bluuebunny/crossref_metadata_2025 huggingface dataset:
Renal Medullary Interstitial Cell‐Specific Knockout of the Endothelin A Receptor Increases Sodium Excretion and Prevents Salt‐Sensitive Hypertension
Authors: Hu Chunyan , Stuart Deborah , Lakshmipathi Jayalakshmi , Harris Raymond C. , Hao Chuan Ming , Kohan Donald E.
Published: June 2021

The renal medullary vasculature and tubules are well established regulators of arterial blood pressure (BP) and body fluid and Na+ balance, however relatively little is known about the role of renal medullary interstitial cells (RMIC). RMIC produce a variety of potentially BP‐regulating factors in response to hormonal and paracrine stimuli; amongst these, endothelin (ET) activation of RMIC ET A receptors (ETAR) may be of particular importance. To test this system in vivo, we bred ETARf/f mice containing loxP‐flanked exons 6–8 of the EDNRA gene with tamoxifen‐inducible tenascin‐C‐CreER2 knockin mice. Tenascin‐C‐CreER2:ETARf/f (RMIC ETAR−/−) mice were injected with tamoxifen (160 mg/kg IP) for 5 consecutive days at 3 months of age. All experiments were performed 2 weeks after the last injection and littermates of the same genotype without injection were used as controls. Within the kidney, medullary, but not cortical, EDNRA gene recombination was observed. Radiotelemetric BP was similar between control and RMIC ETAR−/− mice on a normal (0.3% Na+) or low (0.03% Na+) salt diet, however 7 days of a high‐salt (3.2% Na+) intake increased BP in control, but not RMIC ETAR−/− mice (RMIC ETAR−/− 124 ± 4 mmHg vs control 133 ± 2 mmHg, n=10, p<0.05). A similar response was found in diastolic BP. Urine volume, urinary Na+ excretion and urinary K+ excretion were higher in high‐salt diet‐treated RMIC ETAR−/− mice compared with control (n=12, p<0.05) particularly during the first 3 days of high salt intake; no apparent differences in food intake were noted. No difference was observed in plasma aldosterone or plasma renin concentration between high‐salt diet fed RMIC ETAR−/− and control mice (n=6). Urinary nitrite/nitrate excretion (UNOx) was increased in high‐salt diet‐treated RMIC ETAR−/− mice (RMIC ETAR−/− 2645 ± 242 nmol/day vs control 1970 ± 251 nmol/day, n=12, p<0.05). We conclude that RMIC‐specific knockout of ETAR prevents high‐salt diet‐induced hypertension and that this is associated with increases in urinary fluid and electrolyte excretion and UNOx. Our data suggest a novel regulatory pathway involving RMIC ETAR in BP and water/electrolyte homeostasis.

Support or Funding Information

NHLBI P01 HL136267

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Regulation of ENaC Expression by Paraoxonase 3
Authors: Shi Shujie , Mutchler Stephanie M. , Marciszyn Allison L. , Kleyman Thomas R.
Published: June 2021

The epithelial sodium channel (ENaC) mediates the rate‐limiting step of Na+ uptake across the apical membrane of specific epithelia. ENaC‐dependent Na+ absorption in the kidney has important roles in regulating extracellular fluid volume, extracellular [K+] and blood pressure. ENaC functional expression is tightly regulated by multiple intracellular and exogenous factors, including molecular chaperones that are implicated in key steps during ENaC biogenesis. We previously showed that ENaC activity was reduced by paraoxonase 2 (PON2) in Xenopus oocytes. PON2–mediated inhibition is associated with reduced surface expression of ENaC, suggesting a role of PON2 in regulating ENaC turnover and/or trafficking. In supporting this notion, PONs are the mammalian orthologues of MEC‐6, a C. elegans ER‐resident chaperone. MEC‐6 is required for the proper assembly and surface expression of the touch‐sensing MEC‐4/MEC‐10 channel in worm touch‐receptor neurons. If the chaperone function is conserved between MEC‐6 and mammalian PONs, we predict other members of this family will also regulate the functional expression of related ion channels. Indeed, we found that PON3 is expressed in principal cells of the distal nephron, where ENaC resides. The expression of PON3 inhibits ENaC activity in Xenopus oocytes. The protease chymotrypsin activates ENaC by releasing the embedded inhibitory tracts, thus increasing channel open probability (Po). We found that chymotrypsin‐mediated ENaC activation was not altered by PON3, suggesting that PON3 does not inhibit ENaC activity by reducing channel Po. In addition, ENaC expression in Fisher rat thyroid cells was reduced by PON3. Future studies will investigate the mechanism by which PON3 regulates ENaC functional expression as well as the potential roles of PON3 in regulating ENaC activity, blood pressure and extracellular [K+] in the Pon3 KO mice. Understanding mechanisms by which PON3 regulates ENaC expression will provide novel insights into both normal and altered ENaC functional states, such as hypertension.

Support or Funding Information

DK103834, DK038470, DK079307

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Functional Implications of Sexual Dimorphism of Transporter Patterns along the Rat Nephron
Authors: Hu Rui , McDonough Alicia A , Layton Anita
Published: June 2021

The goal of this study is to investigate the functional implications of the sexual dimorphism in transporter patterns along the rodent nephron as reported by Veiras et al. (J Am Soc Nephrol 2018). To do so, we developed sex‐specific computational models of water and solute transport from Bowman space to the papillary tip of a superficial nephron of the kidneys of the male and female rats. The models account for the sex differences in expression levels of the apical and basolateral transporters, in single‐nephron glomerular filtration rate, and in tubular dimensions. Model simulations predict that ~70 and 40% of the filtered volume is reabsorbed by the proximal tubule of the male and female rat kidneys, respectively. The lower fractional volume reabsorption in the female rat can be attributed to their smaller transport area and lower aquaporin‐1 expression level. The latter also results in a larger contribution of the paracellular pathway to water transport. Similar fractions of the filtered Na+ are reabsorbed by the male and female proximal tubule models, respectively. The lower fractional Na+ reabsorption in female is due primarily to their smaller transport area, lower Na+/H+‐exchanger (NHE3) activity, and lower claudin‐2 abundance. This results in significantly larger delivery of water and Na+ to the downstream nephron segments in female. Unlike in the proximal tubule, the distal nephron of the female rat exhibits higher expression levels in key Na+ transporters, including Na+‐K+‐Cl− cotransporter, Na+‐Cl− cotransporter, and epithelial Na+ channel. Not only do the higher expression levels of these transporters account for the enhanced water and Na+ transport along the female distal nephron, relative to male, but they may also contribute to the female rat's ability to excrete a saline load more rapidly than male.

Support or Funding Information

This research was supported by the Canada 150 Research Chair program and by the National Institutes of Health: National Institute of Diabetes and Digestive and Kidney Diseases, grant R01DK106102.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cerebral Blood Flow Autoregulation in Hypertensive Models of Pregnancy
Authors: Maeda Kenji J , Younes Subhi T. , Garrett Michael R , Ryan Michael J , Sasser Jennifer M
Published: June 2021

Cerebral blood flow (CBF) autoregulation is a robust protective mechanism that maintains constant blood flow during changes in perfusion pressure. Although it is well known that major cardiovascular adaptations occur during normal pregnancies, the cerebral vascular adaptations that occur during pregnancy, especially in the setting of hypertensive pregnancies, are poorly understood. Hypertension is typically associated with normal autoregulatory function in the cerebral circulation, although the upper and lower limits of the autoregulatory curve are shifted to higher blood pressures. Previous studies in pregnant Sprague Dawley (SD) rats show that the cerebral autoregulatory curve is shifted to higher pressures compared to nonpregnant rats. The impact of preexisting hypertension on cerebral blood flow autoregulation has not been examined. The current study was performed to test the hypothesis that CBF autoregulation is impaired during pregnancy in the presence of underlying hypertension.

Dahl salt‐sensitive (Dahl S) and spontaneously hypertensive (SHR) rats, 19–22 weeks of age, were used because they spontaneously develop hypertension without additional challenges. Female rats were mated with same strain males, and gestational day 1 (G1) of pregnancy was defined by the detection of sperm histologically from a vaginal swab. CBF was assessed on G18–20 in rats anesthetized with Inactin. Briefly, catheters were placed in the femoral artery and vein to monitor blood pressure and continuously and administer phenylephrine, respectively.

Laser Doppler flow probes were positioned above two closed cranial windows 3–4 mm lateral and 2mm distal to Bregma to assess relative changes in CBF in response to increases in blood pressure.

Linear regression analysis of CBF autoregulatory curves shows significant differences between pregnant and virgin rats across strains (★★, p<0.0001) and between strains in the pregnant rats (★★, p<0.0001, Figure). No significant differences in slopes were observed between strains in the virgin rats.

Contrary to our hypothesis, these data show that CBF autoregulation was enhanced during pregnancy in two different models of spontaneous hypertension. Previous studies from our laboratory showed increased vascular and blood brain barrier permeability in pregnant Dahl S rats when compared to virgin littermates. Therefore, these data suggest an uncoupling of cerebral vascular permeability from CBF autoregulation in this model of superimposed preeclampsia.

Future studies will investigate the role of pregnancy specific circulating factors that contribute to these changes and their implications on cerebrovascular function.

Support or Funding Information

KJM was supported by 5T32HL105324‐09. Work was supported by 1R01HL137673 to MRG, HL136684to MJR, and R01HL134711 to JMS.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Male Mice Heterozygous LSD1 Knockout Gene Prevented the Effect of High Sodium Intake on Renal Fibrosis and AT1 Conformational Status
Authors: Nascimento Mariana Moura , Katayama Isis Akemi , Pojoga Luminita H , Heimann Joel Claudio
Published: June 2021

Aim

To evaluate the effects of dietary salt overload or restriction on renal structure and AT1 conformational status in wild type and LSD1 (lysine specific demethylase 1) deficient mice.

Methods

LSD1 deficient (Het) and wild type (WT) mice were maintained on high (HS), low (LS) or normal (NS) sodium intake, for 12 months. Renal fibrosis, glomerular volume, glomerular diameter and AT1 and AT2 receptor conformational status (activated or not activated by agonist ligation) were evaluated at the end of the study.

Results: (n=6–17/group)

LS‐WT
LS‐HET
NS‐WT
NS‐HET
HS‐WT
HS‐HET

Renal Fibrosis (%)
1.08±0.03*
1.13±0.04
1.14±0.03*
1.20±0.03
1.23±0.02
1.22±0.02

Glomerular Volume (×106 μm3)
0.25±0.01
0.27±0.01
0.25±0.01
0.23±0.01
0.27±0.01
0.27±0.01

Glomerular Diameter (μm)
82.4±1.78
84.3±2.92
84.0±1.63
81.0±1.31
84.1±1.58
84.7±2.98

Conformational Status AT1R (FI)
16.9±0.81
19.8±1.43
18.8±0.83
21.2±2.10
19.8±0.84&
21.4±0.67

Conformational Status AT2R (FI)
7.08±0.33
7.27±0.38
6.71±0.28
7.12±0.50
7.69±0.32
6.97±0.38

Data are reported as mean±SEM.

p<0.05 vs. HS‐WT and

p<0.05 vs. LS‐WT

Conclusions

Interestingly, high salt intake stimulated renal fibrosis only in WT mice. The same phenomenon was observed in AT1 conformational status. In addition, the dietary salt content did not influence the glomerular volume and diameter in both WT and Het models suggesting that: renal fibrosis development and AT1 conformational status in response to dietary salt overload occur through different mechanisms in WT and Het animals stimulating further studies to elucidate this phenomenon.

Support or Funding Information

Supported by FAPESP, CAPES and American Heart Association

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Deficiency in Pappa2 expression reduces nephron endowment and associates with hypertension and chronic kidney disease in rats
Authors: Kumar Vikash , Yang Chun , Kurth Terry , Geurts Aron , Cowley Allen W
Published: June 2021

Introduction

Recently, a congenic Dahl salt‐sensitive (SS) 26‐P rat strain containing Pappa2 allele of salt‐insensitive Brown Norway (BN) rat strain was identified which found to exhibit significant protection from salt‐induced hypertension and kidney injury. Pappa2 (pregnancy associated plasma protein A2) was found to be differentially expressed in the kidney and altered by dietary salt intake. Pappa2 is a metalloproteinase which releases IGF by cleaving IGFBP5 and has been found to play an important role in the development of bone size and angiogenesis. It has not been previously been implicated to play a role in kidney development nor contribute to cardiovascular function and hypertension. The present study determined whether Pappa2 contributes to kidney development, particularly nephron number and the relationship of these findings with hypertension and chronic kidney disease in rats.

Methods

The localization of Pappa2 in embryonic E20.5 and postnatal P0 kidneys of SS and 26‐P was determined by immunohistochemistry. CRISPR/Cas9 was used to knockout the Pappa2 gene in 26‐P strain. Acid‐maceration method was used to determine glomeruli per kidney at P50 of Pappa2‐WT and Pappa2‐KO rats. Pappa2‐WT and Pappa2‐KO rats (male; 9 wk age) were implanted with arterial catheters and radiotransmitters to record mean arterial pressure (MAP; 24/d). Urine was collected to assess micro‐albumin excretion rate, as an index of renal injury.

Results

Immunohistochemistry studies found that Pappa2 was localized in the ureteric bud at E20.5 kidneys of 26‐P embryos suggesting that Pappa2 could be important for ureteric branching and nephron endowment. CRISPR‐Cas9 mutation of Pappa2 in the 26‐P rat resulted in a severe deficit in the number of mature nephrons in rats at day 50 (26833 ± 892 glomeruli/kidney in Pappa2‐KO vs 36508 ± 1276 glomeruli/kidney in Pappa2‐WT). MAP of 10 weeks Pappa2‐KO rats which were maintained on a 0.4% NaCl diet since weaning averaged 121 ± 2.3 mmHg compared with Pappa2‐WT rat which averaged 113 ± 1.3 mmHg (P<0.05). Albumin excretion rates (UalbV) averaged 151 ± 42 and 51 ± 26 mg/day in 6 month old Pappa2‐KO and Pappa2‐WT rats fed a 0.4% NaCl diet respectively, and progressively increased to 270 ± 85 and 97 ± 28 mg/day at 9 months of age.

Conclusion

Pappa2 determines nephron number which is closely associated with hypertension and kidney injury in rats.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
An Upstream Short Open Reading Frame in the Angiotensin Type 1 Receptor Regulates the β‐Arrestin Signaling Pathway
Authors: Kadam Parnika S , Ji Hong , Pai Amrita V , Speth Robert , Mueller Susette , Sandberg Kathryn
Published: June 2021

The angiotensin type 1 receptor (AT1R) plays a critical role in blood pressure control and thus furthering our understanding of its regulation has physiological and clinical significance. We previously showed a short open reading frame (sORF) in exon (E)2 of the 5' leader sequence of the AT1aR mRNA inhibits AT1R mRNA translation and receptor internalization. In this study, we investigated the role of this sORF in AT1R signaling. The sORF start codon in E2 was disrupted by site‐directed mutagenesis [E1,2(−108T),3‐AT1aR] (MT), which along with the wild type unmutated sORF [E1,2,3‐AT1aR] (WT) were cloned into an expression plasmid [EGFPN2]. Human embryonic kidney‐293 cells were then transfected with WT or MT plasmids. No significant differences were found in the time course of Ang II‐induced ERK1/2 activation between 0–80 min at angiotensin doses of 10 nm (n=3) and 100 nM (n=3); however, the peak response to 10 nM Ang II at 5 min was 1.9‐fold higher in cells transfected with MT compared to WT plasmids (p<0.05; n=4). Furthermore, peak ERK1/2 activation was 1.3‐fold higher in cells transfected with MT compared to WT plasmids after stimulation with Sar1,Ile4,Ile8‐Ang II, a biased AT1R agonist which selectively activates the β‐arrestin‐dependent pathway [pERK1/2/total ERK1/2 normalized to AT1R Bmax: WT, 1.8E‐05 ± 9.5E‐07 vs. MT, 2.3E‐05 ± 3.4E‐06, p<0.05; n=3]. A time course of Sar1,Ile4,Ile8‐Ang II‐induced ERK1/2 activation from 0–80 minutes at 30 μM also showed greater ERK1/2 activation in cells transfected with the MT compared to WT plasmids [p<0.05; n=4]. Since β‐arrestins are implicated to play a role in apoptotic signaling, we are also studying the effect of this sORF on Ang II‐induced cell proliferation and both early and late apoptosis by flow cytometry. This study supports our previous findings that a seven amino acid peptide (PEP7) encoded by this sORF is a selective inhibitor of AT1R‐mediated ERK1/2 activation. These findings not only have implications for the regulation of AT1Rs in physiological and pathological conditions but also for the superfamily of G protein coupled receptors, many of which possess sORFs of unknown function in their 5’ leader sequence.

Support or Funding Information

NIH/NHLBI R01‐HL121456 to KS NIH TL1 Award TL1TR001431

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Myosin Quality Control Systems in Zebrafish Skeletal Muscle Development
Authors: Carlisle Casey , Prill Kendal , Pilgrim David
Published: June 2021

In order to maintain homeostasis, defective proteins must be identified, removed, and replaced. In skeletal muscle, failures in this process can lead to a variety of incurable muscle diseases (myopathies), many of which are fatal. While aspects of protein turnover have been studied widely, the protein turnover process in skeletal muscle is still incompletely understood, especially with respect to the motor protein, myosin. By studying the assembly and maintenance of the basic unit of striated muscle, the sarcomere, we aim to elucidate the components involved and the mechanism controlling muscle myosin quality control in skeletal muscle.

Zebrafish make an excellent model system to study sarcomere protein turnover due to their ability to survive with myopathies that are fatal in other organisms. Using two different zebrafish lines, steif, and herzschlag, we and others show that steif sarcomeres fail to assemble myosin thick filaments due to a loss of a critical component (Etard et al., 2007), while herzschlag sarcomeres degrade over time as a consequence of muscle contractions. Transmission electron microscopy coupled with H&E staining revealed a slow muscle atrophy specific to herzschlag embryos. To identify factors involved in response to myosin damage during assembly or maintenance, we have screened through candidate genes, myosin chaperones and muscle specific E3 enzymes, using in situ hybridization and qPCR. Our data indicates a division of function where specific E3 enzymes and myosin chaperones respond to myosin damage exclusively during assembly, while other E3 enzymes respond to myosin damage during sarcomere maintenance. Pharmaceutical inhibition of the proteasome shifts this response in steif embryos, but not herzschlag, suggesting an interconnected network of protein quality control during myosin assembly. Together, this work helps highlight potential therapeutic targets to treat myosin turnover related myopathies.

Support or Funding Information

NSERC

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
In vitro testing of fluticasone drug delivery system for inflammatory injury and repair
Authors: Tipparaju Srinivas , Kelly Shannon , Weigel Robert , Halasz Kathleen , Tur Jared , Brotto Marco , Sutariya Vijaykumar
Published: June 2021

Glucocorticoids such as dexamethasone and fluticasone have potent anti‐inflammatory properties, but they also have serious side effects when used for extended periods or even at higher doses. For enhanced drug delivery and to test whether glucocorticoids are able to provide benefits at lower doses we developed a new nano‐drug delivery system (called Nano‐Gluco) and tested it in vitro in a model of muscle cell injury and repair. We characterized Nano‐Gluco for its physical and chemical characteristics. We utilized zeta potential, particle size analysis, scanning electron microscopy and differential scanning calorimeter to evaluate Nano‐Gluco. To test the efficacy of the system, we used the LPS induced cellular injury model. We demonstrated that Lactate dehydrogenase (LDH) release was significantly lower in positive controls glucocorticoid treated cells compared with LPS alone. In support for the benefits Nano‐Gluco, it allowed similar protection of cells in vitro at a much lower concentration than glucocorticoids alone. We next utilized the LPS induced injury model with C2C12 (murine myoblasts) cells to identify the effects injury and repair, as well as inflammation signaling. At the molecular level, we tested key inflammatory markers such as NFKb, TNFα, IL1β along with other cell markers such as p27, FOXO for the beneficial effects of steroids in muscle cell repair and inflammation. These markers confirmed the beneficial effects of employing Nano‐Gluco. Overall, we demonstrate that our new Nano‐Gluco system provides a highly efficient method for delivery of glucocorticoids to cells for achieving anti‐inflammatory properties at a much lower concentration compared with current glucocorticoid preparation. We are now moving into animal pre‐clinical studies to advance the use Nano‐Gluco in humans.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Deletion of p16 expressing senescent cells improves muscle mass and age‐dependent outcomes in multiple organ systems
Authors: Judge Jennifer , Shigdar Shahjahan , Davis Carol , McArdle Anne , Richardson Arlan , Brooks Susan
Published: June 2021

Background

Cellular senescence is defined as permanent cell cycle arrest due to upregulation of cyclin dependent kinases such as p16. Senescent cells accumulate with age and can contribute to aging related pathologies. Recently, deletion of p16 expressing senescent cells in vivo has been shown to increase healthspan and lifespan, and ameliorate some tissue specific pathologies in mice. However, it is unknown whether cellular senescence plays a role in age associated declines in skeletal muscle function. We hypothesized that deletion of p16 expressing senescent cells will preserve age‐induced declines in muscle function and mass.

Methods

To eliminate senescent cells in vivo, we utilized p16‐3MR transgenic mice in which administration of the drug ganciclovir will induce cell death in p16 expressing cells. Mice were treated with 25 mg/kg ganciclovir or saline (control) intraperitoneally for 5 days at monthly intervals from 20–26 months of age. Animals were sacrificed at 26 months of age, muscles were weighed, and gastrocnemius muscles were tested for force production in vivo, while extensor digitorum longus and soleus muscles were tested in vitro. Muscle fiber cross sectional areas and fiber type were determined by immunohistochemistry. Additional organs were snap frozen for downstream protein, cytokine, and RNA analyses. mRNA expression was measured using qPCR.

Results

Mice that were treated with ganciclovir lived longer and had a lower incidence of cancer than control mice. Muscle masses of the soleus, tibialis anterior and gastrocnemius were greater in ganciclovir‐treated mice compared with controls. The higher muscle mass in treated mice was not associated with any shift in fiber type distribution, increase in fiber cross‐sectional area, or any expansion of the volume of extracellular matrix, suggesting the higher muscle mass was due to preservation of fiber number. There was no effect of p16 deletion on muscle force production. However, upon examining muscle, spinal cord, serum and lung tissue, p16 deletion impacted age‐dependent changes in gene expression and cytokines.

Conclusions

Differential effects of senescent cell deletion on skeletal muscle mass and function suggests the role of senescent cells in skeletal muscle aging may be context specific. Further studies are needed to more thoroughly determine tissue‐specific benefits, but our work suggests that deletion of senescent cells even late in life may provide protection against global aging processes.

Support or Funding Information

AG051442K12GM111725

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Skeletal Muscle Function is Impaired in Mice Following Surgical Weight Loss
Authors: Stephenson Erin J , Gomes Charles K , Stayton Amanda S , Puchowicz Michelle A , Han Joan C , Pierre Joseph F
Published: June 2021

Limb skeletal muscle mass losses in the year following bariatric surgery are far greater than the loss of lean mass experienced by inactive persons during the typical aging process. This is a clinical concern, since loss of skeletal muscle mass may have long term effects that attenuate many of the benefits associated with marked weight loss (such as sarcopenia, frailty and loss of mobility). Here, we use a mouse model of bariatric surgery (gastric sleeve) to test the hypothesis that surgical weight loss impairs skeletal muscle function. Sixteen‐week‐old male C57BL6/J mice with diet‐induced obesity (DIO; 5.21 kCal/g, 60% fat, 20% carbohydrate, 20% protein; D12492 Research Diets) were randomized into groups receiving either gastric sleeve surgery (GS; 80% resection of the greater curvature of the stomach) or sham surgery (DIO‐sham; laparotomy). A third group of age‐matched lean mice were included as a non‐surgical, non‐obese comparison group (3.82 kCal/g, 10% fat, 70% carbohydrate, 20% protein; D12450J Research Diets). Following surgery, GS mice rapidly lost 20% of their pre‐surgery weight, reducing their initial fat mass by 53% and lean mass by 20%, as measured by EchoMRI. Loss of lean mass was associated with a reduction in grip strength six‐weeks post‐surgery (−10%, p=0.04), compared to DIO‐sham, despite regain of most of the initial lean mass losses. GS mice had reduced total energy expenditure compared to DIO‐sham, as measured by indirect calorimetry at both 22°C (−7% during the light phase, p=0.0015 and −11% during the dark phase, p=0.009) and at 28°C (−7% during the light phase only, p=0.05). No differences in ambulatory activity were observed between groups. These findings, although preliminary, suggest that bariatric surgery causes molecular adaptations in the skeletal muscle that may not be easily reversed once initial lean mass losses are restored. Further work will identify whether surgical weight loss causes alterations in protein breakdown/synthesis and other metabolic adaptations that affect long‐term skeletal muscle function.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Region‐Specific Metabolic Regulation of Intestinal Stem and Progenitor Cells
Authors: Stine Rachel , Seale Patrick
Published: June 2021

Distinct metabolic programs regulate intestinal stem cell renewal and differentiation. However, the mechanisms that rewire metabolism during differentiation are poorly defined. Additionally, cells in intestinal stem cell niches have not been studied in the context of specific intestinal regions to determine if crypts along the length of the intestine are regulated by common or specialized metabolic programs. We predict that transcriptional manipulation of metabolic programming in stem cells and progenitor cells will affect the ability of these cells to properly differentiate and regenerate tissue. Here, we identify the transcription factor PRDM16 as a critical regulator of intestinal metabolism and differentiation. PRDM16 is known to affect the metabolic programming of adipocytes in thermogenic adipose tissue. Surprisingly, acute deletion of Prdm16 in adult mice causes intestinal wasting, apoptosis, and an accumulation of poorly differentiated cells in the small intestinal crypt. RNA‐seq and tracer metabolomic studies show that PRDM16 controls fatty acid oxidation (FAO) in the intestinal crypt. FAO is the process by which fatty acids are broken down to produce acetyl CoA, which can enter the TCA cycle to produce energy and other cellular building blocks. PRDM16 binds, along with its protein partners PPARγ and PPARα, to the promoter and enhancer regions of many FAO genes. The loss of Prdm16 or inhibition of FAO impaired the differentiation of stem and transit amplifying cells into mature cells. Notably, PRDM16 expression is highest in the duodenum and declines distally along the intestine. This gradient of PRDM16 levels controls region‐specific expression of the FAO program and underlies the differential reliance of region‐specific stem and progenitor cells on FAO. We also show that there is a region‐specific sensitivity to FAO inhibition in ex vivo enteroids isolated from different regions. Regional reliance of cells on FAO may have arisen due to the abundance of fats present in the proximal small intestine, where the majority of fat digestion and absorption occurs. Interestingly, this specification persists in culture where nutrients are not differentially available, raising the possibility that over time, cells have more permanently adapted to their environment. Altogether, this study establishes PRDM16 as a region‐specific regulator of intestinal metabolism and stem cell differentiation. We speculate that distinct regional metabolic profiles of stem cells could contribute to determining the character of the mature epithelial cells in different regions. Future experiments will focus on how diet affects regional metabolic profiles of intestinal stem cells and whether alterations in metabolic programming could lead to disease.

Support or Funding Information

F32 DK10574303 to R. Stine, P30 DK050306 Pilot Grant to P. Seale, R01 DK10300802 to P.

Seale

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Rotavirus Infection Activates Intercellular Calcium Waves through Purinergic Signaling
Authors: Chang‐Graham Alexandra , Perry Jacob , Engevik Melinda , Danhof Heather , Kellen Joseph , Hyser Joseph
Published: June 2021

Rotavirus (RV) remains the leading viral cause of diarrheal disease in children worldwide. The life‐threatening diarrhea from RV infection is potentially exacerbated by the release of signaling molecules from infected villus epithelial cells. RV increases cytosolic calcium in infected cells, which is necessary for RV replication and activation of secretory pathways. Thus, characterizing RV‐induced calcium signaling is needed to understand RV pathogenesis. However, the activation of a calcium signal from infected to uninfected cells has not been directly observed. Therefore the signaling mechanisms that facilitate widespread dysregulation of fluid secretion remain undefined. To address this, we conducted live cell fluorescence imaging in cell lines and jejunum human intestinal enteroids (jHIEs) to measure calcium signaling during RV infection.

We generated African monkey kidney MA104 cells and jHIEs that stably express GFP‐based genetically encoded calcium indicators to measure calcium dynamics during RV infection. RV significantly increases the number and magnitude of calcium transients in infected wells. Further, we observed that single RV‐infected cells triggered long‐distance intercellular calcium waves that encompassed surrounding uninfected cells. These calcium waves are mediated by extracellular ATP/ADP since treatment with the ectoNTPase apyrase or the P2Y1 purinergic receptor inhibitor BPTU blocked the waves and also RV replication. We created P2Y1 receptor genetic knockouts using CRISPR/Cas9, and there were reduced calcium waves in RV‐infected knockout cells. In RV‐infected HIEs, blocking calcium waves with apyrase and BPTU decreased fluid secretion in the enteroid swelling assay and decreased serotonin secretion, which exacerbates RV diarrhea and vomiting via the enteric nervous system. Furthermore, we identified that broad anion channel blockers significantly inhibited calcium waves, supporting that ATP/ADP is released via anion channels.

Our results demonstrate that RV‐infected cells release extracellular purines to cause calcium signaling in uninfected cells important for RV replication and secretory pathways. Our data points to purinergic signaling as a therapeutic target for developing anti‐diarrheal drugs. Furthermore, we are the first to show that viruses can exploit purinergic signaling and calcium waves to potentially amplify pathophysiological signaling important for diarrhea.

Support or Funding Information

NIH F30 DK112563, R01 DK115507, R03 DK110270, K01 DK093657, BCM New Faculty Seed Grant and Funding, Augustana College TMC Summer Research Internship Program, TMC DDC Enteroid Core (P30DK056338)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Expression of Circadian Clock Components PER2 and BMAL1 are Altered During Infection of Helicobacter pylori
Authors: Teal Emma L , Li Jing , Chakrabarti Jayati L , Broda Taylor , Steele Nina , Rosselot Drew , Sundaram Nambirajan , Hawkins Jennifer , Hong Christian , Helmrath Michael A , Wells James , Diwan Tayyab L , Zavros Yana L
Published: June 2021

Background/Hypothesis

Several gastrointestinal functions have been demonstrated to be under circadian clock control and colorectal cancer tissues have been found to have a disruption in core circadian clock components BMAL1 and PER2. Those suffering from chronic Helicobacter pylori (H. pylori) infection are at a heightened risk of developing gastric adenocarcinoma. Compared to young individuals, the aged population suffer from an increased susceptibility to H. pylori infection as well as diminished expression of circadian clock components which could further predispose them to the development of gastric cancer. We therefore hypothesize that chronic H. pylori infection induces a hyperproliferative response in the gastric epithelium via BMAL1 activation resulting in a susceptibility to gastric cancer.

Methods

Gastric organoids (hFGOs) derived from primary gastric tissue of either aged (> 55 years of age) or young (14 – 20 years) individuals were generated and subsequently infected with H. pylori. Analysis for proliferation and expression of circadian clock components BMAL1 and PER2 was performed for a period of 24 hours post‐infection. Organoids infected with H. pylori were analyzed for gastric cancer markers by qRT‐PCR.

Results

Gastric fundic tissue obtained from individuals infected with H. pylori were found to have a significantly upregulated expression of cytoplasmic BMAL1. hFGOs exhibited a robust circadian expression of Bmal1, Per2, and phosphorylated (inactive) GSK‐3β. In comparison to uninfected controls, hFGOs infected with H. pylori displayed a heightened expression of BMAL1, phosphorylated GSK‐3β, and epithelial proliferation. Gastric organoids derived from of elderly patients was found to have a diminished rhythmic expression of clock genes Per2 and Bmal1. This dampening in expression of circadian clock components correlates with an increased expression of markers for intestinal metaplasia.

Conclusions

Persistent infection by H. pylori inactivates GSK‐3β resulting in the activation of the BMAL1:CLOCK complex leading to a hyperproliferation in gastric epithelial cells. This mechanism could heighten the risk of developing gastric cancer in the aged population and in individuals with chronic H. pylori infection. Per2 knockout mice have been generated and infected with H. pylori in order to further understand this mechanism in vivo. Results are ongoing and pending.

Support or Funding Information

NIH 1U19AI116491‐01

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Impact of FADS Variants on Responses to Diet in Patients With ALI
Authors: Dosso Beverly , Waits Charlotte Mae , Simms Kelli , Sergeant Susan , Files D. Clark , Chilton Floyd , Langefeld Carl , Rahbar Elaheh
Published: June 2021

Nutritional support in the intensive care unit (ICU) is a vital element of patient care. Polyunsaturated fatty acids (PUFAs) like γ‐linolenic acid (GLA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have been recommended in immunomodulatory diets because of their anti‐inflammatory effects. However, clinical trials have failed to show unanimous benefit with PUFA‐rich diets. Metabolic conversion of these PUFAs are dependent on fatty acid desaturase (FADS) and elongase (ELOVL) enzymes, which are unequal across humans. In fact, several genetic variants within FADS and ELOVL genes have been shown to impact PUFA metabolism. Select variants are also differentially expressed in African Americans. We hypothesize that these genetic variants may help explain the differential responses to PUFA‐rich diets. In this study, we aimed to investigate gene‐diet interactions in patients with acute lung injury (ALI).

Banked DNA and plasma samples from the OMEGA randomized clinical trial (NCT00609180) were used to conduct a secondary analysis on genetic variants within FADS and ELOVL regions; 43 SNPs were genotyped. Plasma PUFA levels were quantified using gas chromatography. Of the 272 enrolled, 143 received omega‐rich vs. 129 placebo diets. All statistical analyses were stratified by race and adjusted for age and gender.

Several SNPs were associated with PUFA levels. Notably, SNP rs174537 had a significant impact on arachidonic acid (ARA). Caucasian T allele carriers on the omega‐rich diet had lower ARA levels (p=0.022, Fig. 1). Interestingly, this SNP had a major impact on PUFAs within African Americans on both placebo and omega‐rich diets. ARA levels were significantly higher and DHA levels were low (p=0.0007) in African Americans. Genotype at rs174537 was associated with patient outcomes; Caucasians with the T allele had significantly more VENT‐free and ICU‐free days (~>12%) than GGs, but this was not observed within African Americans. Instead, a significant interaction between gender and diet was seen, with African American women on placebo diet having 40% fewer VENT‐free (p=0.009) and ICU‐free days (p=0.022) than African American men. Mortality was highest in GGs receiving omega‐rich diets in both races, however this was not statistically significant. These data suggest strong ethnic, gender and genetic factors influencing the response to omega‐rich diets. Further investigation on other gene‐gene interactions (e.g. between FADS and ELOVL variants), and epigenetic modifications are needed to understand the underlying mechanisms regulating PUFA metabolism and biosynthesis in critically injured and ill patients.

Support or Funding Information

NIH K25 HL133611

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Unique Skeletal Muscle Gene Expression Signature Induced by Acute Alcohol Intoxication in mice
Authors: Steiner Jennifer , Veid Cynthia , Lang Charles
Published: June 2021

High levels of alcohol intake lead to skeletal muscle myopathy and subsequent dysfunction both acutely and chronically. Our previous work has shown that a single dose of alcohol, as well as chronic intake, decreases rates of protein synthesis concurrent with suppression of the mTOR signaling pathway. Although evidence exists for the contribution of other anabolic and catabolic factors (myostatin, inflammation, insulin like growth factor, etc.), to the induction of alcoholic myopathy, no definitive mechanism(s) or pathway(s) has been identified. Therefore, the objective of this work was to identify novel pathways for future therapeutic targeting in the treatment of alcoholic myopathy via an unbiased screen of skeletal muscle specific genes differentially regulated by acute alcohol intoxication. Male, 12 week old, C57BL/6 mice were fasted overnight prior to receiving an intraperitoneal injection of 3g/kg BW alcohol or saline (Control group). The gastrocnemius/plantaris muscle complex was excised 1 hour post alcohol intoxication for isolation of RNA and preparation of cDNA prior to RNA deep sequencing. The data were mapped to the mouse genome (mm10, GRCm38) using TopHat 2 software. Using DESeq2 software, differentially expressed genes were determined based on an FDR‐adjusted p‐value of <0.05. Under these parameters, 48 transcripts were identified in response to acute alcohol intoxication. Using the DAVID Bioinformatics Resource, 8 functional groups were recognized including Collagen, Extracellular matrix, Secreted, Hydroxylation, Cell Adhesion, Cytoplasm, Signal and Glycoprotein. Of the 48 genes, 15 were suppressed by alcohol intoxication including Regulator of Cell Cycle (Rgcc), previously shown to be upregulated in models of skeletal muscle hypertrophy and regrowth; Leiomodin2 (Lmod2) which is important in actin polymerization; and xin actin‐binding repeat containing 1 (Xirp1) which protects actin filaments from depolymerization and may be a marker of muscle damage. The remaining 32 genes were increased by acute alcohol intoxication including EMILIN1 which relies on the inhibition of TGF‐β; Aquaporin7 which is implicated in lipolysis and glycerol efflux via regulation of mTOR‐mediated leptin expression; and MAP3K14, also known as NF‐κB inducible kinase, indicating potential modulation of the NF‐κB pathway by acute alcohol. Of the differentially expressed gene transcripts, several do not appear to have a known functional role in skeletal muscle and will therefore provide novel targets for the further characterization of the effects of alcohol on skeletal muscle.

Support or Funding Information

Funds provided by Florida State University

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Copper Supplementation Promotes Myocardial Vascular Regeneration from Bone Marrow‐derived Stem Cells in Mouse Model of Ischemic Heart Disease
Authors: Meng Xia , Yin Wen , Li Chen , Song Xin , Feng Xiaorong , Kang Y. James
Published: June 2021

Copper (Cu) supplementation improves myocardial structural and functional recoveries from chronic ischemic injury in both monkey and mouse models, and myocardial vascular regeneration plays a critical role in this process. The present study was undertaken to test the hypothesis that the homing and differentiation of stem cells make an important contribution to myocardial vascular regeneration in mouse model of ischemic heart disease. Male C57 mice of 8–12 weeks old were subjected to left anterior descending (LAD) coronary artery ligation to induce myocardial ischemic infarction. Four weeks after the surgery, a cardiac‐specific ultrasound‐mediated Cu‐albumin microbubble (Cu‐MB) Cu delivery procedure was used to supplement Cu to the heart twice a week for two weeks, and the controls were treated with the same procedure but with Cu‐absent albumin microbubble. All of the mice were then injected with PKH26‐labeled bone marrow‐derived stem cells (BMSCs), and the hearts were harvested at 1st, 3rd, 7th, or 14th day after the cell transplantation. One day after cell transplantation, the homing of PKH26‐labeled BMSCs in the ischemic infarct area was significantly increased in the Cu‐MB and ultrasound treated mice, but not in the ultrasound untreated mice. Three days after cell transplantation, the number of BMSC‐derived endothelial cells, as defined by the cells dually‐labeled with PKH26 and CD31 were significantly increased in the ischemic infarct area of the mice treated with ultrasound and Cu‐MB. The capillary densities in the ischemic infarct area of the mice treated with ultrasound and Cu‐MB were significantly elevated in comparison with that of mice treated with ultrasound and MB without Cu at 7th or 14th day after cell transplantation. Myocardial structure and contractile function were also significantly improved 14 days after cell transplantation in the ultrasound and Cu‐MB treated mice. These results thus demonstrate that Cu supplementation promotes myocardial vascular regeneration through at least in part enhancing stem cells homing and differentiation to vascular endothelial cells in the infarct myocardium, improving myocardial structural and functional recovery from chronic ischemic injury.

Support or Funding Information

Scientific research funds of West China Hospital, Sichuan University

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Anti‐inflammatory Effect of Table Grapes in Apc(Min/+) Mouse Model of Intestinal Tumorigenesis
Authors: Indukuri Vijaya , Mason Madeline , Reddivari Lavanya , Vanamala Jairam , Lambert Joshua
Published: June 2021

The link between inflammation and cancer is well‐studied. Epidemiological and experimental data suggests that chronic inflammation plays a very important role in the promotion and progression of colon cancer, and that diet can play an important role in mitigation of chronic inflammation. Previously, our laboratory has found that freeze‐dried whole table grape powder (FDGP) can reduce tumorigenesis in APC(Min/+) mice. It has been reported that inflammation plays an important role in tumorigenesis in this model. The objective of the current study is to assess the anti‐inflammatory activity of FDGP in the Apc(Min/+) mouse model. Male Apc(Min/+) mice (4 wks, n= 8/group) were fed diets supplemented with 3% or 6 % FDGP or control diet for 12 wks. At the end of dietary intervention, expression of inflammation‐related markers in the ileum and the plasma were measured by qRT‐PCR and multiplex ELISA, respectively. FDGP supplementation suppressed the levels of the pro‐inflammatory cytokines IL‐1β, IL‐6, IL‐12, IL‐17 (by 30–70%), and enhanced the levels of the anti‐inflammatory cytokine IL‐10 (by ~50%) in both the ileum and serum compared to control‐fed animals. FDGP‐ fed animals also had significantly lower plasma lipopolysaccharide levels (3–5 fold) and reduced mRNA expression of Claudin 2 (~2 fold) in the ileum, suggesting that FDGP improved gut barrier function compared to the control diet. Overall, our results show the potential anti‐inflammatory effect of table grapes in an animal model of intestinal tumorigenesis and indicate the need for further preclinical mechanistic studies.

Support or Funding Information

California Table Grape CommissionDepartment of Food Science, Pennsylvania State University

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.