compared with all genetic controls on a chow diet or Western diet (WD) for 4 to 12 weeks. Insulin treatment did not affect intestinal size, intestinal resistance, or metabolic genes, but it reduced proximal-colon crypt depth and acutely increased colonic serine/threonine-specific protein kinase B (AKT) activation. Feeding with a WD increased body weight and fasting insulin level and decreased oral glucose tolerance in C57Bl/6J and IE-irKO mice. However, although the overall responses of the IE-irKO mice were not different from those of Villin-Cre (Vil-Cre):IRfl/+ and IRfl/fl controls, profound differences were found for female control Vil-Cre mice, which demonstrated reduced food intake, body weight, jejunal glucose transport, oral glucose tolerance, and fasting insulin and cholesterol levels. Vil-Cre mice also had smaller intestines compared with those of IE-irKO and IRfl/fl mice and greater insulin-mediated activation of jejunal IR and AKT. In summary, gain- and loss-of-function studies, with and without caloric overload, indicate that insulin did not exert remarkable effects on intestinal metabolic or morphologic phenotype except for a small effect on the colon. However, the transgenic control Vil-Cre mice displayed a distinct phenotype compared with other control and knockout animals, emphasizing the importance of thoroughly characterizing genetically modified mouse models.
The SNARE Protein Syntaxin-1a Plays an Essential Role in Biphasic Exocytosis of the Incretin Hormone Glucagon-Like Peptide 1
(American Diabetes Association, 2017-09) Wheeler, Sarah E.; Stacey, Holly M.; Nahaei, Yasaman; Hale, Stephen J.; Hardy, Alexandre B.; Reimann, Frank; Gribble, Fiona M.; Larraufie, Pierre; Gaisano, Herbert Y.; Brubaker, Patricia L.
Exocytosis of the hormone glucagon-like peptide 1 (GLP-1) by the intestinal L cell is essential for the incretin effect after nutrient ingestion and is critical for the actions of dipeptidyl peptidase 4 inhibitors that enhance GLP-1 levels in patients with type 2 diabetes. Two-photon microscopy revealed that exocytosis of GLP-1 is biphasic, with a first peak at 1-6 min and a second peak at 7-12 min after stimulation with forskolin. Approximately 75% of the exocytotic events were represented by compound granule fusion, and the remainder were accounted for by full fusion of single granules under basal and stimulated conditions. The core SNARE protein syntaxin-1a (syn1a) was expressed by murine ileal L cells. At the single L-cell level, first-phase forskolin-induced exocytosis was reduced to basal (P < 0.05) and second-phase exocytosis abolished (P < 0.05) by syn1a knockout. L cells from intestinal-epithelial syn1a-deficient mice demonstrated a 63% reduction in forskolin-induced GLP-1 release in vitro (P < 0.001) and a 23% reduction in oral glucose-stimulated GLP-1 secretion (P < 0.05) in association with impairments in glucose-stimulated insulin release (by 60%; P < 0.01) and glucose tolerance (by 20%; P < 0.01). The findings identify an exquisite mechanism of metered secretory output that precisely regulates release of the incretin hormone GLP-1 and hence insulin secretion after a meal.

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