bluuebunny/crossref_metadata_2025 huggingface dataset:
Intermittent Fasting Suppresses Activity‐Associated Energy Expenditure and Enhances Weight Loss in Obesity‐Prone Rats
Authors: Titus Amber R. , Davis Ashley E. , Britton Steven L. , Koch Lauren G. , Novak Colleen M.
Published: June 2021

Weight and fat loss occur when total energy expenditure (EE) is higher than energy input. This can be achieved by lowering caloric input through altering diet, or by increasing total EE. Activity‐associated EE is suppressed during weight loss and maintenance of reduced weight; this occurs through decreased physical activity levels along with increased fuel economy of activity. These adaptations take time to occur. Both activity‐associated EE and resting EE have been shown to decrease during calorie restriction‐induced weight loss in rats. Recently, attention is being paid to alternatives to calorie‐restriction, such as time‐restricted feeding approaches including intermittent, or alternate‐day, fasting. We have found that, unlike daily calorie‐restriction, intermittent fasting achieves a superior result in obesity‐prone rats compared to their lean counterparts. Here, we investigate the potential contribution of physical activity‐associated EE to the altered energy balance contributing to weight loss during intermittent fasting. We examined female high‐and‐low‐capacity runners (HCR and LCR, respectively), rats artificially selected for high and low intrinsic aerobic capacity. HCR are lean and more physically active than their obesity‐prone LCR counterparts. We induced equivalent weight loss within phenotype using intermittent fasting and daily calorie‐restriction, and measured activity‐associated EE during treadmill walking. Calorie‐restriction and intermittent fasting induced equivalent suppression of activity‐associated EE in both HCR and LCR. As we saw previously, intermittent fasting was significantly more effective in inducing weight loss in the obesity‐prone LCR than in HCR. Interestingly, our data do not support the hypothesis that the enhanced weight loss occurs through a release from adaptive thermogenesis in activity‐associated EE. There was also no difference in activity‐associated EE between fed and fasted days in the intermittent‐fasted rats. This implicates intermittent fasting as a superior means of weight loss in obesity‐prone rats, and suggests that the efficacy of intermittent fasting might also vary between people.

Support or Funding Information

Funding: NIH NIDDK R15‐DK‐097644 and R15‐DK‐108668 to Colleen M Novak; P40 OD021331 to Lauren G Koch. Resource for Rat Genetic Models of Aerobic Capacity: P40 OD021331(Lauren G Koch, Principal Investigator; Steven L Britton, Co‐Investigator)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Inactivation of Wnt/β‐catenin signaling pathway by SIRT 6 inhibits tumor growth and proliferation in hepatocellular carcinoma
Authors: Jin Hua , Kim Soo Mi
Published: June 2021

Sirtuins are a family of NAD+‐dependent protein involved in regulating the lifespan and metabolic homeostasis. Sirtuin 6 (SIRT6) has been reported to have tumor suppressing effect in liver. However, the precise molecular mechanism of SIRT6 in human hepatocellular carcinoma (HCC) has not been clearly understood. Therefore, we have investigated the importance of SIRT6 function in HCC cell lines, HepG2 and SNU449. SIRT6 was highly expressed in human HCC cells. Overexpression of SIRT6 significantly diminished the viability of HCC cells whereas silencing of SIRT6 stimulated the viability of HCC cells. Overexpression of SIRT6 increased expression of cleaved‐PARP and cleaved‐caspase9 and decreased the PARP, caspase9, and caspase3. Knockdown of SIRT6 increased the number and size of colonies. In addition, overexpression of SIRT6 significantly inhibited the invasion and metastasis of HCC cells whereas silencing of SIRT6 increased the invasion and metastasis abilities of HCC cells in a time dependent manner. Moreover, overexpression of SIRT6 inhibited vimentin, UPA, and MMP9 protein levels while silencing of SIRT6 in HCC cells increased the protein levels of vimentin, UPA, and MMP9. P‐β‐catenin levels was increased by overexpression of SIRT6 and was decreased by silencing of SIRT6. These data suggest that SIRT6 inhibits the proliferation, invasion and metastasis of HCC cells and may play as a tumor suppressor in HCC cells.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Deletion of claudin‐7 disrupts epithelial cell self‐renewal in mouse colon
Authors: Xing Tiaosi , Benderman Lesley Jasmine , Sabu Stephiya , Chen Yanghua
Published: June 2021

Tight junctions are one of the physical barriers protecting the body from invasion of intestinal lumen pathogens. Disruption of tight junctions has been reported in a variety of intestinal disorders, such as intestinal bowel disease, necrotizing enterocolitis, and colon rectal cancer. Claudin‐7 is one of the tight junction membrane proteins and is essential in maintaining intestinal epithelial integrity. However, the function of claudin‐7 in colonic epithelial homeostasis remains to be determined. Here we report that claudin‐7 is required for maintaining the balance of intestinal epithelial cell proliferation and differentiation. To dissect the role of claudin‐7 in colonic epithelium, we used our global (gCldn7−/−) and intestinal‐specific (cCldn7−/−) claudin‐7 knockout (KO) mice that are generated recently as our model systems. Using the histological analysis, we found that enterocytes and enteroendocrine cells are significantly reduced in postnatal day 4 gCldn7−/− and 2–4 months old cCldn7−/− mouse colons. Although the absolute number of Goblet cells and tuft cells are reduced in claudin‐7 KO colon, the ratio of Goblet cells or tuft cells versus the total number of epithelial cells remains unchanged. These results are confirmed by qRT‐PCR and Western blotting, suggesting that epithelial cell differentiation was significantly altered after claudin‐7 deletion. In addition, the ki67 and PCNA positive proliferative cells are significantly increased in claudin‐7 KO colons and no longer limited to the basal‐lateral crypt region, where epithelial stem cells and fate‐committed progenitor cells are located. Genome‐wide gene‐expression microarray analysis of Cldn7+/+ and gCldn7−/− colons identified a list of gene expression changes related to cell proliferation and differentiation. Notch and Hippo signaling pathway are suppressed after claudin‐7 deletion. These findings suggest that claudin‐7 may have a novel, unidentified function regulating epithelial cell differentiation and self‐renewal in mouse colon.

Support or Funding Information

This study is supported by the National Institute of Health grant DK103166.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
H1/H2 histamine receptor blockade lowers substrate‐dependent mitochondrial H2O2 emission in deep gastrocnemius muscle following a bout of prolonged exercise
Authors: Davidson Laura R. , Wallace Helen Elizabeth , Bell Mackenzie CW. , Brebner Karen , Kane Daniel A.
Published: June 2021

Prolonged exercise increases oxidative metabolism in skeletal muscle mitochondria, which is facilitated, in part, by increased blood flow and delivery of nutrients and oxygen. Histamine mediates many acute exercise responses, including sustained vasodilation and associated delivery of substrates to muscle. We tested the hypothesis that H1 and H2 histamine receptor blockade would attenuate exercise‐induced changes in skeletal muscle mitochondrial function. Adult male Wistar rats (75–94 days old; 441.2 ± 39.5 g) were randomly assigned to one of three groups (n = 12/group): exercise, in the form of one hour of continuous treadmill running at 20 cm/sec (Group 1); mass‐specific oral gavage with H1 antagonist fexofenadine (7.11 mg/kg) and H2 antagonist ranitidine (3.95 mg/kg) one hour prior to the same exercise protocol (Group 2); control, which received neither antihistamines nor exercise (Group 3). An hour post exercise, mitochondrial function was assessed in saponin‐permeabilized myofibers from deep gastrocnemius (mixed fiber types) and soleus (oxidative) skeletal muscle. A sequential substrate‐inhibitor titration protocol was used to examine multiple mitochondrial metabolic pathways (i.e., malate + octanoyl‐carnitine, ADP, lactate + NAD+, pyruvate, glutamate, succinate, inhibitors of antioxidant defense glutathione peroxidase and thioredoxin). Substrate‐dependent oxygen consumption (JO2; high‐resolution respirometry) was not different between treatments; however, H1 and H2 histamine receptor blockade significantly decreased hydrogen peroxide emission (mH2O2; fluorometric monitoring of Amplex Red oxidation) in deep gastrocnemius fibres (P < .05). Given the widespread and routine use of antihistamines, further research into the effects of these antagonists on mitochondrial function, in the context of exercise, is warranted.

Support or Funding Information

The Natural Sciences and Engineering Research Council of Canada (NSERC), The Nova Scotia Health Research Foundation (NSHRF), and Canada Foundation for Innovation (CFI)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Inhibition of Pathological Mitochondrial Fission Restores DSS Associated Respiratory Impairments in an Intestinal Epithelial Cell Line
Authors: Goudie Luke J. L. , Mancini Nicole , Blote Karen R. , Wang Arthur , McKay Derek M. , Shearer Jane
Published: June 2021

Significance

Recent evidence has shown that mitochondrial dynamics and more specifically, mitochondrial fission, play an important role in numerous disease of energy intensive tissues. In particular, mitochondrial fission mediated by the enzyme dynamin related protein‐1 (DRP1) and the receptor fission protein‐1 (Fis1) is associated with reduced ATP production, ROS generation and inflammation. Examination of this interaction and corresponding pathological mitochondrial fission has yet to be examined in the colon in the context of inflammatory bowel disease.

Objective

To identify if a common murine colitis agent, dextran sodium sulfate (DSS), induces mitochondrial respiration dysfunction and if the novel peptide (P110), can restore these deficiencies by inhibiting DRP1 and Fis1 mediated fission.

Design

The murine intestinal epithelial cell line, IEC4.1, was separated into control, P110, DSS and DSS + P110. Cell were seeded and allowed to incubate for 24 hrs before receiving their respective treatments and further incubation for another 24hrs.

Methods

Optimization of both DSS (1–2w./v.%) and P110 (0.5–1.5 uM) dosage and exposure time was performed with the AlamarBlue cell proliferation assay. Basal, mitochondrial complex and maximal respiratory activity were assessed using the Oxygraph‐2k (OROBOROS Instruments, Austria). Respiratory function of cells under various substrate pathways was also examined.

Results

DSS (2%) significantly reduced cell proliferation compared to control and P110 groups (p<0.05). Administration of P110 in 2% DSS cells dose dependently increased cell proliferation, with 1.5 uM P110 exhibiting cell proliferation similar to control. Analysis of mitochondrial complexes revealed that DSS reduced basal, complex I, II, IV and maximal activity (p<0.05). P110 was found to restore DSS associated respiratory deficiencies in complex I, II and IV (p<0.05).

Conclusion

This study highlights novel findings regarding the role of mitochondrial fission in a model system of epithelial dysfunction. Importantly, we show i) evidence that DRP1 and Fis1 mediated fission hold potential; pathological consequences in energetically demanding tissues like the colon and ii) P110 treatment reduces DSS induced damage and its associated respiratory deficiencies. These results suggest mitochondrial dysfunction could be intricately involved in gut barrier deterioration seen in inflammatory bowel disease.

Support or Funding Information

Crohn's and Colitis Canada. The authors wish to thank Daria Mochly‐Rosen for donating P110.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Deep Proteomic Quantification of Inner Medullary Collecting Duct Protein Phosphorylation: Response to Vasopressin
Authors: Deshpande Venkatesh , Chou Chung‐Lin , Knepper Mark
Published: June 2021

Progress in protein mass spectrometry instrumentation and labeling methods have led to dramatic increases in the depth of phospho‐peptide identification and quantification. Here we used tandem mass tag (TMT) labeling and LC‐MS/MS for deep identification of phospho‐proteomic responses to the vasopressin analog dDAVP (1 nM for 30 minutes) in inner medullary collecting duct (IMCD) suspensions from rats. We analyzed three pairs of samples (dDAVP vs. vehicle) on different days. The analysis yielded 16 GB of spectral data mapping to 11,658 phospho‐peptides in 1758 distinct proteins. Vehicle vs. vehicle comparisons allowed assessment of the variability of the method and permitted dual criteria to be applied for stringent identification of abundance changes (based on t‐statistic and z‐score). Among all phospho‐peptides quantified, only 93 were increased in abundance, and only 49 were decreased, indicating very selective regulation within the broad range of phospho‐peptides quantified. A substantial number of the increases were in aquaporin‐2 and the UT‐A urea channel, including a previously unidentified phosphorylation site at Ser918 of UT‐A1 (sequence RAS*MITK) that was more than doubled in abundance. Abundance changes in aquaporin‐2 peptides confirmed prior findings. Of interest, phosphorylation changes in Raf1 (increased phosphorylation at Ser259, a kinase inhibitory site) and A‐Raf (decreased phosphorylation of Ser255, a kinase activating site) provide an explanation for findings that vasopressin decreases ERK activation and AQP2 phosphorylation at Ser261 in the collecting duct. Finally, vasopressin significantly increased phosphorylation of protein kinase D1 (Prkd1) at Ser255 and of phosphatidylinositol 4‐kinase beta at Ser551. Both proteins play central roles in regulated exocytosis of ENaC, and we speculate that they could play similar roles in vasopressin‐stimulated exocytotic apical insertion of aquaporin‐2 and UT‐A1.

Support or Funding Information

V.D. was a member of the 2017 Biomedical Engineering Student Internship Program at the National Institutes of Health.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Role of Myosins II and VI in Mediating Hormonal Regulation of NHE3 Activity in the Rat Renal Proximal Tubule
Authors: Crajoinas Renato Oliveira , Castelo‐Branco Regiane Cardoso , Polidoro Juliano Zequini , Ralph Donna Lee , Malnic Gerhard , McDonough Alicia Ann , Girardi Adriana Castello Costa
Published: June 2021

Rapid regulation of NHE3 activity may occur through redistribution of the transporter between the body and the base of the proximal tubule microvilli (PT MV). Myosins are motor proteins that move along actin filaments. The present study tested the hypothesis that myosins II (myo II), which moves cargo away from cell center, and VI (myo VI), which moves cargo toward cell center, may mediate NHE3 redistribution within the MV in response to stimuli. Renal PT NHE3 activity was evaluated in Wistar rats by net bicarbonate reabsorption (JHCO3−) in the presence or absence of 10−10 M angiotensin II (Ang II), 10−4 M forskolin (FSK), a cAMP‐elevating agent, 10−5 M blebbistatin (a specific myo II inhibitor) or 4×10−6 M 2,4,6‐triiodophenol (TIP) (a myo VI inhibitor). As expected, Ang II increased [4.36 ± 0.18 nmol/cm2.s (N = 5)] whereas FSK reduced [1.26 ± 0.10 nmol/cm2.s (N = 5)] JHCO3− compared to CTRL [2.74 ± 0.13 nmol/cm2.s (N = 4)]. Simultaneous tubular perfusion of Ang II with blebbistatin prevent Ang II‐induced NHE3 stimulation [2.92 ± 0.14 nmol/cm2.s (N = 5), P < 0.001]. Simultaneous tubular perfusion of FSK with TIP reduced the inhibition with FSK alone [1.78 ± 0.15 (N = 4) vs. 1.26 ± 0.10 nmol/cm2.s (N = 5), P < 0.05] on JHCO3−. The localization of NHE3 and myosins IIB and VI was assessed by confocal immunofluorescence in Wistar rats kidney after a 30 min pressor dose of Ang II (50 ng/kg/min). NHE3 and myo VI shift from base to body of MV after Ang II; myo IIB remains mostly enriched at the MV base. To assess NHE3 activity in OKP cells, experimental pH recovery points were fitted to simple exponential decay curves (y = A·e(−kt) + y0) in order to predict the rate constant (k) of each recovery curve. FSK reduced NHE3 activity [0.025 ± 0.003 s−1 (N = 4)] compared to CTRL [0.048 ± 0.007 s−1 (N = 5)]. siRNA for myo VI efficiently reduced its expression by 64 %; the inhibitory effect of FSK on NHE3 activity was completely abolished by siRNA myo VI [0.043 ± 0.005 s−1 (N = 4)]. These results provide further evidence for myosin II and VI regulation of NHE3 redistribution in the rat kidney proximal tubule.

Support or Funding Information

FAPESP

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
cGMP Stimulates K48‐linked Poly‐ubiquitination of NKCC2 in Rats Thick Ascending Limb
Authors: Ares Gustavo
Published: June 2021

NaCl reabsorption by the thick ascending limb (TAL) is mediated by the Na/K/2Cl cotransporter NKCC2. The second messenger cyclic guanosine monophosphate (cGMP) decreases NKCC2 activity by decreasing surface NKCC2 levels. We showed that NKCC2 is constitutively ubiquitinated and degraded by the proteasome, and that cGMP enhances the rate of ubiquitination. Ubiquitination is a post‐translational modification in which the C‐terminus of the ubiquitin is attached to a substrate protein. Poly‐ubiquitination occurs when the C‐terminus of another ubiquitin, is then linked to the previously added ubiquitin, creating a chain. There are 7 lysine (k) in the ubiquitin molecule where poly‐ubiquitination can occur. K48‐linked poly‐ubiquitination on ubiquitin is associated with proteasomal degradation. Therefore, we hypothesized what cGMP stimulates K48‐linked poly‐ubiquitination of NKCC2 in rats TAL. To test our hypothesis we immunoprecipitated K48‐linked poly‐ubiquitin using Tandem Ubiquitin Binding Entities (TUBEs) and western blot against NKCC2 in Sprague‐Dawley rat TAL suspensions. First, we performed a k48‐TUBEs concentration‐response curve against K48‐linked poly‐ubiquitination of NKCC2 to ensure linear relationship. TALs suspension were aliquoted in 4 samples and placed at 37°C for 30 minutes in the presence of the proteasomal inhibitor (MG132 20μM) to stop protein degradation. TALs were lysed at 4°C and excess amount of protein lysate (150 μg) was incubated with increasing concentration of k48‐TUBEs. We obtained a linear increase in K48‐linked poly‐ubiquitination of NKCC2 with increasing concentration of k48‐TUBEs. Next, to study the effect of cGMP on k48‐ubiquitination of NKCC2. The conditions were set to fit within the linear range (80 μg of protein) to ensure complete immunoprecipitation of K48‐linked poly‐ubiquitination of NKCC2. We found that the membrane‐permeant cGMP analogue dibutiryl cGMP (db‐cGMP) stimulates K48‐ubiquitination on NKCC2 in a concentration‐dependent manner (baseline: 100.0%; db‐cGMP 100 μM: 115.3 ± 3.7%; db‐cGMP 250 μM: 127.8 ± 5.2%; db‐cGMP 500 μM: 148.3 ± 18.2% p<0.05). These data indicates that cGMP stimulates K48‐linked poly‐ubiquitination of NKCC2, and suggest a triggering mechanism for NKCC2 degradation by the proteasome. We conclude that cGMP increases K48‐linked poly‐ubiquitination of NKCC2. To our knowledge, this is the first piece of evidence showing that cGMP may trigger a signal for NKCC2 degradation through stimulation of k48‐ubiquitination.

Support or Funding Information

AHA Post‐Doctoral (14POST20110009)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Chronic High Potassium Diet Decreases Thiazide‐Sensitive Sodium Excretion in High Fat‐Fed Mice
Authors: Nizar Jonathan , Vo Vianna , Bhalla Vivek
Published: June 2021

High fat‐fed mice recapitulate several features of the Metabolic Syndrome, including impaired sodium excretion and increased blood pressure. In this model, post‐thiazide sodium excretion is increased compared to low fat‐fed controls, providing evidence that increased Na‐Cl co‐transporter (NCC)‐mediated transport may contribute to both impaired sodium excretion and hypertension in the Metabolic Syndrome. We sought to test whether a chronic, high potassium diet regulates NCC differently in high fat‐fed mice compared to low fat‐fed controls.

We fed high fat‐fed mice either a normal (0.7% w/w) or high (5% w/w) potassium diet for 1 week then performed an intraperitoneal injection either vehicle or 30 mg/kg hydrochlorothiazide and collected urine for 4 hours. We then switched the dietary potassium content of both groups and rechallenged the mice with the same drugs. After a washout period, we sacrificed mice for tissue analysis.

While on a high potassium diet, post‐thiazide urinary sodium excretion was no different from vehicle and significantly lower than excretion on a normal potassium diet (0.164 +/− 0.024 vs. 0.026 +/− 0.005 μmol/min, normal vs. high potassium diet, respectively; p<0.001). Post‐thiazide potassium excretion was no different on either diet (0.138 +/0 0.017 vs. 0.109 +/− 0.006 μmol/min normal vs. high potassium diet, respective; p = 0.28) and higher than vehicle. Urine flow rate was no different while on either normal or high potassium diet after vehicle or thiazide injection. Membrane preparations of whole kidney lysate blotted for both total and phosphorylated NCC were not significantly different in high fat‐fed mice on a normal vs. high potassium diet. High fat‐fed mice do not demonstrate the high potassium diet‐induced repression of NCC phosphorylation that is well‐documented in low fat‐fed mice, and have detectable, albeit diminished sodium excretion. These data support a role for attenuated repression of the potassium‐NCC axis in engendering impaired sodium excretion and hypertension in this model of Metabolic Syndrome.

Support or Funding Information

JMN receives support from the NIH (1K08 DK114567‐01). JMN was previously supported by an American Heart Association Postdoctoral Fellowship (16POST27770003). VB receives support from the NIH (1R01 DK091565).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
TRPV4 inhibition protects against hypokalemia during low K+ intake
Authors: Tomilin Viktor N. , Zaika Oleg , Mamenko Mykola , Pochynyuk Oleh M.
Published: June 2021

Tight regulation of K+ balance is fundamental for normal physiology. Reduced dietary K+ intake, which is common in Western diets, often leads to hypokalemia and associated cardiovascular‐ and kidney related pathologies. Distal renal tubule and specifically the collecting duct (CD) is the only place of controlled K+ reabsorption via H+‐K+‐ATPase in the state of dietary K+ deficiency. We previously demonstrated that TRPV4 Ca2+ channel, abundantly expressed in the CD, is a potent kaliuretic factor essential for flow‐induced K+ secretion. Here, we investigated a potential role of TRPV4 in controlling H+‐K+‐ATPase‐dependent K+ reabsorption in the CD. Treatment with low K+ diet (<0.01% K+) for 7 days reduced plasma K+ levels in WT mice from 4.3±0.2 mM to 3.3±0.2 mM, but not in TRPV4 −/− mice (4.3±0.1 mM and 4.2±0.3 mM, respectively). Furthermore, we detected significant reduction in urinary K+ levels in balance studies upon switching to low K+ diet in the presence of TRPV4 inhibition. By using freshly isolated split‐opened CDs, we detected greatly accelerated H+‐K+‐ATPase‐dependent pHi extrusion in TRPV4 −/− mice when compared to WT mice. Finally, TRPV4 −/− animals had significantly more acidic urine (6.2±0.1 vs 6.7±0.2 in WT) on low K+ diet, which is consistent with increased H+‐K+ ATPase activity. In summary, we propose that TRPV4 inhibition could be a novel strategy to manage certain hypokalemic states in clinical setting.

Support or Funding Information

This research was supported by NIH‐NIDDK DK095029 (to O. P.), AHA 17GRNT33660488 (to O. P.), and ASN Ben J. Lipps Research Fellowship (to V. T.).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Response of intercalated cell BKα knock‐out mice to a high K diet
Authors: Ray Evan C. , Carrisoza‐Gaytan Rolando , Marciszyn Allison L. , Wu Peng , Liu Leah C. , Subramanya Arohan R. , Wang Wenhui , Flores Daniel , Kohan Donald E. , Kleyman Thomas R. , Satlin Lisa M.
Published: June 2021

Flow‐induced K secretion (FIKS) in the cortical collecting duct (CCD) is mediated by apical Ca2+−/stretch‐activated BK channels. These channels also contribute to the renal adaptation to dietary K intake. BK channels are expressed in both intercalated (ICs) and principal cells (PCs) in CCDs. Several lines of evidence suggest that BK channels in ICs are responsible for FIKS: (i) the density of BK channels in ICs exceeds that in PCs and (ii) dietary K loading upregulates BKα expression predominantly in the apical membrane of ICs in rabbit CCDs. We generated a mouse with targeted deletion of BKα, the pore forming subunit of the BK channel, in ICs (IC‐BKα‐KO) by crossing floxed BKα mice with B1 V‐ATPase Cre mice, after confirming that the B1 Cre was expressed primarily in ICs in the kidney. As the targeted deletion in exon 7 of the BKα sequence was not expected to lead to a frame shift, immunodetectable BKα was identified in ICs of IC‐BKα‐KO CCDs using an antibody directed against the BKα C terminus. Perforated whole‐cell recordings performed at +60 mV in 7 CCD ICs in an IC‐BKα‐KO fed a HK diet revealed that 4 ICs had no BK currents and 3 ICs had low charybdotoxin‐sensitive K currents (37, 65, 90 pA) vs. readily detectable currents in 2 ICs in WT mice fed a control K diet (600–800 pA). IC‐BKα‐KO and floxed controls were fed a HK diet × 10 days to maximize BK channel expression and received isotonic saline IP to induce a high urine flow rate. Over 6 hr following volume expansion, rates of urinary Na (39.1±11.1 vs. 36.0±15.2 μmol/hr) and K (165.5±50.1 vs. 171.9±76.0 μmol/hr) excretion were similar in IC‐BKα‐KO (n=10) and floxed (n=17) mice of both sexes. KO mice exhibited a higher blood [K] (5.5±0.8 vs. 5.0±0.7 mM; p<0.05) and BUN (25.3±5.7 vs. 20.6±5.1 mg/dL; p<0.03) vs. floxed controls although BW did not differ (21.0±2.7 vs. 22.6±3.4. gm, respectively). The difference in blood [K] between male IC‐BKα‐KO and floxed mice was highly significant (5.7±0.7 vs. 5.0±0.7 mM; n=14 and 16, p<0.02). In the absence of volume expansion, rates of urinary Na (1.3±0.8 vs. 1.3±1.3 μmol/hr) and K (50.9±23.7 vs. 40.4±22.5 μmol/hr) excretion were similar in the two groups over a 6 hr period (n=9 and 15, respectively). These results suggest that in the absence of IC BKα, mice have a limited capacity for adaptation to a HK diet, as evidenced by the higher blood [K], but still demonstrate FIKS.

Support or Funding Information

K08 DK110332, P30 DK079307, R01 DK038470, R01 DK098145, I01 BX002510

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Sex Differences in Urinary Exosomes from Salt‐sensitive 129Sv Mice
Authors: Liu Lauren , Tuna Kubra , Chacko Kevin , Sanchez Victor , Schramm Whitney , Ahmed Hamzah , Alli Abdel
Published: June 2021

129Sv mice are susceptible to the development of salt‐induced hypertension and renal damage. We and others observed that female 129Sv mice have lower systolic blood pressure compared to male 129Sv mice. We did not find differences in electrolyte excretion or osmolality between female and male 129Sv mice. Urinary exosomes originate from different cell types such as renal epithelial cells. Their cargo is a source of biomarkers in the pathogenesis of hypertension and renal disease. We recently showed kidney exosomes may play a role for intercellular communication in the nephron. We hypothesized that there are differences in the production of exosomes and their cargo from female and male salt‐sensitive 129Sv mice. Exosomes were characterized by size using NanoSight Analysis and by Western blotting for the exosomal markers flotillin‐1, annexin V, and Hsp70. Using nanoparticle tracking analysis, we found a greater concentration of urinary exosomes from salt‐loaded female 129Sv mice compared to age‐matched salt‐loaded male 129Sv mice. On a normal‐salt diet, the size of the urinary exosomes from the female mice was smaller compared to the male mice, but the opposite was noted when a high‐salt diet was maintained. Importantly, we found urinary exosomes from salt‐loaded female 129Sv mice are enriched in annexins compared to males. Future studies will be performed to determine if the enrichment of annexins in the kidney of female mice contributes to the up‐regulation of urinary exosomes and altered exosomal cargo.

Support or Funding Information

This research was supported by a National Institutes of Health grant K01 DK099617 (to A. A. Alli).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Astrocyte‐specific deletion of Kir4.1 increases normoxic ventilation after acclimatization to chronic sustained hypoxia.
Authors: Moya Esteban A. , Hawkins Virginia E. , Yip Gigi , Mulkey Daniel K. , Powell Frank L.
Published: June 2021

Chronic sustained hypoxia (CH) produces ventilatory acclimatization to hypoxia (VAH), which involves time‐dependent increases of ventilation in normoxia and the hypoxic ventilatory response (HVR). Previous results show that glia are activated by CH and contribute to VAH but the contribution of astrocytes involved in central CO2‐sensitivity to VAH is not known. We tested the hypothesis that inwardly rectifying K+ 4.1 channels in astrocytes contribute to VAH using conditional gene deletion in transgenic mice. Conditional knockout (cKO) Kir4.1 mice had loxP sites at both ends of the entire Kir4.1 open reading frame and WT mice did not. WT and cKO Kir4.1 mice expressed tdTomato reporter and Cre‐recombinase/ERT2 receptors in astrocytes under the control of hGFAP promoter. Immunofluorescence against markers of astrocytes and Kir4.1 confirmed that tamoxifen treatment decreased Kir4.1 expression in astrocytes in CO2‐senstivie areas of the medulla of cKO Kir4.1 but not WT mice. After CH (PiO2 = 70 mmHg for 1 week), ventilation measured with barometric pressure plethysmography increased in normoxia, acute hypoxia and acute hypercapnia relative to chronically normoxic control mice. Ventilation was greater in normoxia after CH in Kir4.1cKO than in WT because of greater respiratory frequency and tidal volume. Ventilation in acute hypoxia or hypercapnia after CH was not significantly different between genotypes and metabolic rate was similar between genotypes at all O2 levels. The results support a role in astrocytes in the persistent hyperventilation in normoxia with VAH.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Assessing Sex‐Specific Effects of Ozone Exposure on Pulmonary Function and Lung Mechanics
Authors: Fuentes Nathalie , Nicoleau Marvin , Cabello Noe , Silveyra Patricia
Published: June 2021

Background

Exposure to ground‐level ozone, an oxidative pollutant, causes lung inflammation, which can lead to pulmonary injury and impair lung innate immunity. There is emerging evidence that pulmonary diseases such as asthma affect women disproportionally with a greater degree of severity than men. In addition, most hospitalizations for asthma in women occur during the luteal phase or around the peri‐menstrual stage. However, the combined effect of ozone and sex hormones in respiratory mechanics is unclear. Here, we hypothesized that circulating estrogen levels can regulate pulmonary function and lung mechanics following ozone exposure.

Methods

We performed gonadectomy and hormone replacement (17β‐estradiol, 2 weeks) in a group of adult male and female C57BL/6 mice. In control females, the stages of the estrous cycle were monitored by daily by vaginal smear, and confirmed by serum sex hormone levels. We exposed animals to 1 ppm of ozone or filtered air (FA) for 3 hours, and we compared lung function 24h after exposure with a methacholine challenge (MCh; 0 – 50 mg/ml) using the FlexiVent system.

Results

We observed significant changes in respiratory parameters (Ers, H, Rrs, RN, G) in males and females, and in females exposed to ozone at different stages of the estrous cycle, at the two highest MCh concentrations. The pressure‐volume curves obtained demonstrated similar MCh concentration‐dependent changes. Gonadectomized males exposed to ozone had higher Rrs and Ers than females and males exposed to FA, and treatment with estradiol ameliorated these effects. Surprisingly, female mice in the metestrus and diestrus stages exposed to FA had higher Rrs and Ers values than when compared to the proestrus and estrus stages. Contrarily, exposure to ozone caused a decrease in parameters (Ers, Rrs) during metestrus and diestrus stages, but a slight increase of these in the proestrus and estrus stages.

Conclusions

Our results indicate that pulmonary function following ozone exposure can be affected by circulating hormone levels. Future studies examining diseases associated with environmental pollutants should consider the women's menstrual cycle.

Support or Funding Information

The authors thank the Pennsylvania State University College of Medicine Genome Sciences Core Facility for Bioanalyzer analysis. The authors also thank Todd Umstead and Susan Di Angelo for assistance with training and experiments, and Dr. Joanna Floros for the ozone equipment. NIH Grant K01HL133520.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effect of Maternal Protein Restriction on the Surfactant System during Early Postnatal Life
Authors: Khazaee Reza , McCaig Lynda , Huang John , Hardy Daniel , Yamashita Cory , Veldhuizen Ruud
Published: June 2021

Introduction

Intra‐uterine growth restriction (IUGR) is defined by a low birth weight and contributes to a variety of adult‐onset diseases with different severities between males and females. There is some evidence for structural and functional changes to the lung during the post‐natal period. However, the specific changes over time and the effects of IUGR on the pulmonary surfactant system are not fully elucidated. Pulmonary surfactant is critical for normal respiration, it is a lipid‐protein mixture which coats the inside of the lung and maintains the lungs' ability to expand easily during respiration. Therefore, in this study we investigated the effects of IUGR on the surfactant system and lung function during early postnatal life. It was hypothesized that IUGR leads to lung dysfunction through alterations of the pulmonary surfactant system.

Materials and Methods

IUGR was induced in a pregnant rat model via a low protein diet. Briefly, pregnant rats were randomized to a control 20% or a low 8% protein diet. Rats at days 1, 7 and 21 were euthanized, and were either lavaged immediately (day 1) or were analyzed for lung compliance and elastance using a FlexiVent followed by lung lavage (day 7 and 21). Lavage was processed to obtain surfactant which was analyzed via phospholipid‐phosphorus measurements.

Results

Weights were significantly lower in IUGR offspring in all age groups compared to control offspring. Surfactant analysis demonstrated a significant decrease in the amount of phospholipid surfactant of IUGR female offspring compared to control female offspring at day 1 (p<0.01). Also, the surfactant phospholipid content at birth (day 1) for both control and IUGR males and females was significantly higher compared to animals at day 7 and 21 (p<0.01). The results showed that lung compliance in 21‐day female IUGRs (but not male IUGRs) was significantly lower compared to female controls (p<0.01), while elastance was significantly higher for this group compare to female controls (p<0.01). Day 7 male and female offspring showed no difference for compliance and elastance compare to the control groups.

Discussion and Conclusion

IUGR resulted in alterations in surfactant content at day 1 and lung compliance at day 21 in IUGR females compared to control females. This shows that IUGR alters the lung function, and the effect was different between males and females. No changes in the extracellular surfactant content in 21‐day female IUGRs suggests that the lower lung compliance is potentially the result of alterations happened to surfactant biophysical properties under IUGR. These potential alterations will be investigated by assessing the reducing surface tension ability of the surfactant.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Differential Ubiquitination of Profilin‐1 in Hypoxia‐Induced Pulmonary Hypertension
Authors: Zhao Jingru , Wade Brandy , Ma Jing , Hart Charles Michael , Sutliff Roy
Published: June 2021

Pulmonary hypertension (PH) is a chronic illness characterized by increased pressure in the pulmonary arteries due to the thickening and constriction of the arterial walls. Vascular thickening is in part due to proliferation of smooth muscle cells. The ubiquitin proteasome system is an important regulator of cellular proliferation that alters protein stability and expression by modifying lysines (K) within target proteins with ubiquitin moieties. Addition of ubiquitin can alter activity of a protein or mark them for degradation. We hypothesized that hypoxia‐induced changes in ubiquitination impact PH development. Mass spectrometry (MS) was used to screen changes in ubiquitination of proteins in lung tissue from mice exposed to chronic hypoxia (10% O2) for 3 weeks. Profilin‐1, a mediator of actin‐polymerization, had decreased ubiquitination on K54 (Fold change −1.86) and K126 (Fold change −1.40). Profilin‐1 is known to play a role in cellular proliferation and its overexpression leads to increased wall thickness in aortas of spontaneously hypertensive rats. However, its role in PH is not well characterized. Proflin‐1 promotes actin polymerization by binding directly to ADP‐actin and catalyzing nucleotide exchange. Actin polymerization plays a key role in cellular proliferation, cell motility, and muscle contraction. We observed no difference in profilin‐1 protein levels in hypoxia‐exposed human pulmonary artery smooth muscle cells (HPASMC) and lungs from chronically hypoxia‐exposed mice when compared to normoxic controls (P>0.05). This lack of an effect of ubiquitination on protein levels suggests that Profilin‐1 ubiquitination could regulate its activity rather than stability. Preliminary fractionate on results show increased association of Profilin‐1 and G‐actin in hypoxia. Furthermore, disrupting actin polymerization using actin polymerization inhibitors decreased HPASMC proliferation. Therefore, our results indicate that reduced levels of ubiquitinated Profilin‐1 may promote actin polymerization which increases proliferation and contributes to PH. In addition, they suggest that changes in protein ubiquitination may contribute to PH progression.

Support or Funding Information

This work was supported in part by Merit Review funding from the Department of Veterans Affairs, Office of Research and Development (1I01BX001910 to CMH), by NIH Grant R01HL102167 (to CMH and RLS), and by support from NIH T32 HL076118 and American Heart Association 16POST30930007 (to BEW). MS work was performed by the Emory Integrated Proteomics Core.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Fast, transient relaxation of rat pulmonary artery by angiotensin II via AT1R‐eNOS signaling pathways
Authors: Kim Hae Jin
Published: June 2021

Angiotensin II (AngII) is conventionally known as a vasoconstrictive agonist in the large conduit arteries. However, our preliminary study showed that the application of AngII induces only a transient, weak contraction in pulmonary arteries (PAs) followed by persistent desensitization. We have previously reported eNOS expression in pulmonary smooth muscle cells (PASMCs). Here we hypothesized that the concomitant activation of the myogenic eNOS by AngII might limit the contractile responses of PA. Myograph study (isometric contraction of vessel rings) of the rat PAs showed biphasic dose‐response to AngII with peak response at 1 μM. When pre‐treated with L‐NAME (NOS inhibitor) or ODQ (guanylate cyclase inhibitor), the transient AngII‐contraction became augmented and sustained. The AngII‐contraction was abolished by losartan (AT1 receptor antagonist), while neither PD123319 (AT2 receptor antagonist) nor A779 (Mas receptor antagonist) affected the AngII‐contraction. In human PASMCs, eNOS expression was confirmed, and the pro‐activating phosphorylation of Ser 473 in Akt and Ser 1177 in eNOS by AngII treatment was consistently observed by immunoblot. Also, the membrane expression of AT1R was sensitively shifted to the cytosol by AngII. Consistent with the AT1R internalization, the second AngII hardly induced any contractile response in PAs. Interestingly, the desensitization was also significantly removed by the treatment of L‐NAME, immediately after the washout of first AngII application, suggesting the involvement of eNOS signaling pathway in the control of AT1R recycling. Taken together, we proposed that the auto‐inhibitory role of the muscular eNOS in PAs play physiological roles of active relaxation under various vasoconstrictive conditions, preventing excessive contractile response of the low‐pressure pulmonary circulation.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Building a Pedigree: Utilization of Patient Encounters to Strengthen Genetics Foundation
Authors: Morrow Kyle Adam , Carter Ronda F.
Published: June 2021

Integration of basic science knowledge with clinical reasoning skills fosters long‐term retention of material. Evidence shows that active learning increases student performance outcomes in nearly every metric studied. However, resources aimed at teaching educators how to actively engage their students, especially within the medical school curriculum are largely absent. Here, we describe a team‐based learning (TBL) strategy intended to hone both patient interviewing skills and the ability to incorporate patient history and presentation into the construction of a genetic pedigree. In addition to other required courses, our first‐year medical students are enrolled in molecular medicine and primary clinical skills (PCS) courses. One week prior to the TBL event, students learned how to take a patient history and summarize key clinical factors in the PCS course. In the molecular medicine course, students learned basic inheritance patterns (dominant, recessive, mitochondrial, and sex‐linked) and completed a case‐based independent assignment designed to enhance their ability to construct patient pedigrees. Within the TBL event, students interviewed standardized patients with suspected genetic disorders in their family in real time, obtaining a thorough family history as indicated by the patient's clinical presentation. After the interview, students worked within their individual teams to construct a likely patient pedigree. Inheritance pattern, recurrence risks, and proper patient counseling were discussed as a class after evaluating one team's representative pedigree. Three days after the TBL event, students were given their normal molecular medicine exam. In addition to a >10% increase in exam performance on the genetics topics over the previous year where this strategy was absent, the students communicated an increased understanding of how to properly take a patient history and an enhanced awareness of potential genetic influences on the larger clinical picture.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Multi‐facet Application of High Impact Practice‐Personal, School, and Community
Authors: Chakraborty Tandra R.
Published: June 2021

High Impact Practices (HIP) are now a nationally recognized form of instruction that enhances student's engagement, active, cooperative and collaborative learning which makes the learning experience of students fun and memorable. In a Neurobiology course offered to the junior and senior college level students (Biology and Neuroscience majors) some of the active learning strategies that I find most beneficial are, implementation of technology using iPads, application formal debate, role‐playing (dramatic presentation, using technology), group work, clarification pauses, and documentary movies related to the topic of interest, peer survey's and Neuroscience fair. Although all of the mentioned teaching strategies have their own benefits some can have an even broader impact at the personal, school and community level. The ‘Neuroscience Fair’ was one of the most effective and well appreciated by both scholars and students. Students work in groups on assigned projects and research on various complex concepts of Neuroscience e.g. mechanism of Alzheimer's disease and then design a model to explain the project in layman's terms in such a way that middle schoolers can understand the concept. Survey questionnaires are made for middle school students, presenters, and judges. Our survey results indicate a positive correlation between student liking and understanding the concepts of the presentation. 100% of the faculty judges thought the concepts were brilliant and creative. Student presenters found it to be hard work but were extremely positive with their performance and acquired knowledge. The Neuroscience Fair is an attempt to create a personal‐school‐community partnership to enhance community awareness of neurodegenerative diseases and concepts by peer‐assisted learning.

Support or Funding Information

Adelphi University

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.