bluuebunny/crossref_metadata_2025_split huggingface dataset:
Nutritional Status Affects Expression of Insulin Receptor mRNA in a Tissue Specific Manner in Channel Catfish
Authors: Leiker Alexyss , Evans Paige , Watkins Brianna , Kobayashi Yass 
Published: November 2023

The objective of this study focused on the influence of nutritional status on the expression of two insulin receptor mRNA (IRa and IRb) in juvenile channel catfish. Obesity is a prevalent problem, affecting over 36% of the American population. Channel catfish could serve as an alternative model organism for the investigation of human metabolic disorders such as obesity due to its widely known genome and genetically increased growth leads to an obese‐like phenotype. However, the role of insulin and its receptor(s) on the development of obese‐like phenotypes in channel catfish is unclear. Juvenile channel catfish were randomly assigned to three treatments (n=4 tanks per treatment, 8 fish per tank). Fish assigned to the control group received food daily for 28 days, whereas fish assigned to the fasted group were fasted for 28 days. Fish assigned to the fasted‐fed group were fasted for 14 days and fed for the remaining 14 days. Brain, liver, and muscle tissues were collected from the fish on day 28 and expression of IRa and IRb mRNA were quantified using real‐time PCR. Expression of both IR mRNA in muscle and brain was similar among all treatments. In contrast, expression of both IRa and IRb was higher (p<0.01) in the liver in the fasted fish compared to the other two treatment fish. The results of this study showed that expression of IR mRNA is influenced by the nutritional status in a tissue‐specific manner regardless of the transcripts. Given the importance of insulin on nutrient metabolism, results of this study could provide insights to the mechanism(s) associated with the development of the obese‐like phenotype in channel catfish.Support or Funding InformationThis study is supported by the Kansas IDeA Network of Biomedical Research Excellence (Grant Number: 20GM103418).
Diabetes Mellitus Influences the Pattern of Distribution of Irisin in Pancreatic Islet Cells
Authors: Adeghate Ernest , D'Souza Crystal , Al Harthi Ahmed Mukhtar 
Published: November 2023

Irisin is a newly discovered myokine, which enhances the turnover of white adipose cells into brown fat cells. Irisin is derived from FNDC5, a membrane‐bound protein. Moreover, irisin has been shown to play an important role in energy regulation especially in muscle cells. Although, irisin has been detected in many organs including the exocrine pancreas, liver, testes and sebaceous gland, a precise localization of this polypeptide in pancreatic islet cells has not been undertaken. Immunohistochemistry was used to determine the pattern of distribution of irisin in streptozotocin‐induced (60 mg/Kg body weight) diabetes in rats. Image J® software was used to quantify the number of irisin‐immunoreactive cells in diabetic and non‐diabetic rat pancreas. Our study shows that irisin is present in a large number of pancreatic beta cells where it co‐localizes with insulin. In contrast, irisin does not co‐localize with glucagon in normal rat pancreas. However, this pattern of distribution changes after the onset of diabetes mellitus (DM). DM is characterized by increased number of glucagon immunopositive cells. The newly formed glucagon‐positive cells now contain irisin as well. The reason why irisin co‐localizes with glucagon in diabetic rat pancreas and not in non‐diabetic rats is unclear. It is possible that in diabetic rats, insulin, the natural inhibitor of glucagon has been removed paving way for glucagon expression in pancreatic beta cells devoid of insulin. The presence of irisin in insulin secreting beta cells suggests that the beneficial effect of irisin on glucose metabolism could indeed be via the endocrine pancreas.Support or Funding InformationUnited Arab Emirates University
Heat Shock Factor Activation in Skeletal Muscle Following Lengthening Contractions
Authors: Pollock‐Tahiri Evan , Locke Marius 
Published: November 2023

Heat shock proteins (HSPs) are integral for the maintenance of proteostasis and allowing cells to cope with episodes of stress. HSPs are regulated by transcription factors known as heat shock transcription factors (HSFs). Given that when skeletal muscles are subjected to lengthening contractions (LCs) both muscle damage and HSPs are increased, it was of interest to investigate the mechanism of the LC‐induced increase in HSPs and assess the role of HSFs in this process. Thus, the purpose of this study was to determine if HSF activation (HSF trimerization and DNA binding) is elevated in rat skeletal muscle following exposure to 60 LCs. To do this, male sprague‐dawley rats were anaesthetized and one tibialis anterior (TA) muscle was subjected to 60 LCs (3 sets of 20 LCs with 5 minutes rest between sets) and removed at 0, 1, 3, or 24 hours after LCs, while the non‐stimulated contralateral TA served as a control. Hsp72 and Hsp25 content, HSF activation, muscle damage, and muscle glycogen content were assessed at various time points. Following 60 LCs, electrophoretic shift assay showed a transient increase in HSF activation (0–3 hours) that was followed by a significant (p<0.001) increase in Hsp72 (3.4±0.21 fold) and Hsp25 (3.2±1.2 fold) content 24 hours later as assessed by western blotting. Histological assessment of the TA muscle revealed evidence of muscle damage (measured as necrotic fibers and inflammatory infiltrates) and glycogen depletion. Taken together, the results suggest LCs induce structural and metabolic stress that results in muscle damage, increased HSF activation and the accumulation of Hsp72 and Hsp25.Support or Funding InformationNo external funding
RNA Next Gen Sequencing of Kidneys from Diabetic Rats Provides Insights Into Reno‐protective Effects Of Vitamin D Receptor Activation
Authors: Prabhakar Sharma S , Kam Katherine , Kottapalli Rao , Yego Chepchumba 
Published: November 2023

IntroductionDiabetic nephropathy (DN) is the leading cause of end stage renal disease. The current therapy of DN remains suboptimal despite many currently available options. Recent experimental studies including from our laboratory and clinical reports support the therapeutic potential of vitamin D receptor activators, particularly paricalcitol. However molecular mechanisms underlying such protective effects are unclear. The goal of this study was to evaluate global gene expression changes using next‐generation sequencing‐based transcriptome analysis following paricalcitol treatment in obese ZSF1 rats, a murine model of metabolic syndrome that develops DN.MethodsAll rats were fed with a high fat diet to maintain hyperglycemia from 8 to 31 weeks. The test group of rats was injected subcutaneously twice a week with 0.2 μg of paricalcitol from 21 to 31 weeks. Following treatment, kidneys were harvested and total RNA was isolated and processed for transcriptome analysis using Illumina HiSeq 2500. Paricalcitol‐ treated ZSF1 rats were compared with obese untreated rats. The differential gene list from this analysis was imported into Ingenuity® Pathway Analysis (IPA®) for global transcriptome analysis and pathway mapping.ResultsWe detected 650 genes that had a significant change of at least 2 fold based on log 2 fluorescence intensity values. Of these 568 transcripts were up‐regulated whereas 82 transcripts were down regulated. Established pathways that were dominantly regulated include the Wnt signaling pathway that is primarily associated with cell cycle regulation. Specific molecules that were regulated with this pathway are Wnt3 Wnt9B and Wnt11 that were up regulated. Wnt 10b was also down regulated. OX40 signaling that involves regulation of the NF kB pathway was also regulated. Vitamin D signaling molecules that significantly changed following paricalcitol include the transcription factor enhancer binding protein α (EBPα), interleukin‐12 (IL‐12), interferon γ (IFNγ) and IP‐10. Several pathways that have previously been linked to diabetic nephropathy were differentially regulated by paricalcitol. These include endothelial nitric oxide synthase (eNOS) signaling, vascular endothelial growth factor (VEGF), protein kinase C and NF kB.ConclusionsParicalcitol treatment induces significant changes of gene expression in the kidney and most of these changes involve up regulation of several genes. Wnt signaling as well as changes in vitamin D signaling are regulated by paricalcitol. Thus the reno‐protective effects by paricalcitol treatment possibly involve inhibition of signaling pathways that have previously been associated with the progression of DN.Support or Funding InformationSupported by Woirhaye Research Endowment
Whole Genome and RNA Sequencing Identifies Genetic Factors involved in Nephron Deficiency in the HSRA Congenital Solitary Kidney Rat
Authors: Wang Xuexiang , Johnson Ashley C. , Garrett Michael R 
Published: November 2023

The HSRA rat is a unique polygenic model of congenital abnormalities of the kidney and urogenital tract, with 50–75% of offspring developing a single kidney. The model also exhibits nephron deficiency (~20% less nephrons) in the remaining kidney, glomerular and kidney hypertrophy, and elevated single nephron GFR, which leads to progressive kidney injury and decline in kidney function with age. A number of animal studies have provided an understanding of the physiological mechanism of injury, but it remains unknown the specific genetic factors that cause failure of one kidney to develop, as well as the mechanism of nephron deficiency in the remaining kidney. A recent genome‐wide association study (GWAS) performed by others using the heterogeneous stock rat (progenitor of the HSRA model), identified genomic loci on chromosomes (RNO) 4, 10, and 14 associated with the solitary kidney trait. The confidence interval for the RNO4 locus is ~4.2 Mb containing 14 genes, the RNO10 interval is ~1.5Mb containing 15 genes, and the RNO14 locus is consistent with locus linked to solitary kidney in the ACI rat. The whole genome was sequenced to a depth of 18X coverage using the Illumina NextSeq500 platform. Sequence alignment and annotation [BN reference genome (UCSC Rn5)], identified 1,776,635 SNP, 314,786 INDEL, and 25,346 structural variants. RNA sequencing of embryonic kidney isolated at key stages of nephrogenesis (E14.5, 15.5, and 16.5) between single kidney and two‐kidney control identified dysregulation of genes linked to cellular movement (p‐value= 2.36E‐06 ‐ 1.49E‐02 n=78/451), cell morphology (p‐value= 1.86E‐05 ‐ 1.49E‐02 n=82/451) and cell‐to‐cell signaling and interaction (p‐value= 8.38E‐05 ‐1.49E‐02 n=68/451). The top canonical pathway was associated with Wnt/b‐catenin (p‐value= 4.89E‐05) which includes Wnt2/Nlk candidates genes and is highly relevant to kidney development. Based on the GWAS, whole genome sequencing, and RNA sequencing, the causative genomic loci were refined to specific genes [c‐Kit (RNO14), Fam58b and Nlk (RNO10), and Met and Wnt2 (RNO4)] that could play a role in the solitary kidney trait and nephron deficiency in the HSRA model. In summary, the identification of the specific gene defect, modifier genes, and signaling pathways that lead to loss of one kidney and nephron deficiency will provide a mechanistic link between the solitary kidney genotype and the development of kidney dysfunction exhibited by the HSRA rat.Support or Funding InformationSupported by HL094446 (Garrett), UMMC, and Robert Hearin Foundation. Genomics Core [P20 GM103476 [MS‐INBRE‐(Elarsi)], P30GM103328 (CPN‐COBRE) and P20GM104357 (Obesity‐COBRE)
Conditional knockout of astrocytic Kir4.1 channels impairs central respiratory drive despite maintaining a subset of Cx26+ astrocytes within the RTN
Authors: Hawkins Virginia E , Kolling Fred W , Nelson Craig E , Mulkey Daniel K 
Published: November 2023

Central chemoreception is the mechanism by which the brain regulates breathing in response to changes in tissue CO2/H+. The retrotrapezoid nucleus (RTN) has been identified as a key locus for central respiratory control. Neurons in this region are intrinsically sensitive to changes in CO2/H+ (i.e. are chemosensitive). RTN astrocytes also function as chemoreceptors by sensing CO2/H+ through inhibition of an inwardly rectifying (Kir4.1‐like) K+ channel, and respond by releasing ATP via connexin 26 (Cx26) hemichannels. This CO2/H+‐dependent purinergic drive from RTN astrocytes has been shown to increase the chemosensitive RTN neuron response by ~30%. However, it is not known whether Kir4.1 in astrocytes is required for RTN chemoreceptor function, and little is known regarding the molecular identity of the chemosensitive astrocyte cell subset. To investigate this we generated an inducible astrocyte specific Kir4.1 channel knockout (Kir4.1 cKO) using GFAP‐CreERT2 and Kir4.1 floxed mouse lines, along with a TdTomato reporter line. Whole‐cell voltage clamp recordings of RTN astrocytes in juvenile brainstem slices from Kir4.1 cKO mice showed the absence of a Kir4.1‐like, CO2/H+ ‐sensitive current. RTN neurons from Kir4.1 cKO brainstem slices show a reduced purinergic component to their CO2/H+ response, suggesting deletion of Kir4.1 from astrocytes may disrupt the ability of these cells to respond normally to changes in CO2/H+. Respiratory activity was measured in adult mice by whole body plethysmography and showed that Kir4.1 cKO mice hypoventilate in 100% O2 and have a reduced tidal volume response to hypercapnia, compared to controls. For examination of gene expression, RTN GFAP‐TdT+ astrocytes from both control and Kir4.1 cKO brainstems were acutely dissociated and single cells were isolated by FACS into 96‐well plates. Single cell qPCR revealed that the majority of astrocytes within the RTN express glial markers Kcnj10 (Kir4.1), Aqp4, Aldh1L1 and Gfap, and NeuN was probed for to exclude neuronal transcript contamination. A subset of RTN astrocytes expressed Gjb2 (Cx26), which unlike Kcnj10 expression, was not greatly reduced in astrocytes from the Kir4.1 cKO mouse RTN. These results demonstrate that Kir4.1 channels in astrocytes contribute to the central drive to breathe, and suggest that RTN astrocytes are a heterogeneous population, with a discrete subset that expresses Cx26. These findings provide insight into the specialized nature of astrocyte chemoreceptors and may identify new targets important for the central control of breathing.Support or Funding InformationNIH HL104101 (DKM) and F32HL126381 (VEH)
Influence of CO2 and HCO3− on Basal Activity and Acid Sensitivity of Chemosensitive Neurons in the Retrotrapezoid Nucleus
Authors: Goncalves Christopher M. , Kuo Fu‐Shan , Daniel Emily , Mulkey Daniel K. 
Published: November 2023

Central chemoreception is the mechanism by which the brain regulates breathing in response to changes in tissue CO2/H+. A region of the brainstem called the retrotrapezoid nucleus (RTN) is an important site of chemoreception. The mechanisms of RTN chemoreception involve direct H+‐mediated activation of chemosensitive neurons, and indirect modulation of chemosensitive neurons by CO2/H+‐dependent ATP‐purinergic signaling. Recent evidence identifies astrocytes as the source of CO2/H+‐evoked ATP release, possibly by a mechanism involving CO2‐mediated opening of connexin 26 hemichannels. However, the extent to which ATP contributes to RTN chemoreception is questionable; some studies report that ATP release from astrocytes is required for chemosensitive RTN neurons to sense changes in CO2/H+, whereas other studies showed that blocking ATP receptors in the RTN decreased (~ 30%) but did not eliminate CO2/H+‐sensitivity of RTN chemoreceptors. Based on evidence that CO2 is required for ATP release by RTN astrocytes, we chose to investigate the contribution of purinergic to RTN chemoreception by comparing the firing responses of chemosensitive RTN neurons to graded acidifications in the presence and absence of CO2 and HCO3−. Cell‐attached recordings were used to identify RTN chemoreceptors in brainstem slices (300 μm thick) isolated from rat pups (P7–11 days postnatal) incubated in a bicarbonate (26 mM) buffered solution by their characteristic response to CO2; i.e., low basal activity in 5% CO2 (pH=7.3; 0.7 ± 0.1 Hz) and high activity in 10% CO2 (pH=7.0; 1.7 ± 0.3 Hz) or 15% CO2 (pH=6.9; 2.2 ± 0.4 Hz). After returning to control conditions (5% CO2; pH=7.3) switching to a bicarbonate‐free HEPES buffered solution (pH=7.3) decreased baseline activity by 0.5 ± 0.4 Hz (T3=5.175, p=0.014; paired t‐test). In HEPES buffer, acidifications to pH=7.0 and pH=6.9 increased activity by 0.65 ± 0.2 Hz and 0.8 ± 0.2 Hz, respectively. In some cases, during prolonged exposure to HEPES baseline activity increased to near control levels, suggesting that removal of CO2/HCO3− from the perfusate only transiently suppressed chemoreceptor activity. These results suggest that RTN chemoreceptors respond more vigorously to hypercapnic acidosis compared to the same acidification in HEPES buffer. This finding is consistent with the possibility that CO2‐evoked ATP release from astrocytes contributes in part to RTN chemoreception. These results also suggest that HCO3− controls baseline activity of RTN chemoreceptors, possibly by influencing intracellular pH or regulating HCO3−‐sensitive K+ channels (i.e., KCNQ).Support or Funding InformationNIH Grant HL104101
Noradrenergic modulation of chemosensitive neurons in the retrotrapezoid nucleus
Authors: Kuo Fu‐Shan , Barna Bárbara F , Chen Dawei , Takakura Ana C , Mulkey Daniel K 
Published: November 2023

Chemosensitive neurons in the retrotrapezoid nucleus (RTN) regulate breathing in response to CO2/H+ changes, and serve as an integration center for other autonomic centers including brainstem noradrenergic neurons. Considering that application of norepinephrine (NE) into the RTN strongly modulates breathing (see abstract by Oliveira et al.) and disruption of NE signaling has been implicated in breathing disorders associated with Rett syndrome, sleep‐disordered breathing and multiple system atrophy, we sought to characterize the effects of NE on chemosensitive RTN neurons. Cell‐attached recordings were used to identify RTN chemoreceptors in brainstem slices (300 μm thick) isolated from rat pups (P7–11 days postnatal) by their characteristic response to CO2; i.e., low basal activity in 5% CO2 (0.2 ± 0.1 Hz) and high activity in 10% (1.8 ± 0.1 Hz) or 15% CO2 (2.1 ± 0.1 Hz ). We found that bath application of NE (1 μM) increased activity by 1.1 ± 0.1 Hz in 48 of 66 cells (73%), decreased activity by 1.0 ± 0.1 Hz in 6 of 66 cells (9%), or had no effect on activity in 12 of 66 (18%) of cells. Interestingly, basal activity but not CO2/H+‐sensitivity differed between each group based on their NE response; e.g., basal activity and firing response to 10% CO2 was 0.4 ± 0.1 and 1.2 ± 0.1 Hz (NE‐activated), 1.1 ± 0.3. and 1.1 ± 0.1 Hz (NE‐inhibited) and 0.03 ± 0.01 and 1.1 ± 0.01 Hz (NE‐insensitive). The excitatory effect of NE on RTN chemoreceptors was dose dependent (EC50 of 235 nM), retained during synaptic blockade (high Mg+2/low Ca2+) and could be mimicked by an α1‐receptor agonist (phenylephrine; 10 μM) and blocked with a α1‐receptor antagonist (prazosin; 1 μM). The inhibitory effect of NE on RTN chemoreceptors was retained during synaptic blockade and blunted by a α2‐receptor antagonist (idazoxan; 1 μM). A subset of NE‐activated cells were inhibited by NE in the presence of prazosin, whereas some NE‐inhibited cells showed an excitatory response to NE in the presence of idazoxan. A third group of RTN chemoreceptors did not respond to NE under control conditions or during hypercapnia, and these cells also did not respond to phenylephrine. In addition, whole‐cell voltage clamp (Vhold= −80 mV; TTX) recordings from CO2/H+‐sensitive RTN astrocytes show the absence of an NE‐sensitive current. These results indicate that i) RTN chemoreceptors are intrinsically sensitive to NE; ii) NE can increase or decrease chemoreceptor activity by α1‐ and α2‐receptor dependent mechanisms, respectively; and iii) NE modulates discrete subsets of RTN chemoreceptors based on basal activity and the differential contributions of α1‐α2‐receptors.Support or Funding InformationNIH Grant :HL104101
Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is Crucial for CO2 Signal Transduction in Avian Intrapulmonary Chemoreceptors (IPC)
Authors: Molloy Rhett Gordon , Low Trevor Michael , Bassett Peter Ross , Lonjaret Jean‐Guillaume , Hempleman Steven Curtis 
Published: November 2023

CFTR is an anion channel and ABC transmembrane protein which also modulates other anion channels and transporters. Since CFTR is widely expressed in cells that transport anions, and we hypothesize that anion transport in IPC is critical for CO2 transduction, we tested for the presence of CFTR in IPC. Glibenclamide, a non‐selective CFTR inhibitor, and CFTR‐172inh, a highly specific CFTR inhibitor were given separately to pentobarbital‐anesthetized mallards (Anas platyrhynchos, n =15) while recording single cell IPC spike train responses to inspired CO2 steps (0%–7%, 11sec period). Both drugs produced similar results, where IPC discharge with the lowest 2 μmol/kg doses was unchanged from control, while the 4 and 8 μmol/kg doses significantly decreased both mean IPC discharge and phasic adaptation (ANOVA, p<0.01). IPC CO2 sensitivity was completely abolished in 4 CFTR‐172inh experiments and 4 glibenclamide experiments. Results suggest that CFTR is a crucial anion regulating channel involved in IPC CO2 signal transduction.Support or Funding InformationSupport: NIH R15 HL087269‐02.