bluuebunny/crossref_metadata_2025 huggingface dataset:
Leptin acts as a metabolic regulator of Angptl6 expression
Authors: Kim Na Hyun , Kim Min Jeong , Kim Dae Sik , Kong In Deok
Published: June 2021

Leptin, an adipocyte‐secreted protein, is one of the crucial regulators of food intake and energy expenditure. Circulating leptin concentration is correlated with energy intake and body fatness in rodents and humans. Recently, it has been known that some angiopoietin‐like proteins (Angptls) regulate lipid, glucose and energy metabolism. We examined the effect of leptin on the level of some Angptls using high‐fat diet and hyperleptinemia model. C57BL/6 mice were fed a HF diet or normal chow for the periods of 12 weeks.

HF diet caused obesity, elevation of serum transaminase levels, and hyperlipidemia. And also HF diet induced an impaired glucose tolerance (IGT). Plasma concentration of leptin was approximately increased 4‐fold; adiponectin and RBP4 level were also increased in HF diet group. Plasma Angptl3 concentration of HF diet mice was lower than that of control mice. Angptl4 was decreased in liver, but no difference in epididyimal fat. Quantitative real‐time PCR analysis revealed that the mRNA expression level of Angptl6/AGF in liver was significantly increased in HF diet group. In leptin‐treated mice, food intake and body weight were decreased time‐dependently. Blood level of fasting glucose and triglycerides were decreased in leptin‐injected mice. Serum Angptl3 concentration of leptin‐treated mice was lower than that of control mice. Quantitative real‐time PCR analysis revealed that the mRNA expression level of Angptl3 and Angptl4 in liver were decreased, Angptl6/AGF was significantly increased in liver and epididymal fat in leptin‐treated mice.

In conclusion, Angptl3, 4 were down‐regulated and Angptl6/AGF was up‐regulated by HF diet model and leptin injection mice. It could be suggested that leptin is an important regulator of Angptl6/AGF expression in hyperleptinemia.
Mechanical Stretch Stimulates Cholangiocyte Proliferation and Profibrotic Gene Expression
Authors: Munshi Md KAMRUZZAMAN , Wise Candace , Yang Fuquan , Dostal David E. , Glaser Shannon S.
Published: June 2021

The angiotensin type 1 receptor (AT1) plays a key role in pressure‐induced vascular growth. AT1 also functions as a mechanoreceptor in addition to a GPCR. Although marked cholangiocyte proliferation occurs during obstructive cholestasis, a condition associated with increased biliary pressure, the role of AT1 in this process remains unclear.

METHODS/RESULTS

In vivo, administration of Ang II (50 ng/Kg/min) to normal and BDL rats resulted in a significant increase in PCNA positive cholangiocytes as compared to saline treated rats. PCNA increased 64% and 30% for normal and BDL treated with Ang II compared to controls, respectively. In vitro, Ang II increased NRIC (normal rat cholangiocyte) proliferation (maximal effect observed after 48 hrs at 10−7M Ang II (1.7‐fold)). This proliferation was mediated by AT1 as it is the only isoform expressed by rat cholangiocytes. To address the role of mechanical stress, NRIC were plated on BioFlex culture plates and static equiaxial strain was applied to the cells for 24 hrs. Mechanical strain increased NRIC growth (2‐fold) and expression of angiotensin converting enzyme (ACE) mRNA levels (19‐fold). Mechanical strain also increased CTGF, fibronectin 1, and NCAM1 profibrotic gene expression and Ang II secretion by 15%.

CONCLUSION

Ligand and mechanical activation of AT1 induces cholangiocyte growth and profibrotic gene expression.
Oxygen‐induced superoxide production in solitary complex neurons in rat medullary slices
Authors: Dean Jay B. , Landon Carol S. , D'Agostino Dominic P.
Published: June 2021

We previously reported that the rate of superoxide (•O2−) production in CA1 neurons in hippocampal slices decreases as O2 is decreased from 95% to 60, 40 or 20% (J. Neurophysiol. 98:1030‐1041, 2007). Here we report the effects of continuous exposure to O2 (95, 40 & 20%) on •O2− production in solitary complex (SC) neurons in medullary slices (400μm, weaned rats). The rate of intracellular •O2− production was measured using the fluorogenic probe, DHE (2.5μM), added to the superfusate (35–37°C). Fluorescence imaging initially classified cells at the start of the experiment as dim versus bright. Dim SC neurons incubated in 95%O2 had a similar rate of •O2− production (increasing fluorescence) over time as that reported previously for CA1 hippocampal neurons. Continuous exposure of dim cells to 20 or 40%O2, however, revealed slightly lower but similar rates of •O2− production that were significantly greater than that reported for CA1 neurons. SC neurons initially identified as bright cells increased their rate of fluorescence during the first 60–90 minutes but thereafter fluorescence reached a plateau indicative of a decreased rate of •O2− production. In bright SC cells, 95% and 20%O2 caused the lowest and highest rate of •O2− production, respectively. We conclude the following: 1) dim SC neurons have a higher capacity for •O2− production during exposure to 20–40%O2 than CA1 hippocampal neurons. 2) Neurons with initially bright DHE fluorescence may be damaged from slicing and lose their •O2− generating mechanisms rapidly, especially in the presence of 95%O2 (ONR N000140110179, NIH R01HL56683).
Application of in vitro methods for studying oxidant stress in lung epithelium of multiple species
Authors: Murphy Shannon R , Chan Jackie K , Edwards Patricia C , Van Winkle Laura S
Published: June 2021

Ozone (O3) is a pervasive oxidant air pollutant that changes pulmonary function and exacerbates respiratory disease. Exposure to O3 increases intracellular levels of reactive oxygen species in airway epithelium, resulting in increased inflammation. Interleukin 8 (IL8) is a proinflammatory cytokine released by epithelial cells undergoing stress. Our objective is to define how epithelial cell populations at different cycle stages or in animals with a previous in vivo exposure react to acute oxidative stress in vitro. We subjected airway explants from rhesus macaques, Swiss Webster mice and a human alveolar Type II‐like epithelial cell line (A549) to acute oxidant stress using H2O2 in vitro for 4 hours. Airway explants were assayed using differential permeability to ethidium homodimer‐1 to define cytotoxicity. IL8 (or MIP2) gene expression was measured using qRT‐PCR. H2O2 induced a 3‐fold increase in IL8 expression in control explants from Filtered Air exposed monkeys. Previous in vivo episodic exposure to O3, House Dust Mite Antigen (HDMA), or O3+HDMA resulted in the attenuation of the IL8 response. In mouse airway explants, H2O2 induced a 2‐fold increase in MIP2 expression. Similarly, H2O2 induced a 7‐fold increase in quiescent A549 cells, however, the response was reduced in proliferating cells. We conclude that quiescent cells and animals without previous oxidant exposure are more responsive to oxidants. Funded by NIH grants ES000628, RR000169, ES012720 and EPA RD832414.
Oxidative metabolism studies on thermal regulation‐Protective mechanism against free radical in zebrafish brain
Authors: TSENG YUNG‐CHE , Chen Ruo‐Dong , Hwang Pung‐Pung
Published: June 2021

Exposure of ectothermic vertebrates to fluctuated temperatures accelerates mitochondrial respiration and has been shown to increase the formation of mitochondrial reactive oxygen species (ROS), such as O2−, H2O2, and O2−·. Excess ROS production by intensively respiring mitochondria is held responsible for cellular damage. The present study was to test whether a protective oxidative‐stress pathway would be initiated in fish central nervous system upon cold stress.

Effects of acute cold exposure (from 28 °C to 18 °C) on brain oxidative stress parameters were investigated in zebrafish (Danio rerio). Concentrations of cellular protein carbonyl groups (biomarkers of oxidative stress) were significantly increased after 1‐hour cold exposure. Increased levels of anti‐oxidative stress parameters, catalase (CAT) and superoxide dismutase (SOD) were observed at 1‐ and 6‐hour cold exposure, respectively. In spite of maintained anti‐oxidant capacity, the increment status of cellular oxidized protein accumulation was still observed. A neuronal defense pathway against ROS, peroxisome proliferator‐activated receptor (PPAR), acts with genes encoding uncoupling proteins (UCPs) was further monitored. Real‐time PCR analysis indicated that ppar‐1αb increased was transcriptionally regulated the expression of zucp2 and zucp2l. Mitochondrial biogenesis activation, mild uncoupling, and reduction of free radicals may result intracellular pO2 levels drop. Moreover, these protective mechanisms in fish brain have been found to cause hypoxia‐induced factor (HIF) stabilization that facilitates an increase in energy supply.
The effects of allopurinol, uric acid sodium salt, and inosine on plasma uric acid and xanthine oxidase activity in broilers
Authors: Settle Tabatha Lynn , Carro Maria , Falkenstein Elizabeth , Klandorf Hillar
Published: June 2021

Xanthine oxidoreductase (XOR) has two forms in the avian, xanthine dehydrogenase (XD) and xanthine oxidase (XO). The purpose of these studies was to determine the effects of allopurinol and inosine on plasma uric acid and enzyme activity. In the first study, Cobb × Cobb (n=25; 7 weeks old) broilers were assigned to five treatments (n=5). Treatments included: Group 1 (control, no allopurinol), Group 2 (allopurinol addition), Group 3(allopurinol and osmotic pump), Group 4 (allopurinol, dietary uric acid addition), and Group 5 (allopurinol, uric acid injection). PUA was measured before the addition of allopurinol and weekly after the onset of the 3 week study. Total XO+XD in the liver was significantly higher (p=0.012) in group1 as compared to group 3 birds. Birds in group 2, group 4, and group 5 had significantly lower (p≤ 0.001) uric acid concentrations in the liver. In the second study, Cobb × Cobb (n=15; 5 weeks old) were separated into three treatments (n=5); control (CON), INO (inosine at 161g/kg feed), or INOAL ( inosine and allopurinol 50mg/kgBW). There was a significant increase (p=0.005) in total enzyme activity of the liver in the INO birds. INOAL birds had a significantly lower (p=0.005) uric acid concentration in the liver. The reduced uric acid concentration in the liver suggests that there is a residual effect of allopurinol, which potentially results in lowered antioxidant activity in this tissue.
Insulin increases the activity of NCC through a PI3K dependent mechanism
Authors: Arroyo Juan Pablo , Vazquez Norma , Gamba Gerardo
Published: June 2021

Obesity and diabetes mellitus are associated with the development of arterial hypertension. One potential mechanism that has been advanced to explain the obesity‐induced hypertension is the effect that insulin may have upon renal salt reabsorption. In this study we assessed the effect of insulin on the activity of the renal NaCl cotransporter, NCC, using the heterologous expression system of Xenopus laevis oocytes. Three days after injection with NCC cRNA the effect of insulin was assessed. NCC activity was measured as the thiazide‐sensitive tracer 22Na+ uptake. We observed that NCC activity is increased by 3‐fold when oocytes were exposed to insulin for 1 to 15 minutes before the uptake. The positive effect was dose and time dependent, with significant effect of insulin as low as with one unit and, as fast as after one minute of exposure. The addition of wortmannin, an inhibitor of phosphatydil‐inositol 3‐kinase (PI3K) completely abrogated the positive effect of insulin on the transporter's activity, without affecting the basal function. The effect of insulin was also abolished by pI103, a PI3K specific inhibitor, and by the specific AKT inhibitor IV. MAP or mTOR kinases inhibitors had no effect. Coinjection of NCC cRNA with WNK4, WNK3, or SPAK cRNA did not affect the activation of NCC by insulin. We conclude that insulin is a powerful activator of NCC and that insulin's effect is translated through the PI3K‐AKT pathway, suggesting that is due to threonine/serine phosphorylation. These observations may have implications to explain the association between obesity and diabetes mellitus with arterial hypertension.
Gender‐dependent regulation of expression of the KCNQ1 channel regulatory β‐subunit KCNE3 throughout the estrous cycle in rat distal colonic crypts
Authors: O'Mahony Fiona , Alzamora Rodrigo , Carter Derek , Thomas Warren , Harvey Brian J.
Published: June 2021

The KCNQ1 potassium channel associates with the KCNE3 (‐subunit in the epithelium of the colon and mediates basolateral K+ recycling that is required for apical Cl− secretion. This study tested whether sexual dimorphism exists in the expression and function of the KCNQ1/KCNE3 channel complex in rat distal colonic crypts. The association of KCNE3 with KCNQ1 and the abundance of KCNE3 mRNA and protein were significantly higher in male compared to female crypts. KCNE3 protein expression in the female rat colon varied throughout the estrous cycle; expression was highest during proestrus and declined 4.8‐fold in estrus (P (0.01, n=3) and became undetectable at metaestrus. Whole‐cell K+ currents in male crypts displayed a linear current‐voltage relationship and were 10‐fold more sensitive to chromanol 293B when compared with K+ currents in female crypts which displayed outward rectification. Basolateral membrane K+ currents recorded in colonic epithelia mounted in Ussing chambers also displayed a 10‐fold higher sensitivity to chromanol 293B in male tissues compared to females. This study demonstrates gender and estrous cycle regulation of the expression of KCNE3 and the properties of the KCNQ1 channel activity required for Cl− secretion.

Funded by HEA Ireland PRTLI NBIP Cycle 4.
IRBIT regulates the WNK/SPAK pathway
Authors: Yang Dongki , Li Qin , Muallem Shmuel
Published: June 2021

IRBIT (inositol‐1,4,5‐trisphosphate [IP3] receptors binding protein released with IP3) was discovered as an inhibitor of IP3 receptors and was later shown to activate the Na+‐HCO3− co‐transporter NBCe1B (also known as pNBC1). More recently, we showed that IRBIT plays a major role in epithelia to stimulate epithelial fluid and HCO3− secretion. IRBIT functions to coordinates epithelial fluid and electrolyte transport by stimulating the transporters NBCe1B and CFTR in the pancreatic duct. Another major regulatory pathway in epithelia is the WNK/SPAK kinases pathway that functions to inhibit epithelial fluid and electrolyte transport. This pathway plays a critical role in the control of systemic electrolyte metabolism and blood pressure. In many cases the WNKs phosphorylate SPAK, which, in turn, acts to phosphorylate several transporters and inhibit their activity primarily by increasing their endocytosis and reducing their surface expression. A central question is the interplay between the relative roles of the stimulatory IRBIT and inhibitory WNK/SPAK pathways in epithelial function. To address this question, we examined the effect of WNKs, SPAK and IRBIT in regulating the activity of NBCe1B and CFTR in model systems and secretory ducts. We found that WNKs acts through SPAK to inhibit both transporters by controlling their surface expression. Remarkably, IRBIT completely inhibits these effects by antagonizing the function of SPAK. IRBIT does not prevent the interaction of SPAK with the transporters, but rather makes the transporters insensitive to SPAK. The molecular mechanism of how IRBIT prevents the effect of SPAK is being explored and will be reported.
Epithelial membrane pumps, channels and transporters as phenotypic markers of equine hoof keratinocytes in situ: an immunohistochemical study
Authors: Asplin Katie , Harris Pat , Mobasheri Ali
Published: June 2021

Several different enzymatic techniques have been published for isolating and culturing primary equine hoof keratinocytes. However, the phenotype of the resulting primary cultures is poorly understood. Freshly isolated hoof cultures consist of a mixture of keratinocytes and fibroblasts, with the latter outgrowing and overtaking the keratinocytes. Although monoclonal antibodies against cytokeratins and vimentin have been used in recent studies to investigate the differentiation of hoof epidermal cells, there has been little progress on the basic cell biology of equine hoof keratinocytes and the markers that they express in vivo. In this study we hypothesized that epithelial transport proteins may be used as phenotypic markers for these cells. To test this hypothesis we employed a panel of monoclonal and polyclonal antibodies against a range of epithelial ion and solute transporters to establish new phenotypic markers for these cells in situ. Equine hoof keratinocytes showed positive and preferential immunoreactivity for several transporters and ion channels compared to other cell types in the hoof. These included Na, K‐ATPase, Na, K, 2Cl co‐transporter, epithelial sodium channels (ENaC), Maxi K (BK) calcium‐activated potassium channels, and aquaporins including AQP3 and AQP9. These results suggest that epithelial membrane transporters are specific and sensitive “metabolic” markers of hoof keratinocytes.
Pufferfish Slc26a5 (prestin) exchanges Cl− for oxalate, sulfate, and bicarbonate
Authors: Hirata Taku , Kato Akira , Chang Min‐Hwang , Hirose Shigehisa , Romero Michael F
Published: June 2021

The solute carrier 26 (Slc26) transporters are anion transporters with diverse substrate specificity. They are expressed mainly in epithelia and play a central role in secretion and absorption of anions such as chloride, oxalate (ox2−), sulfate (SO42−), bicarbonate (HCO3−), iodide, formate and hydroxyl. In mammals the Slc26a6 protein is expressed in kidney and gut epithelia, and mediates electrogenic Cl−/ox2−, Cl−/nHCO3−, Cl−/SO42− exchange. Recently, chicken and zebrafish prestin (Slc26a5) were shown to mediate electrogenic Cl−/ox2− and Cl−/SO42− exchange rather than being an auditory sensor. We have cloned and characterized the Slc26a5 (fPrestin) transporter from the euryhaline pufferfish Takifugu obscurus (mefugu). This fPrestin mRNA is expressed ubiquitously unlike fSlc26a6A‐C which is found in kidney and gut. While the fSlc26a6A‐C mRNAs are increased in response to a freshwater to saltwater transition, fPrestin expression is unchanged. We next expressed fSlc26a5 in Xenopus oocytes and evaluated transport using microelectrodes. These experiments reveal that fPrestin mediates electrogenic Cl−/ox2−, and Cl−/SO42− as previously shown for chicken and zebrafish Slc26a5. Furthermore, fPrestin also can mediate electrogenic Cl−/nHCO3− exchange. These transport features are quite similar to mammalian Slc26a6 which has been implicated in renal Ca‐oxalate, stones.
Luminal ATP release is impaired in intestinal alkaline phosphatase knockout mice duodenum
Authors: Akiba Yasutada , Watanabe Chikako , Nakano Takanari , Narisawa Sonoko , Millan Jose Luis , Kaunitz Jonathan D
Published: June 2021

Brush border intestinal alkaline phosphatase (IAP) pH‐dependently regulates the concentration of luminally‐released ATP, which stimulates duodenal bicarbonate secretion (DBS) via P2Y receptors. We hypothesized that lack of IAP increases luminal [ATP], enhancing DBS. We measured DBS with pH and CO2 electrodes and ATP with luciferin‐luciferase in a perfused duodenal loop in wild type (WT) or knockout (KO) mice lacking Akp3 (duodenal IAP equivalent) expression, or in SD rats. ATP degradation was impaired in KO duodenum, consistent with the role of IAP in ATP degradation. AP inhibition with glycerol phosphate (GP) increased DBS, inhibited by the P2 receptor antagonist suramin, with increased luminal [ATP] in WT mice and rat duodenum. In contrast, GP or suramin had no effect on DBS and ATP release in KO duodenum. Real‐time PCR revealed that the expression of multidrug resistant‐associate protein 2 (MRP2) was selectively downregulated with no change in the expression of other MRPs, multidrug resistance protein (MDR) 1a, ecto‐nucleoside triphosphate diphosphohydrolase isozymes or cystic fibrosis transmembrane conductance regulator. Furthermore, the MRP inhibitor MK571 inhibited GP‐induced ATP release in rat duodenum. These results suggest that lack of IAP impairs ATP release via downregulation of MRP2, which may participate in the epithelial ATP release.

NIH‐NIDDK R01 DK54221
Osmotic stress (OS)‐Induced Tight Junction (TJ) Disruption in Caco‐2 Cell Monolayers Involves Calcium‐Mediated JNK Activation and JNK‐Mediated MLCK activation
Authors: Samak Geetha , Rao R. K.
Published: June 2021

We investigated the signaling mechanism involved in OS‐induced TJ disruption in the intestinal epithelium. Caco‐2 cell monolayers were subjected to OS by exposure to 0.4 M mannitol. TJ integrity was evaluated by measuring transepithelial electrical resistance (TER), inulin flux and immunofluorescence localization of TJ proteins, occludin, ZO‐1 and claudin‐4 (Cldn‐4). OS‐induced decrease in TER, increase in inulin flux and redistribution of TJ proteins was attenuated by BAPTA and thapsigargin (TG), the calcium blockers, and by siRNA‐mediated knockdown of JNK1/2. OS rapidly increased phospho‐JNK1/2 (active) levels, which was attenuated by BAPTA and TG. OS also induced a rapid and transient activation of MLCK (increased phospho‐MLC levels), which was attenuated by SP600125 (JNK inhibitor), BAPTA and TG; JNK activation was unaffected by ML‐7. OS‐induced TJ disruption was prevented by ML‐7 (the MLCK inhibitor). OS‐induced TJ disruption was associated with the Tyr‐phosphorylation of occludin, ZO‐1 and Cldn‐4. Inhibition of JNK or MLCK abrogated OS‐induced Tyr‐phosphorylation of TJ proteins. BAPTA and TG also attenuated OS‐induced Tyr‐phosphorylation of TJ proteins. These studies demonstrate that OS‐induced disruption of intestinal epithelial TJ involves elevation of intracellular calcium, calcium‐mediated JNK activation, JNK‐mediated MLCK activation and Tyr‐phosphorylation of TJ proteins.
Protection of the intestinal barrier in hyperthermia: defining the role of 12/15‐lipoxygenase.
Authors: Novosad Veronica Lea , Phillips Neil A. , Oliver S. Ryan , Clanton Thomas L.
Published: June 2021

Hyperthermia can increase intestinal permeability, often contributing to severe heat illness. Previous work has shown that baicalein is protective of barrier integrity in hyperthermia. Since baicalein is both a 12/15‐lipoxygenase (LOX) inhibitor and an antioxidant, the mechanism of action is not understood. This study tested whether other LOX inhibitors, ETYA and LOXBlock‐1 were protective. LOXBlock‐1 does not have antioxidant properties. Intestinal segments of adult mice were isolated and everted to create ~8 × 2 cm sacs. These were treated with the LOX inhibitors, ETYA (0, 10, 20, and 50 μM) or LOXBlock‐1 (0, 10, 20, and 40 μM). All tissues were first incubated at 37± 0.5°C for 30 min with the drugs. Then temperature was increased to 42± 0.5°C for 90 min. Permeability was measured by accumulation of fluorescent labeled dextran (4kDal) that diffused into the sacs. Compared to control segments (42°C), ETYA significantly increased permeability (P<0.02). A similar trend was seen for LOXBlock‐1. Thus, in contrast to baicalein, no protection of barrier integrity was observed during hyperthermia with 12/15 LOX inhibitors and in most cases LOX inhibition worsened barrier function. This suggests that normal LOX function is critical for maintaining intestinal barrier in hyperthermia and that baicalein's protective effects must be related to other mechanisms such as its known antioxidant actions. NHLBI 53333
Epidermal growth factor treatment prevents intestinal injury in weanling mice with septic peritonitis
Authors: Dominguez Jessica A. , Coopersmith Craig M.
Published: June 2021

Sepsis is common in infants with a mortality rate of >30%. Neonates are susceptible to intestinal injury due to an immature gastrointestinal tract. Epidermal growth factor (EGF) has trophic and maturational effects on the intestine. The aim of this study was to determine if EGF treatment prevents sepsis‐induced intestinal injury and improves survival in weanling mice. Three‐week old mice were subjected to 2×25 cecal ligation and puncture or sham laparotomy and were treated postoperatively with or without EGF (150 μg/kg/d i.p.). At 24 hr, intestines were evaluated for apoptosis, proliferation, and villus length. Survival studies were done in a separate cohort of mice. Compared to shams, septic mice had increased intestinal apoptosis (72 ± 14 vs. 6 ± 1 cells/100 crypts; p<0.001) and decreased proliferation (629 ± 54 vs. 1207 ± 37 cells/100 crypts; p<0.001), while EGF normalized both to sham levels (12 ± 1 vs. 6 ± 1 and 1064 ± 44 vs. 1207 ± 37 cells/100 crypts, respectively; p=ns). Septic mice had shorter villi compared to shams, and EGF resulted in normalization to shams (216 ± 3 vs. 398 ± 22 vs. 456 ± 26 μm; p<0.001 and p=ns). EGF treatment resulted in improved 7‐day mortality (50% vs. 83%), but the difference was not statistically significant. EGF preserves intestinal integrity in a weanling model of septic peritonitis, and thus may be a novel therapeutic agent for the treatment of neonatal sepsis. NIH GM66202 & GM082008.
Hnf1α is required for proper gut epithelial endocrine cell specification and controls the mTOR signalling pathway in mice
Authors: Boudreau Francois , Lussier Carine R , Brial Francois , Rivard Nathalie , Perreault Nathalie
Published: June 2021

Although roles for Hnf1α are well characterized in the pancreas and liver, its precise function within the maintenance of intestinal homeostasis remains mostly unknown. We aimed to determine the role for this transcriptional regulator in this context. Loss of Hnf1α in mice results in a disturbance of intestinal epithelial cell lineages functions, increased crypt proliferation and intestinalomegaly during adult life. We show that Hnf1α is an important regulator of the mucosal barrier function and lumen‐to‐blood glucose delivery. The intestine of Hnf1α null mice display impaired endocrine cell differentiation with a decrease in gastric inhibitory polypeptide and an increase in ghrelin expression, both gut specific hormones directly involved in glucose and energy homeostasis. A similar impact is observed on proper Paneth cell differentiation with abnormalities in the granule exocytosis pathway. Finally, we identify the Ddit4l/REDD2 inhibitor of the mammalian target of rapamycin (mTOR) to be down modulated in the small intestine of adult Hnf1α mutant mice. This observation correlates with the abnormal sustained activation of the mTOR signalling pathway at the reach of adulthood. Together, these results unravel a novel role for Hnf1α in sustaining the maturation of secretory cell lineages and in regulating intestinal growth through the mTOR signalling pathway. This work was supported by the CIHR
The SWI/SNF chromatin remodeling complex is required for GI smooth muscle development and differentiation
Authors: Zhang Min , Chen Meng , Zhou Jiliang , Herring B. Paul
Published: June 2021

We have shown that myocardin related transcription factors require Brg1 or Brm containing SWI/SNF chromatin remodeling complexes to induce the transcription of smooth muscle‐specific genes in nonmuscle cells. In addition, expression of dominant negative Brg1 or knockdown of Brg1 attenuated expression of SRF dependent smooth muscle‐specific genes in primary cultures of smooth muscle cells. The goal of our current studies is to elucidate the role of SWI/SNF complexes in regulating gastrointestinal smooth muscle development in vivo. Mice harboring a smooth muscle‐specific knockout of Brg1 on a wild type or Brm null background were generated and analyzed. Both single and double KO mice exhibited a significantly shorter gut with attenuated contractility and altered smooth muscle cell organization. In Brg1/Brm double KO mice we also observed decreased expression of smooth muscle‐specific contractile proteins that contributes to the lack of GI motility and postnatal lethality of these mice. Although the Brg1 KO mice are viable they exhibit impaired GI motility and many of these mice develop megacolon. The GI phenotypes of the smooth muscle specific Brg1 KO mice, in the presence of normal levels of Brm, suggests that Brg1‐containing SWI/SNF complexes play specific roles in GI development that cannot be compensated for by Brm‐containing complexes.

Supported by an AHA fellowship to MZ, DK61130 to BPH, and an AHA SDG to LZ.
17β Estradiol relaxes tension in male guinea pig gallbladder strips
Authors: Kline Loren , Karpinski Edward
Published: June 2021

In this study we have shown that 17β estradiol (E2) caused relaxation of male guinea pig gallbladder strips precontracted with either 1nM cholecystokinin octapeptide (CCK) or 40mM KCl. E2 relaxation was concentration dependent in both cases. The purpose of this study was to determine whether E2 relaxation was due to inhibition of intracellular Ca2+ release by IP3 or extracellular Ca2+ entry. Paired t‐tests were used. The KCl and CCK produced similar amounts of tension (0.84±0.04 vs. 0.88±0.05g, n=7). The E2 induced relaxation after CCK was significantly (p<0.001; 46.1±10.8 vs. 74.9±8.1%, n=7) less than observed with KCl. 2‐APB (125 μM), significantly (p<0.001; 0.87±0.06 vs. 0.41±0.07g, n=4) decreased CCK induced tension, and significantly (p<.0.01; 57.5±5.5 vs. 71.2±5.6%, n=4) increased E2 induced relaxation. Similar results were observed when KCl was used with 2‐APB (p<0.001; 0.8±0.07 vs. 0.61±0.06g, n=4). The E2 induced relaxation was increased in the presence of 2‐APB (p<0.01; 73.7±8.3 vs. 100.2±5.5%, n=3). E2 induced relaxation of KCl induced tension suggested that the E2 induced relaxation is mediated by inhibiting extracellular Ca2+ entry. The use of 2‐APB and the increase in E2 induced relaxation supports this conclusion. E2 relaxed CCK induced tension; 2‐APB increased the E2 relaxation. E2 induced relaxation is primarily mediated by blocking extracellular Ca2+ entry.
Characterization of Melatonin Receptors and Signaling Pathways in Gastrointestinal Smooth Muscle
Authors: Ahmed Rashad , Mahavadi Sunila , Grider John R , Murthy Karnam S
Published: June 2021

Melatonin, a close derivative of serotonin, is involved in physiological regulation of circadian rhythms. In the gastrointestinal (GI) system, melatonin exhibits endocrine, paracrine and autocrine actions and is implicated in the regulation of GI motility. Generally, melatonin actions oppose the stimulatory actions of serotonin on motility. However, it is not known whether melatonin can also act directly on GI smooth muscle cells.

AIM

To determine the expression of melatonin receptors in smooth muscle and identify their signaling pathways.

METHODS

Muscle cells were isolated from rabbit stomach by enzymatic digestion. Expression of melatonin receptors, MT1 and MT2, was determined by RT‐PCR and Western blot. G protein activity was measured by melatonin‐induced increase in Gα binding to [35S]GTPγS. PLC‐β activity was measured by ion‐exchange chromatography. Cytosolic Ca2+ was measured in fura‐2 loaded cells and muscle contraction by scanning micrometry.

RESULTS

MT1 was detected by RT‐PCR and western blot. Melatonin activated Gαq, but not Gαs, Gαi1, Gαi2, or Gαi3. Consistent with activation of Gαq, melatonin stimulated PI hydrolysis, increased cytosolic Ca2+, and elicited muscle contraction.

CONCLUSION

. Gastric smooth muscle cells express receptors for melatonin (MT1) coupled to Gq. The receptors mediate stimulation of PLC‐β activity and increase in cytosolic Ca2+, and elicit muscle contraction.
Ghrelin enhances the regenerative potential of stem cells
Authors: Fahim Mohamed A , Karam Sherif , Al‐Ramadi Basel
Published: June 2021

Little is known about the mechanisms involved in the regenerative potential of stem cells following damage. Ghrelin is a peptide that promotes growth hormone secretion and in the thymus, inhibits apoptosis and increase cell proliferation.

Presently we examine the influence of ghrelin on the regenerative potential of stem cells in bone marrow and gut. Cell degeneration was induced in mice by 2 doses of doxorubicin (10 and 6 mg/kg, iv). Some treated mice received continuous infusion of ghrelin (1.25 μg/hr) by using an implanted osmotic pump. To label dividing stem cells, all mice received a single ip injection of BrdU two hours before sacrifice. Blood was collected for differential cell counts and the gut was processed for histology and immunohistochemistryg using anti‐BrdU antibody. The results showed that the numbers of WBCs, RBCs and platelets were substantially reduced by 3 days after administration of chemotherapy and began to recover by day 10. These findings tend to occur in parallel with the dramatic damage and recovery in mucosal gut. In ghrelin‐treated mice, a dramatic recovery in blood cell counts and gut damage was evident as early as day 3. Immunohistochemistry depicted an increase in the number of BrdU‐labeled cells in response to damage, which upon recovery were reduced, suggesting that ghrelin enhances the regenerative potential of stem cells in both the bone marrow and gut.
Activation of proteinase activated receptor‐2 (PAR‐2) inhibits spontaneous activity in rat colon
Authors: Kerr Paul M , Koshka Amanda , Plane Frances
Published: June 2021

Proteolytic cleavage of proteinase activated receptor‐2 (PAR‐2) reveals a tethered ligand which binds to and activates the receptor. Rat colonic smooth muscle shows spontaneous contractile activity which is generated by pacemaker cells. This study investigated the effects of PAR‐2 activation on spontaneous contractile activity in the isolated rat colon.

Strips of rat colon longitudinal muscle were suspended in organ baths and increasing concentrations of trypsin, or the mimetic peptide FLIGRL, were applied. In some experiments carbachol was used to increase smooth muscle tone.

Trypsin and FLIGRL each induced concentration‐dependent inhibition of spontaneous contractile activity of rat colon. Trypsin‐evoked relaxations (66.6±3.3; n=8) were abolished by apamin, an inhibitor of small conductance Ca2+‐activated potassium channels (SKCa). Similar results were obtained with FLIGRL (n=5). Cyclopiazonic acid, an inhibitor of sarcoplasmic reticulum Ca2+‐ATPase, also abolished the effects of trypsin (n=7). Neither trypsin (n=4) nor FLIGRL (n=4) reduced carbachol‐induced tone.

In conclusion, activation of PAR‐2 inhibits spontaneous contractile activity in the rat colon via release of Ca2+ from intracellular stores and activation of SKCa channels. PAR‐2 activation is limited to pacemaker cells since no effect was observed on carbachol stimulated smooth muscle.

Supported by NSERC
Translational Regulation of CUG‐BP1 by Polyamines through the RNA‐binding Protein HuR
Authors: Cui Yuhong , Xiao Lan , Rao Jaladanki N. , Zou Tongtong , Liu Lan , Gorospe Myriam , Wang Jian‐Ying
Published: June 2021

All mammalian cells depend on polyamines for growth and division, but the exact roles of polyamines at the molecular level remain unclear. CUG‐BP1 protein interacts with CG‐rich element of many labile mRNAs and regulates decay and translation of its target transcripts. HuR modulates mRNA turnover and translation and its activity is tightly regulated by cellular polyamines. This study tests if polyamines regulate CUG‐BP1 expression through HuR in normal intestinal epithelial cells (IEC‐6 line).

Methods

Polyamine levels were depleted by inhibiting ODC (key enzyme for polyamine biosynthesis) with DFMO but increased by overexpressing ODC gene. CUG‐BP1 expression was examined by newly translated protein and reporter assay using chimeric CUG‐BP1 3′‐UTR. RNA‐binding was examined by biotin pull‐down and RNP‐IP assays.

Results

Decreased polyamines by DFMO increased CUG‐BP1 protein, whereas elevated polyamines through ODC overexpression reduced CUG‐BP1 protein, neither intervention changed CUG‐BP1 mRNA levels. Polyamine depletion also increased CUG‐BP1 translation, but it failed to alter CUG‐BP1 protein stability. HuR bound the CUG‐BP1 mRNA and this association increased following polyamine depletion. HuR silencing repressed CUG‐BP1 translation in polyamine‐deficient cells, thus reducing CUG‐BP1 expression levels.

Conclusions

These results indicate that polyamines repress CUG‐BP1 translation by reducing HuR interaction with the CUG‐BP1 mRNA.
PGC‐1 (Peroxissome‐proliferator activated receptor gamma co activator 1) α/β and melanoma growth: a possible energy‐mediated relation.
Authors: Passos Luís Augusto , de Souza Heraldo Possolo
Published: June 2021

The uncontrolled cell proliferation is a remarkable feature of cancer and it is known to require a large amount of energy. PGC‐1α/β were described as co activators capable of promoting mitochondria biogenesis and increase its respiratory function. Our hypothesis is that PGC‐1α/β may be over expressed in melanoma cells, increasing its capability of proliferation. We cultured normal melanocytes (Ma) and two cancer lineages (Tm1 and Tm5) and mRNA expression of PGC‐1α/β and TFAM (Mitochondrial Transcription Factor A) were analyzed by RT‐PCR. Cell growth was determined by counting in Neubawer's chamber at every 24 hours, within 5 days, using Tripan Blue exclusion to check cell viability. PGC1α mRNA was increased in Tm1 and Tm5 compared to Ma (Ma=1,23±0,24 vs Tm1=2,67±0,23 vs Tm5=3,05±0,43, p<0,01). PGC‐1β (Ma=0,47±0,02 and Tm1=0,67±0,02 vs. Tm5=0,81±0,03, p<0,05) and TFAM (Ma=0,91±0,07 e Tm1=0,89±0,03 vs. Tm5=1,17±0,06, p<0,05) expression were increased only in Tm5. Cancer cells duplication time was 24 hours whereas Ma was duplicated at 72 hours and all lineages presented 95% to 100% viability during the experiment. In conclusion, exponential growth in cancer lineages may be related to increasing energy availability mediated by PGC‐1 α/β expression. Financial support: FAPESP and DIREX‐LIM.
The regulation of intestinal iron transport in a rat model of chronic renal failure
Authors: Srai Surjit Kaila , Sharp Paul A , Marks Joanne , Unwin Robert J , Debnam Edward S
Published: June 2021

The hormone erythropoietin (Epo) is produced by the kidney and stimulates erythropoiesis. In chronic renal disease Epo levels are reduced and patients develop anemia, which is treated by administering a combination of iron and recombinant Epo. The mode of action by which Epo improves iron homeostasis is still unclear, but it may involve suppression of the iron regulatory peptide hepcidin. To study the effects of Epo on iron metabolism in chronic renal failure (CRF) we used the well established 5/6th nephrectomy rat model. CRF animals were treated with human recombinant Epo (100 U/kg body weight) for 5 weeks before experimentation. CRF increased hepatic hepcidin expression and decreased intestinal iron absorption. Following Epo treatment, hepcidin expression significantly decreased and iron absorption was restored to levels found in sham‐operated rats. In intestinal Caco‐2 cells, Epo stimulated iron absorption, suggesting a direct role for Epo in the intestinal handling of iron. In the context of treating anemia in CRF, Epo down‐regulates hepcidin, which in turn permits the release of iron from reticuloendothelial macrophages; in addition Epo stimulates intestinal iron absorption. Together these pathways can normalize iron balance. This work was funded The Roche Foundation for Anemia Research (RoFAR) and Kidney Research UK.
Identification of hepcidin antagonists through a high throughput screening approach
Authors: Fung Eileen , Hsu Jason , Damoiseaux Robert , Ganz Tomas , Nemeth Elizabeta
Published: June 2021

Hepcidin is a peptide hormone with a key role in iron homeostasis. It acts by causing degradation of its receptor, the cellular iron exporter ferroportin, which results in decreased iron supply to plasma. Elevated hepcidin is a mediator of anemia of inflammation (AI), a condition which develops in a wide variety of infectious and inflammatory diseases and contributes to their morbidity. During inflammation, increased hepcidin levels leads to a restriction of iron supply for erythropoiesis, and eventually anemia. We developed a high‐content screen using cells expressing ferroportin‐GFP fusion protein to identify small molecules acting as hepcidin antagonists. We identified compounds which disrupt the hepcidin‐ferroportin axis and allow continuous iron export from cells in the presence of hepcidin. The hepcidin antagonists include thiol modifiers and cardiac glycosides. Thiol modifiers inhibit hepcidin binding to ferroportin while cardiac glycosides inhibit ferroportin internalization. These hits are undergoing further characterizations in cellular studies and animal models of AI. The studies will answer fundamental questions about the pathogenesis of AI and the related problem of resistance to erythropoiesis‐stimulating agents. The screen may also provide lead compounds for further development as therapeutics. This work is supported by NIH/NIDDK and NRSA.
Effect of stress on mucin‐producing cells in the intestinal tract of mice
Authors: Habiyambere Bupe Martha , Onyango Edward M
Published: June 2021

Mucins are heavily glycosylated proteins that form part of the protective mucosal barrier of the gastrointestinal tract. This study investigated the effects of chronic stress on mouse intestinal histology especially that of mucin‐producing cells. Sixteen ICR male mice were divided into two groups of eight, a test and a control group. Test mice were restrained for 6 hours daily for 21 days. The control group had no access to feed or water during the 6‐hour restraint period of test mice. After the study period, all mice were euthanized and samples were collected. Blood was collected by retro‐orbital puncture and centrifuged to obtain serum for a corticosterone assay. Duodenal and jejunal intestinal portions were removed and fixed in Methacarn, embedded in paraffin, sectioned and stained using Alcian Blue‐Periodic acid Schiff to visualize the mucin‐producing goblet cells and measure villi length. As expected stressed mice showed higher corticosterone levels compared to control animals (P<0.05). Histology showed a decrease in the number of goblet cells in both the duodenum and jejunum of stressed mice compared to control mice (P<0.05). Villi length was lower in stressed mice compared to control animals (P<0.05). The findings suggest that stress may affect the dynamics of mucin production in the intestinal tract of mice.

Research was supported by East Tennessee State University.
Segmental expression of H,K‐ATPase‐α2, KCC3, KCC4, and DRA in mouse distal colon is upregulated by dietary Na+ restriction
Authors: Lytle Christian , Evans Adam , Payne John A. , Talbot Colleen
Published: June 2021

The proximal and distal segments of the colon are known to differ considerably in their ion transport functions and their responses to Na+ depletion. We examined the distribution of absorptive ion transporters in surface epithelial cells (SEC) of the cecum and colon (proximal, middle, distal) by confocal IF microscopy and Western blot of female CD‐1 mice maintained on a standard or low‐Na+ diet for 8 days. Mucosal layers were probed with antibodies selective for H,K‐ATPase‐α2 (H,K), K‐Cl cotransporter‐3 and ‐4 (KCC3, KCC4) and anion exchangers AE1, AE2, and DRA. H,K was restricted to the apical margin of SEC in the mid and distal colon, and its abundance increased >15‐fold with Na+ deprivation. Apical DRA and basolateral KCC3 and KCC4 expression increased distally along the colonic axis with augmented expression (~40%) in the mid to distal colon of Na+ restricted mice. Cecal SEC also highly expressed DRA, KCC3 and KCC4, and Na+ deprivation strongly reduced expression. AE1 was highly expressed in the distal colon and AE2 in the proximal colon; Na+ deprivation diminished expression of both these transporters. Our findings implicate coordinate expression of apical H,K and DRA together with basolateral KCC3/4 in distal colon transport (KCl absorption and/or NH4+ secretion), and establish the cecum as a target of divergent aldosterone control.
Taste receptor‐mediated duodenal bicarbonate secretion is associated with incretin release in rats
Authors: Wang Joonho , Akiba Yasutada , Engel Eli , Guth Paul H , Kaunitz Jonathan D
Published: June 2021

Luminal nutrient chemosensing during meal ingestion is mediated via intestinal endocrine cells to regulate secretion and motility. We have reported that luminal L‐glutamate (L‐Glu) with inosinate (IMP) stimulates duodenal bicarbonate secretion (DBS) possibly via taste receptor heterodimers T1R1/R3. To further study this mechanism, we hypothesized the involvement of gastric inhibitory peptide (GIP) or glucagon‐like peptide (GLP) release in L‐Glu/IMP‐induced DBS. We measured DBS with a pH‐stat or with pH and CO2 electrodes through a rat duodenal loop. Rat GIP, GLP‐2, or exendin‐4 (GLP‐1 receptor agonist, Ex‐4) was continuously injected iv (0.1 – 1 nmol/kg/hr). Furthermore, L‐Glu and/or IMP was luminally perfused with or without iv injection of ProGIP (GIP receptor antagonist) or exendin‐3‐(9–39) (GLP‐1 receptor antagonist). GIP and GLP‐2 dose‐dependently increased DBS, whereas Ex‐4 decreased DBS. Perfusion of L‐Glu with IMP increased DBS, with no effect of ProGIP or exendin‐3‐(9–39). Luminal perfusion of L‐Glu with IMP increased portal venous [GLP‐1], with no effect on [GIP]. These results suggest that L‐Glu/IMP‐induced DBS is possibly mediated via GLP‐2 release.

A grant from Ajinomoto Inc, NIH R01 DK54221
Increased mitochondrial expression of copper/zinc superoxide dismutase (SOD1) following adenoviral‐mediated gene transfer inhibits angiotensin II (AngII) intra‐neuronal signaling
Authors: Li Shumin , Yang Rui‐Fang , Yin Jing‐Xiang , Schultz Harold D. , Zimmerman Matthew C.
Published: June 2021

Adenoviral‐mediated overexpression of the superoxide (O2•−) scavenging enzymes SOD1 or SOD2 similarly inhibit AngII intra‐neuronal signaling. Although SOD1 is commonly thought to reside only in cytoplasm, it is also known to be present in mitochondria. We hypothesized that overexpressing SOD1 via adenoviral‐mediated gene transfer (AdSOD1) increases mitochondrial levels of SOD1 and, as a result, attenuates AngII intra‐neuronal signaling. In mitochondria isolated from AdSOD1‐infected CATH.a neurons, SOD1 protein and activity are significantly elevated compared to AdEmpty (control vector)‐infected neurons. As measured by MitoSOX Red fluorescence, AngII (100 nM, 20 min) increased mitochondria O2•− levels (4.3 ± 0.3 and 4.8 ± 0.3 fold increase, respectively; P<0.05 vs. baseline fluorescence), but this response was attenuated by AdSOD1 (3.3 ± 0.2 fold increase; P<0.05 vs. control and AdEmpty). As expected, overexpressing the mitochondrial‐targeted SOD2 also decreased mitochondrial O2•−. Further, SOD1 overexpression significantly blunted the AngII‐induced inhibition of K+ current in neurons (11 ± 3% vs 45 ± 4% inhibition in control neurons, P<0.05). These data indicating that adenoviral‐mediated overexpression of SOD1 increases functional protein in mitochondria provide a rational explanation why SOD1 and SOD2 overexpression similarly inhibit AngII‐dependent responses. Support: AHA 0930204N