bluuebunny/crossref_metadata_2025 huggingface dataset:
An Essential Role for Nuclear Factor of Activated T Cells in the regulation of Cyclin D1/CDK4 and Cyclin A2/CDK2 Activities in Vascular Smooth Muscle Cells and their Cell Cycle Progression
Authors: Karpurapu Manjula , Wang Dong , Rao Gadiparthi N
Published: June 2021

PDGF‐BB induced cyclins D1 and A2 expression and CDK4 and CDK2 activities in vascular smooth muscle cells (VSMCs). Interference with activation of NFATs by CsA and VIVIT completely suppressed PDGF‐BB‐induced cyclins D1 and A2 expression and CDK4 and CDK2 activities, resulting in blockade of VSMC in G1 phase. Bioinformatics analysis of cyclins D1 and A2 promoters revealed the presence of NFAT binding elements, and PDGF‐BB induced the binding of NFATs to these elements in CsA and VIVIT‐sensitive manner. Consistent with NFAT binding to their promoter elements, blockade of NFAT activation by CsA and VIVIT suppressed PDGF‐BB‐induced cyclins D1 and A2 mRNA levels. ChIP analysis showed that NFATc1 binds to both cyclins D1 and A2 promoters in response to PDGF‐BB treatment in vivo in CsA and VIVIT‐sensitive manner. Balloon injury (BI) induced cyclins D1 and A2 expression and CDK4 and CDK2 activities in rat carotid arteries and these responses were completely blocked by adenovirus‐mediated transducion of VIVIT. Adenovirus‐mediated expression of VIVIT in the arteries also attenuated BI‐Induced PCNA expression resulting in reduced neointima formation. Together, these findings show for the first time that NFATs play a crucial role in the regulation of cell cycle‐dependent genes, cyclins D1 and A2 both in vitro and in vivo and thereby in injury‐induced vascular wall remodeling. Based on these observations, NFATs may be used as potential target transcriptional factors for the development of drugs against cardiovascular diseases such as restenosis.
The effect of glutathione on the gastric H,K‐ATPase in vitro
Authors: Nagatani Gerald , Okamoto Curtis
Published: June 2021

OBJECTIVE

The gastric H,K‐ATPase is a heterodimeric P‐type ATPase transporter responsible for acid secretion in the stomach. Glutathione (GSH) is an antioxidant that has been shown to regulate the activity of another P‐type ATPase transporter, the sarcoplasmic/endoplasmic reticulum Ca‐ATPase. Here we examine a potential role that glutathione might play in the regulation of the gastric H,K‐ATPase in vitro.

METHODS

Gastric microsomes, purified by standard subcellular fractionation techniques from hog stomach parietal cells, were used in all assays. Para‐nitrophenylphosphate (pNPP) hydrolysis due to H,K‐ATPase activity was measured in the presence of varying concentrations of GSH. GSH‐treated microsomal proteins were separated by SDS‐PAGE and Coomassie‐stained and immunoblotted for the α‐subunit (HKα) or the β‐subunit (HKβ) with their respective monoclonal antibodies. HKα was immunoprecipitated and immunoblotted with a monoclonal GSH antibody. The interaction of glutathione‐S‐transferase (GST) with HKα was tested using a blot overlay assay on microsomal proteins transferred to a nitrocellulose membrane as well as a cosedimentation assay with microsomes and GST.

RESULTS

H,K‐ATPase acitivty as measured by pNPP hydrolysis was inhibited in a dose dependent manner by GSH. Treatment of gastric microsomes with 10 mM GSH resulted in a loss of HKα, but not HKβ, immunoreactivity as shown by Western blots and Coomassie staining. HKα is glutathiolated as shown by immunoblotting immunoprecipitated HKα with the GSH antibody. HKα is bound by GST in overlay assays. GST cosediments with gastric microsomes.

CONCLUSION

Glutathione not only affects H,K‐ATPase activity and HKα protein levels, but also appears to be a post‐translational modification on HKα. GST may bind to HKα via the glutathione modification.
The reactive nitrogen species peroxynitrite is a potent inhibitor of Na,K ‐ ATPase activity
Authors: Reifenberger Matthew Stanton , Milanick Mark
Published: June 2021

Peroxynitrite (ONOO−) is a potent oxidant formed by nitric oxide (NO) reacting with superoxide (O2−). Peroxynitrite modifies tyrosine, cysteine and other residues and affects protein function which might contribute to pathophysiological responses. Peroxynitrite reacts with tyrosine to form 3‐nitrotyrosine which is elevated in a number of diseases. We investigated the effects of peroxynitrite on the Na‐pump and show that a single bolus addition of peroxynitrite inhibited Na, K – ATPase activity with an IC50 of 107 ± 9 uM. To mimic cellular/physiological production of peroxynitrite (chronic, low levels) a syringe pump was used to slowly release (≈0.1 uM / second) peroxynitrite. This treatment was equally effective at inhibiting Na,K ATPase activity as a single bolus addition of equal cumulative concentration. Peroxynitrite led to Na pump nitrotyrosine formation and preliminary data showed modification to cysteines as detected by 5‐(iodoacetamido)fluorescein. Glutathione (GSH) and dithioreitol (DTT) were unable to reverse inhibition. The flavonoid epicatechin which prevented tyrosine nitration enhanced ATPase inhibition. The presence of Na and Mg ATP during peroxynitrite treatment increased the IC50 to 145 ± 10 uM while the presence of K and Mg ATP increased the IC50 to 255 ± 13 uM. This result suggests that the E‐PNa conformation of the pump is more sensitive to peroxynitrite than the E (K) conformation.
Caloxin 1c2‐ A Novel Engineered Selective Inhibitor of Plasma Membrane Ca2+ ‐ Pump Isoform 4, Effects on Coronary Artery
Authors: Szewczyk Magdalena M. , Pande Jyoti , Kuszczak Iwona , Grover Ashok K.
Published: June 2021

Coronary artery smooth muscle expresses two isoforms of plasma membrane Ca2+ pumps: PMCA4 and PMCA1. The previously reported peptide inhibitor caloxin 1b1 was obtained by using extracellular domain 1 (amino acids 115–131) of PMCA4 as the target. Here, we report its mutagenesis by creating a phage display library of caloxin 1b1‐like peptides. The library was screened by affinity chromatography with the PMCA purified from the human erythrocyte ghosts that contain mainly PMCA4 isoform. This yielded caloxin 1c2 with the following key properties: a) Ki = 2.3 ± 0.3 μM which corresponds to a 20x higher affinity for PMCA than caloxin 1b1, and b) it is isoform selective since it has 10x higher affinity for PMCA4 than for PMCA1, 2 or 3. It had the following functional effects on the coronary artery smooth muscle: a) it increased the basal tone of the de‐endothelialized arteries: the increase being similar at 10, 20 or 50 μM, and b) it enhanced the increase in the force of contraction at 0.05 but not at 1.6 mM extracellular Ca2+ when Ca2+‐extrusion via the Na+‐Ca2+‐exchanger and the sarco/endoplasmic reticulum Ca2+ pump were inhibited. We conclude that PMCA4 is pivotal to Ca2+ homeostasis in coronary artery smooth muscle. We anticipate caloxin 1c2 to aid in understanding the role of PMCA4 in signal transduction and Ca2+ homeostasis due to its isoform selectivity and ability to act when added extracellularly.

Supported by Heart & Stroke Foundation of Ontario.
Differential contributions of α and γ ENaC subunit cleavage to channel activity
Authors: Carattino Marcelo D , Maarouf Ahmad B , Hughey Rebecca P , Kleyman Thomas R
Published: June 2021

Maturation of the epithelial sodium channel (ENaC) involves furin‐dependent cleavage at two extracellular sites within the α subunit and at a single site within the γ subunit. Channels carrying mutations at the furin cleavage sites within the α subunit of mouse ENaC (αR205/A, R231/Aβγ) are uncleaved and have very low activity. We recently identified a putative prostasin cleavage site (γRKRK186) that is distal to the furin cleavage site (γRKRK143) in the γ subunit. We also demonstrated that the tract αD206‐R231, between the furin cleavage sites in the α subunit, as well as the tract γE144‐K186, between the furin and prostasin cleavages sites in the γ subunit, are inhibitory domains. ENaC cleavage by furin, and subsequently by prostasin, leads to a stepwise increase in the open probability of the channel as a result of release of α and γ subunit inhibitory tracts, respectively. We used the Xenopus laevis oocyte expression system to examine, at steady state level, the functional contribution of the presence and absence of the α and/or γ inhibitory domains. Co‐expression of either wild type channels (αβγ) or channels carrying mutations in the furin cleavage sites of the α subunit (αR205/A, R231/Aβγ) with prostasin leads to an increase in currents to a similar level. Mutation of both furin and prostasin cleavage sites in the γ subunit impaired prostasin activation of wild type and mutant ENaCs. Oocytes expressing either wild type or furin‐resistant mutant α subunits with furin‐resistant γ subunits lacking the inhibitory tract (αβγR143/AΔE144‐K186 and αR205/A, R231/AβγR143/AΔE144‐K186, respectively) exhibited currents 2–3 fold larger than oocytes expressing wild type channels. Our results indicate that release of the γ inhibitory tract has a dominant effect with regard to activating ENaC.
A high‐throughput assay of Kir1.1 function for molecular library screening: implications for hypertension and Bartter syndrome pharmacotherapy
Authors: Denton Jerod , Lewis L. Michelle , Banerjee Sreedatta , Fallen Katherine , Weaver David
Published: June 2021

Kir1.1 is expressed in the kidney tubule where it promotes fluid reabsorption and thus positively regulates blood volume and pressure. Loss‐of‐function mutations in Kir1.1 cause Bartter syndrome, a renal tubule disorder accompanied by salt and water wasting and hypotension. Selective pharmacological inhibitors and activators of Kir1.1 may therefore be beneficial in management of hypertension and treatment of individuals with Bartter syndrome, respectively. However, the molecular pharmacology of Kir1.1 and other inward rectifier K+ channels is virtually undeveloped. We have therefore developed a fluorescence‐based assay of Kir1.1 K+ channel function for high‐throughput screening of small‐molecule libraries. We demonstrate that the assay is robust, reproducible, DMSO tolerant, fully automatable, and can be used to screen more than 1,500 compounds per hour. With a simple modification, the assay can be used to screen for inhibitors or openers of the pH/K+‐inactivated channel. The latter will be critical for identifying compounds to correct the gating defect induced by several different Kir1.1 mutations identified in Bartter patients. We are currently using the assay to screen a 130,000 member compound library for novel modulators of Kir1.1.
dDAVP increases surface NKCC2 levels via activation of PKA in rat thick ascending limbs
Authors: Caceres Paulo S , Ortiz Pablo A
Published: June 2021

cAMP stimulates NaCl absorption in the thick ascending limb (THAL) by increasing surface NKCC2. Arginine vasopressin (AVP) stimulates NaCl absorption and increases cAMP in the THAL via V2 receptors (V2R). It is not known whether AVP increases surface NKCC2 by activation of cAMP‐dependent protein kinase A (PKA). We hypothesize that stimulation of V2R in the THAL increases surface NKCC2 levels via PKA. We used surface biotinylation and Western blot to measure NKCC2 in the plasma membrane of rat medullary THAL suspensions. We found that 30 minutes stimulation of V2R with dDAVP (1 μM) increased surface NKCC2 by 23 ± 9 % (p <0.05). In the presence of the phosphodiesterase inhibitor IBMX (0.5 mM), dDAVP increased surface NKCC2 by 44 ± 11 % (p <0.05). However, in the presence of the PKA inhibitor H‐89 (10 μM), addition of dDAVP/IBMX did not significantly change surface NKCC2 (basal: 100%, dDAVP/IBMX +H‐89: 108 ± 16%, n = 5). In control experiments, H‐89 alone did not significantly affect surface NKCC2 (basal: 100%, H‐89: 92 ± 4%, n = 6). The total NKCC2 pool was not affected by dDAVP/IBMX (basal: 100%, dDAVP/IBMX: 109 ± 11%, n = 5) or by 10 μM H‐89 (basal: 100%, H‐89: 107 ± 10%, n =5). The intracellular protein GAPDH was not detected in the surface fraction, indicating that intracellular proteins are not biotinylated. We concluded that acute V2R stimulation increases surface NKCC2 levels in the THAL via PKA activation.
Microarray Analysis of Potassium‐Induced Genes in the Mouse Kidney
Authors: Jackson Cynthia Ann
Published: June 2021

The kidney maintains potassium (K) homeostasis in the body by excreting the excess potassium intake rapidly and efficiently. In the process of excreting the excess K‐load, many genes are either activated or deactivated to achieve the goal of maintaining potassium homeostasis. In order to understand some these underlying renal mechanisms involved in K homeostasis, RNA was isolated from mice fed a control diet (1.1 % K, NK) and mice fed a high K diet (5% K, HK) for seven days. Transcriptional profiles of the kidneys from NK and HK mice were compared using mouse Affymetrix GeneChip 430 2.0. Relevant gene expressions were confirmed by real time RT‐PCR. Several genes relating to potassium homeostasis were detected. The genes for the potassium channels increased six‐fold in ROMK (KCNJ‐1), four fold in KCNK‐1 or TWIK‐1, decreased two fold in KCNK‐5 or TASK‐2. Gene expression also increased in other components coupled to potassium homeostasis, such as SGK‐1 (2.5 fold), AQP‐4 (3 fold), RGS‐2 (4 fold), IGF‐2 (9 fold), PRIP (4 fold), GBP3 (6 fold), and CDKN1c (2 fold). This analysis indicates that other physiological functions that are not directly associated with the potassium channel, such as G‐protein and aquaporin‐4 may be required to achieve potassium homeostasis. A more comprehensive analysis is needed.
Mathematical model of interactions between cell volume regulation and transport in cortical TAL cells
Authors: Nieves‐Gonzalez Aniel , Clausen Chris , Layton Harold E , Moore Leon C
Published: June 2021

We developed a mathematical model of a cortical thick ascending limb (cTAL) cell to study interactions between ion transport and cell volume (CV) regulation. Because the NKCC2 transporter mediates apical Na+ uptake, and its activity is regulated by CV‐dependent kinases, we hypothesize that transport‐related CV changes should result in compensatory regulation of NKCC2 activity and Na+ reabsorption. The cTAL model is based on mass conservation and includes models of Na+‐K+‐ATPase, the NKCC2 (A‐isoform) and KCC1 cotransporters, and passive ion fluxes. CV‐sensitive regulation of the NKCC2 and KCC1 activities is represented with empirical functions. Model parameters were chosen to yield potentials, cytosolic [ion], and ionic fluxes consistent with published data. To test our hypothesis, we simulated published experiments which measured the relationship between short‐circuit current (Isc) and luminal [NaCl]. Simulations were done with and without NKCC2 and KCC1 regulation. The agreement between the predicted and measured responses was significantly improved, particularly at lumen [NaCl] > 75 mM, by inclusion of NKCC2 and KCC1 regulation. Moreover, the gain that provided the best fit to the Isc curve yielded regulatory volume decreases consistent with published data. These findings support the concept that CV regulatory responses modulate Na+ transport in cTAL cells.

Supported by NIH grant DK‐42091.
Microinjection of a selective kappa opioid agonist into the supraoptic nucleus (SON) increases inducible cAMP element repressor (ICER) expression
Authors: Gottlieb Helmut , Martinez Michelle , Ji Lisa , Cunningham JT
Published: June 2021

Administration of kappa opioid (κ) agonists in conscious rats produce diuresis via central inhibition of vasopressin (AVP) neurons in the SON and paraventricular nucleus of the hypothalamus, but the molecular mechanism is unknown. Activation of κ receptors produces an inhibition of cAMP accumulation via Gi/o proteins. However, pre‐treatment with pertussis toxin (an inhibitor of Gi/o) fails to block the κ effects in the SON. This suggests that activation of other transcriptions factors may participate in cAMP inhibition (i.e., ICER). This study examined changes in ICER staining following direct microinjection of a selective κ opioid agonist, U50‐488H (U50), into the SON of conscious rats. The forebrain was processed for ICER using polyclonal anti‐ICER antibody raised in rabbit. Microinjection of U50 elicited significant increases in ICER staining in the SON (C, 35 ± 4; U50, 64 ± 5; P < .05). In addition, double labeling shows significant increase in AVP/ICER staining as compared to control (C, 11 ± 1; U50, 22 ± 2; P<.05). This is the first study to show direct activation of ICER by κ. In addition, U50 preferentially increased ICER expression in AVP neurons and not in oxytocin neurons. These results suggest a novel pathway for κ opioid‐mediated suppression of cAMP accumulation, AVP neurons inhibition, and suppression of AVP secretion that is independent of Gi/o. Supported by NIH HL62579 & DK57822.
NO produced by endothelial NO synthase (eNOS) promotes diuresis and natriuresis
Authors: Perez‐Rojas Jazmin Marlen , Beierwaltes William H , Garvin Jeffrey L
Published: June 2021

Vascular NO reduces blood pressure (BP) by decreasing peripheral resistance. Renal NO reduces BP by increasing glomerular filtration and inhibiting water and Na absorption along the nephron. Renal nNOS plays a major role in water and Na excretion, but iNOS's is minor. The role that eNOS plays is unclear. We hypothesized that NO produced by eNOS promotes excretion of water and Na, and this is primarily a renal effect. To test our hypothesis we gave conscious wild‐type (WT) and eNOS knockout (−/−) mice a volume load and measured urinary volume (UV), Na excretion (UVNa) and excretion of osmoles (UVOsm) over 3 hrs. We also measured BP and plasma renin concentration (PRC). To give the volume load we trained mice to drink a large volume of water in 3 hrs while in metabolic cages. On the day of the experiment water was replaced with 1% sucrose, and mice only had access to it for 1 hr. Intake was similar in both groups. Over 3 hrs WT excreted 62 ± 10 % of intake, but eNOS −/− excreted only 42 ± 5 % (p ≤ 0.05). Na excretion was 49.3 ± 7.0 in WT vs. 37.8 ± 6.4 umoles/3 hrs in eNOS −/− (p ≤ 0.05). UVOsm was also decreased by 25% in eNOS −/− (p ≤ 0.05). Pressure natriuresis was not the cause because BP in eNOS −/− was 118 ± 3 compared to 110 ± 2 mm Hg in WT (p ≤ 0.05). PRC was similar in both strains. We conclude that NO produced by eNOS promotes water and Na excretion. This is likely due to renal eNOS because the effects of NO on the systemic vasculature promote Na and water retention.
Seasonal rhythm in neuroendocrine and thermoregulatory system in humans
Authors: Kanikowska Dominika Patrycja , Sato Maki , Inukai Yoko , Shimizu Yuuki , Nishimura Naoki , Iwase Satoshi , Sakurai Hiroki
Published: June 2021

Seasonal variations have been reported in a wide range of physiological parameters. In the present study we investigated the seasonal changes on hormonal and thermoregulatory systems. 8 volunteers were subjected for the experiment at four times of the year (2007) – around the vernal and autumnal equinoxes and the summer and winter solstices. Seasonal changes in the thermoregulatory responses are assessed by measuring core and skin temperatures, sweat rate and blood flow during immersion of the leg in water (mild heat at 40–42°C) for 30 min. The following parameters have been analyzed: metabolites of catecholamine (VMA, HVA, 5‐HIAA), angiotensin II, aldosterone, antidiuretic hormone (ADH), cortisol, ACTH, GH, TSH, fT3, fT4, leptin, IL‐6. Tympanic temperature increased significantly in winter compared with the same condition in spring and summer. Sweating rate and concentration of ADH were significantly higher in summer season compared with winter and spring. Concentration of VMA, ACTH and cortisol were significantly higher in summer compared with other seasons. Concentration of IL‐6 was significantly lower in spring and summer compared with winter. The results of this study demonstrated in ADH, angiotensin II, VMA, Il‐6, and core body temperature and sweat rate with the season statistically significant changes.
Identification and functional significance of a brain aquaporin‐4/Na+, K+‐ATPase/mGluR5 macromolecular complex
Authors: Borisovna Illarionova Nina , Gunnarson Eli , Zelenina Marina , Kamali‐Zare Padideh , Brismar Hjalmar , Bondar Alexander , Zelenin Sergey , Aperia Anita
Published: June 2021

The water channel aquaporin‐4 (AQP4) is expressed in astrocytes, which play a major role in clearing the narrow extracellular space (ES) between neurons and astrocyte endfeet from K+ and glutamate released during neuronal activity. Compelling evidence suggests that AQP4 may play a role in the K+ clearance. Here we show that AQP4 co‐immunoprecipitates with Na+, K+‐ATPase and inwardly rectifying K+ channel Kir4.1; transporters of key importance for the K+ clearance. Using GST pull down assay, we identified the MVAFKGVWTQ of the AQP4 N‐terminus as the site for interaction with Na+,K+‐ATPase. The metabotropic glutamate receptor mGluR5 was also pulled down by AQP4. Using rubidium uptake and astrocyte water permeability measurements, we found that the mGluR agonist DHPG increased both Na+,K+‐ATPase‐mediated K+ transport and AQP4‐mediated water transport. Finally, we created a mathematical model, of K+ transport across the plasma membrane in the ES. This model predicts that AQP4‐driven water transport will facilitate K+ clearance and stabilize the membrane potential. Thus we have demonstrated a multiprotein K+‐transporting microdomain in the astrocyte that includes AQP4. This microdomain is functionally regulated by glutamate and serves to optimize the clearance of K+ from the ES.
Acute dehydration increases tyrosine kinase B receptor (TrkB) phosphorylation in the supraoptic nucleus (SON) of the rat
Authors: Carreno Flavia Regina , Acosta Jessica , Ji Lisa , Cunningham J Thomas
Published: June 2021

Recent studies indicate that the brain neutrophic factor (BDNF) mRNA expression and release is increased in the SON by osmotic stress. The molecular mechanism of action of BDNF and its physiological significance for the vasopressin secretion after dehydration remains to be clarified. Our overall hypothesis is that BDNF release in the SON may produce its responses by phosphorylation of its cognate receptor TrkB. Following 48h water deprivation (48hWD), vasopressin positive neurons in the SON showed increased immunoreactivity for both c‐fos and the phosphorylated form of TrkB (pTrkBY515; abcam, ab51187) as compared to control, euhydrated rats. Brain punches taken from the SON were homogenized in modified RIPA buffer and 5 μg of the total lysate was subjected to Western Blot analysis of both TrkB (abcam, ab18987) and pTrkBY515 expression. Densitometric measurements of the immunoreactive bands for TrkB and pTrkBY515 were normalized using β‐actin as a standard. Although no changes were observed for the TrkB expression, pTrkBY515 expression was increased by 5 fold (P<0.01) in the SON of 48hWD animals compared to euhydrated animals. In conclusion, our data show that increased TrkB phosphorilation in the SON following acute dehydration may play a role in the intracellular signaling associated with vasopressin secretion. The increased TrkB phosphorilation may be at least in part mediated by BDNF. (HL62579)
Identificaiton of Non‐Alcoholic Steatohepatitis (NASH) Using Plasma Metabolome in Humans
Authors: Kalhan Satish C. , McCreedy Bruce , Edmison John , Berger Alvin , Hu Yun‐Fu , Dasarathy Srinivasan , Hanson Richard W. , McCullough Arthur J.
Published: June 2021

The plasma metabolomic profile of subjects with non‐alcoholic fatty liver disease (NAFLD), hepatic steatosis and steatohepatitis (NASH), was examined to identify specific disease‐related patterns and to identify physiological and biochemical perturbations. Plasma samples were obtained after an overnight fast from histologically confirmed subjects with NASH (N=24) or steatosis (N=11), and compared with healthy age and sex‐matched controls (n=25). Subjects with steatosis and NASH were obese and were insulin resistant, as determined by HOMA. Using GC‐MS and LC‐MS, 345 metabolites were detected in the plasma samples, of which 137 compounds were different amongst the groups (p<0.05, q<0.1). Significant differences between NASH and controls were observed for branched chain amino acids, serine, threonine, glutamine, glutamate and aromatic amino acids. Other changes were related to glutathione, purine and fatty acid metabolism. Plasma concentrations of hexanoylcarnitine, cholate and glycocholate were higher in NAFLD when compared with controls. Random forest analysis and recursive partitioning could separate subjects with NASH from controls with an error rate of ∼13%. A recursive partitioning model using body mass index (BMI) and an unnamed compound had an excellent predictive value for subjects with NASH. We conclude that the plasma metabolome can be used to identify subjects with NASH, generate metabolic pathway related hypotheses, and used to follow response to therapeutic interventions without the need for repeat liver biopsies.
Suppression of 11b‐Hydroxysteroid Dehydrogenase Type 1 with RNA Interference Substantially Attenuates 3T3‐L1 Adipogenesis
Authors: Liu Yong , Park Frank , Pietrusz Jennifer L. , Jia Guangfu , Singh Ravinder J. , Netzel Brian C. , Liang Mingyu
Published: June 2021

Overexpression of 11b‐hydroxysteroid dehydrogenase type 1 (11b‐HSD1) in adipose tissue leads to obesity in mice. We hypothesized that endogenous 11b‐HSD1 played a significant functional role in adipogenesis. Stable transfection of 3T3‐L1 cells with small hairpin RNA (shRNA), compared to a nucleotide‐substituted control, silenced 11b‐HSD1 expression and substantially attenuated the accumulation of lipid droplets and the expression of adipogenesis marker genes induced by a mixture containing either corticosterone or dexamethasone. 11b‐HSD1 silencing significantly elevated the concentration of 11‐dehydrocorticosterone in the culture supernatant, but did not affect the levels of corticosterone or dexamethasone, measured by LC‐MS/MS. 11b‐HSD1 silencing significantly attenuated the translocation of glucocorticoid receptors to the nucleus and reduced the number of cells entering the S phase in response to glucocorticoids (13 ± 1% vs. 30 ± 1% in control, n=5, P<0.05), a critical event in the initiation of adipogenesis. 11b‐HSD1 shRNA delivered by lentiviral vectors after the induction of differentiation reduced 11b‐HSD1 expression, but did not affect the further progression of adipogenesis. Our results established a functional and stage‐dependent role of 11b‐HSD1 in the initiation of 3T3‐L1 adipogenesis, and provided new insights into the development of obesity and related diseases.
Proteomic identification of peculiar targets regulated by upstream kinases of Snf1/AMP‐activated protein kinase in yeast Saccharomyces cerevisiae
Authors: Kim Myoung‐Dong , Cho Jea‐Won , Park Eun‐Hee , Chung Byung‐Hee , Kim Jong‐Dai , Lee Hyeon‐Ju , Kim Eun‐Ae , Choe Myoen
Published: June 2021

In budding yeast Saccharomyces cerevisiae, Snf1 (sucrose non‐fermenting) protein kinase of the Snf1/AMP‐activated protein kinase family is required for growth on non‐fermentable carbon sources and non‐preferred sugars. Three kinases, Elm1, Sak1 and Tos3, activate Snf1 by phosphorylation of its activation‐loop threonine in stress condition such as low glucose level. The absence of all three upstream kinases causes the Snf‐ phenotype. In this study, substantial role of each upstream kinase of Snf1 was explored by proteomic analysis of S. cerevisiae strains grown in glucose and glycerol. MALDI‐TOF analysis indicated that each protein kinase regulates expressions of peculiar targets even in non‐stress condition.
Mitochondrial protein import and assembly dynamics in response to chronic contractile activity in skeletal muscle of young and aged animals
Authors: Joseph Anna‐Maria , Huang Julianna , Adhihetty Peter J. , Ljubicic Vladimir , Hood David A.
Published: June 2021

Mitochondrial protein import adapts to perturbations in cellular energy status by altering the rate at which nuclear‐encoded proteins are translocated into mitochondria. The purposes of this study were to determine whether the import and assembly of precursor proteins are similar in muscle from young and aged F344XBN animals, and to assess the effect of chronic contractile activity (CCA) on the import pathway in subsarcolemmal (SS) mitochondria. Import of Tom40 into the outer membrane increased (p<0.05) over time in both young and aged animals, but was not different between the two groups. However, assembly of the multisubunit TOM complex containing Tom40, as assessed by Blue Native‐PAGE, was 2.2‐fold greater (p<0.05) in aged, when compared to young animals. Following 7 days of CCA (3hr/day), Tom40 assembly into the TOM complex was accelerated by 75%, compared to non‐stimulated muscle in young animals. This increase due to CCA was only 65% in aged animals (p<0.05). CCA also resulted in an 85% increase in the import of the matrix protein OCT in SS mitochondria from young animals, while only a 28% increase in OCT import was observed in aged animals (p<0.05). Our findings suggest that aged muscle has a greater capacity to assemble Tom40 into a functional TOM complex, when compared to young animals. CCA can further accelerate the rate of import and assembly in both groups, however this effect is attenuated with age.
Thyroid hormone effect on activity levels and tissue mitochondrial biogenesis and function in young and old rats
Authors: Klaus Katherine A , Walrand Stephane , Morse Dawn , Schimke Jill M , Short Kevin R , Novak Colleen , Levine James A , Nair K. Sreekumaran
Published: June 2021

Triiodothyronine (T3) has a major impact on the regulation of energetics and mitochondrial (mt) function. We determined whether chronic T3 administration alters the age‐related decline in mt function. Ten young (Y, 8 mo) and ten old (O, 27 mo) F344/BN rats were randomized to receive T3 (200 mg/d) or placebo for 14d via subcutaneous osmotic pump. Because OT3 rats decreased food consumption by 39%, we added ten pair fed rats (OPF). T3 treatment caused a significant weight loss (Y, −11.97%; O, −17.49%), while energy expenditure increased in both Y (50.86%) and O (54.40%), but decreased in PF (17.55%). Fat and muscle loss were similar in Y and O in response to T3 (fat, −52.53%(Y) vs −51.69% (O); muscle, −21.50% (Y) vs −21.03% (O) in soleus and −16.70% (Y) vs −17.35% (O) in plantaris), whereas these changes were minor with OPF (fat, −10.4%; soleus −1.69% ; plantaris 7.69%). While mt ATP production and citrate synthase activity in heart and soleus muscle were lower in O, these measures increased equally in Y and O in response to T3. In the soleus, mt DNA abundance increased in Y (54.39%) and O (23.36%) and UCP3 expression increased 10‐fold in both age groups. Gene transcripts levels of PGC1α, COX3&4 and NADH4, UCP2& 3 increased in soleus of T3 rats. In conclusion, T3 similarly increased energy expenditure, weight loss and mt biogenesis in both Y and O rats, despite differences in food intake.

NIH grant PO1 AG09531 and Mayo Foundation
Elevated serum advanced glycation end products and poor grip strength in older community‐dwelling women
Authors: Dalal Mansi Sunil , Ferrucci Luigi , Sun Kai , Fried Linda , Varadhan Ravi , Walston Jermey , Guralnik Jack , Semba Richard
Published: June 2021

Background:

Aging is characterized by loss of muscle strength, but its etiology is not well understood. Advanced glycation end products (AGEs) are bioactive compounds that are implicated in the pathogenesis of diabetes, cardiovascular disease, and renal failure. However, their relationship with skeletal muscle has not been well characterized.

Objective:

The objective of the study was to examine the relationship between serum AGEs, circulating receptor for AGEs (sRAGE), endogenous secretory RAGE (esRAGE), and grip strength in older women.

Methods:

Serum AGEs, sRAGE, esRAGE, and grip strength were measured in 559 women, age ≥65 years, in the Women's Health and Aging Study I.

Results:

The mean (SD) grip strength among women in the highest quartile of serum AGEs compared with the lower three quartiles was 18.2 (6.4) and 20.1 (6.2) kg, respectively (P = 0.004). Women in the highest quartile of serum AGEs had decreased grip strength (beta = −1.31, SE = 0.61, P = 0.03) in a multivariate linear regression model, adjusting for age, race, BMI, cognitive dysfunction, and depression. Serum sRAGE and esRAGE were not significantly associated with grip strength.

Conclusion:

Women with high serum AGEs have greater muscle weakness. Further studies are needed to determine whether AGEs, a potentially modifiable risk factor, are associated with physical performance and disability in older adults.
Prolonged Exercise Training does not Preserve Mitochondrial Enzyme Activity in Senescent Rats
Authors: Betik Andrew C , Thomas Melissa M , Wright Kathryn J , Hepple Russell T
Published: June 2021

Skeletal muscle aerobic function (VO2max) and mitochondrial oxidative capacity decline with aging, particularly in senescence (SEN). Since exercise training improves muscle VO2max and oxidative capacity in young adult skeletal muscle, our objective was to determine if treadmill training from late middle age (LMA) into SEN would minimize the declines in skeletal muscle aerobic function and oxidative capacity seen in SEN. 62 male rats at late middle age (29 mo) were randomly separated into control and exercise groups. Exercise consisted of 60 m/day treadmill running, 5 d/week for 7 weeks and then decreased to 4 d/week for a total of 5 and 7 mo (until 34 and 36 mo of age respectively). Although running capacity was significantly higher in the trained groups (13% at 34 mo and 21% at 36 mo), muscle VO2max at matched muscle O2 delivery was not significantly greater in the trained groups compared to sedentary controls at either age. Similarly, citrate synthase and cytochrome oxidase activity in the plantaris, soleus and the heart muscle were not different in the trained vs the sedentary groups, suggesting no benefit of training for muscle oxidative capacity. The superior exercise capacity suggests a better overall level of function of the senescent rats; however, contrary to our hypothesis and prior observations in LMA rats, treadmill exercise training between LMA and SEN does not benefit muscle aerobic function or mitochondrial oxidative capacity.

Supported by CIHR (AB, RT), CIHR Provincial Training Program in Bone and Joint Health (AB), AHFMR (RT) and NSERC (MT).
Oral Administration of EGCG, an Antioxidant Found in Green Tea, Inhibits Tumor Angiogenesis and Growth of Breast Cancer in Female Mice
Authors: Gu Jian‐Wei , Young Emily , Covington Jordan , Johnson James Wes , Tan Wei
Published: June 2021

Oxidative stress has been implicated in promoting growth and malignancy of cancers including breast cancer. We evaluated whether oral administration of epigallocatechin‐3‐gallate (EGCG), an antioxidant found in green tea, inhibits vascular endothelial growth factor (VEGF) expression, tumor angiogenesis, and growth of breast cancer in female mice. 7‐wk‐old female mice (C57BL) were given EGCG 25mg/50ml in drinking water for 5 wks, which was around 50–100 mg/kg/day. The control mice received regular drinking water. In the 2nd wk, ~1×10^5 E0771 (mouse breast cancer) cells were injected in 4th mammary gland of the mice. Tumor size was monitored by measuring tumor cross section area (TCSA). In the end, the tumors were isolated for measuring tumor weight, intratumoral microvessel (IM) density using CD31 immunohistochemistry staining, and VEGF protein levels using ELISA. Oral EGCG treatment caused the significant decreases in TCSA (66%), in tumor weight (68%), in IM density (155±6 vs. 111±20 IM#/mm^2), and in VEGF protein levels (59.0±3.7 vs. 45.7±1.4 pg/mg) of breast tumors, compared to the control, respectively (N = 8; P<0.01). VEGF plasma levels were lower in EGCG mice than control mice (40.8±3.5 vs. 26.5±3.8 pg/ml; P<0.01). The results suggest that antioxidant treatment with EGCG significantly inhibits breast cancer progression via reducing VEGF expression and tumor angiogenesis. (NIH/AA013821 & NIH/HL 51971)
In vitro acute effects of free fatty acids on protein degradation in young rat skeletal muscle
Authors: Góis Lidiany , Zanon Neusa Maria , Migliorini Renato Helios , Kettelhut Isis do Carmo
Published: June 2021

Previous reports from literature have suggested that plasma free fatty acids (FFA) may reduce the rates of proteolysis, sparing muscle proteins in situations like fasting and diabetes. Here we investigated the acute effects of palmitic acid on total protein degradation and the activity of proteolytic pathways in rat skeletal muscle incubated in vitro. Soleus muscles were removed from Wistar male rats (70–80g) and incubated in Krebs‐Ringer buffer in the presence of palmitic acid (0,3–2,0mM) bound to albumin 1–2%. The rates of total proteolysis and proteolytic pathways were determined by release of tyrosine in the medium. The presence of palmitic acid (1,0mM) induced a clear reduction of total proteolysis (nmol Tyr/mg.2h) (0,340±0,014; 0,242±0,007; n=8), which was accompanied by a 50% reduction in the activity of calcium‐dependent proteolytic process. No changes in lysosomal and ATP‐Ub‐proteasome processes were observed. The inhibition of palmitic acid oxidation (50%) by etomoxir (75μM) and dichloroacetate (7mM) or the presence of 2‐bromopalmitate, a non‐metabolizable analog of fatty acids, did not alter the antiproteolytic effect observed. The present results show that palmitic acid exerts an acute inhibitory effect on total proteolysis by reducing the activity of calcium‐dependent pathway in rat skeletal muscle and this effect seems not depend on fatty acid oxidation.

Financial Support: FAPESP and CNPq
Metformin restores aortic ring responses of obese‐diabetic Zucker rats
Authors: Prasad Raju Y , Yang Wei‐min , Woodlief Tracey L , Cortright Ronald N , Neufer P. Darrell , Cathey Brook , Wingard Christopher J
Published: June 2021

Metformin (MET) has been used to treat diabetes and shown to reduce blood pressure, restore endothelial function and microvascular reactivity in diabetic animal models. However, MET's effect on vascular reactivity in obese‐diabetic rat has not been studied. We sought to determine the effects of MET on male Zucker rat aortas divided into lean and obese‐diabetic controls (LC, OC) or metformin treated (LM, OM). Aortic rings were subjected to cumulative dose response protocols for phenylephrine (PE) and acetylcholine (Ach), norepinephrine (NE), serotonin (5‐HT) and a rho kinase inhibitor (H1152P). The PE response decreased with MET treatment in the obese‐diabetics (EC50 OM = 226.4, OC = 94.9 nM) and improved the Ach response (OM = 29.4, OC = 43.8 nM). NE showed a decreased sensitivity in the obese‐diabetic (OM = 5.6, OC = 2.2 nM) while MET caused an increase in sensitivity in the leans (LM = 0.9, LC = 7.6 nM). The H1152P response was improved with MET (LM = 1.2, LC = 0.9, OM = 1.0, OC = 0.5 uM). We conclude that MET restored aortic responsiveness from the obese‐diabetic through improvement of endothelial dependent relaxation and reversal of contraction by Rho‐kinase. Supported NIH DK061314 and ECU.
Central and peripheral leptin treatment produce similar increase in muscle and bone mass in ob/ob mice
Authors: Bartell Shoshana M , Gaddam Dhanunjaya R , Ambati Suresh , Hartzell Diane L , Hamrick Mark , She Jin‐Xiong , Della‐Fera Mary Anne , Baile Clifton A
Published: June 2021

Leptin administered either centrally or peripherally reduces body weight (BW), food intake (FI), & body fat in leptin deficient ob/ob mice. However, there are contradictory findings when assessing effects of leptin on bone mass in rodents. The objective of this study was to determine the effects of peripheral & central administration of leptin on bone metabolism in the axial & appendicular skeleton in ob/ob mice (15 wk old, init. BW=61.3g). Leptin was delivered subcutaneously (SQ, 0 or 10μg/d) or intracerebroventricularly (ICV, 0 or 1.5μg/d) via osmotic pumps for 12 days (n=10). There were no significant differences between ICV and SQ leptin. Regardless of mode of administration, leptin decreased BW (51.0 vs 62.2g, p=0.003; mean of leptin treatments vs controls), FI (3.85 vs 5.69g, p=0.03), body fat (46.6 vs 56.0g, p=0.001), & relative total fat pad wt (13.1 vs 12.9g/g BW p=0.02) & increased gastrocnemius muscle wt (97.8 vs 110.4mg, p=0.01), relative muscle mass (242.7 vs 173.4mg/g BW, p<0.01), bone mineral density (0.0554 vs 0.0510g/cm2, p=0.001), & bone mineral content (0.418 vs 0.376g, p=0.02). These results show that both modes of leptin administration increased bone & muscle mass equally in ob/ob mice. Supported by the Georgia Research Alliance (GRA) Eminent Scholar endowment (CAB) & a GRA Challenge Grant (CAB & JXS).
Development of Diabesity in Mice with Pan‐Neuronal Deletion of Shp2
Authors: Krajewski Stan , Banares Steven , Huang Xianshu , Scadeng Miriam , Jhala Ulupi S. , Feng Gen‐Sheng , Zhang Eric E. , Krajewska Maryla
Published: June 2021

Obesity and diabetes are increasingly serious health problems. However, molecular mechanisms and neuronal regulatory role in the metabolic disorders are not fully understood. We show here that Shp2, a widely expressed Src homology 2‐containing tyrosine phosphatase, indeed plays a critical role in adult brain controlling food intake, energy balance and metabolism. Conditional mutant mice with Shp2 selectively deleted in neurons, generated by crossing a pan‐neuronal Cre‐line (CRE3) with Shp2flox/flox mice developed obesity and diabetes associated with pathophysiological complications that resemble those encountered in humans, such as hyperglycemia, hyperinsulinemia, hyperleptinemia, insulin and leptin resistance, vasculitis, nephropathy, urinary bladder infections, prostatitis, gastric paresis, and impaired spermatogenesis. This mouse model may help to elucidate molecular mechanisms that lead to diabesity in humans. It also provides a tool to study in vivo complications of uncontrolled diabetes. Supported by the NIH grants: NS36821 for SK and DK73945 to GSF
The effects of nesfatin‐1, an anorexigenic peptide, on blood pressure and lights out locomotor activity
Authors: Yosten Gina Louise Cecilia , White Meghan M. , Samson Willis K.
Published: June 2021

Here we describe the effects of Nesfatin‐1 (NES) on mean arterial blood pressure and locomotor activity in normotensive rats. Cannulas were inserted into the right lateral ventricle of male rats. After 5 days, the left ascending carotid artery was cannulated with PE‐50 tubing. The next day, mean arterial pressure (MAP) was recorded in conscious, unrestrained animals for 30 minutes following intracerebroventricular (i.c.v.) injection of 20, 60, or 180 pmol NES or saline. For behavior, naïve rats with i.c.v. cannulas were habituated in a behavior box (Opto‐Max, Columbus Instruments) for 1 hour (16:00). At 17:00, 20, 60, or 180 pmol NES or saline was administered. The lights were turned off at 18:00 and activity was recorded until 19:00.

There was an initial significant increase in MAP after i.c.v. injection of all three doses of NES which lasted approximately 15–20 min. Behavioral readings indicated that the rats were very active in the initial 15 minutes after i.c.v. injection of NES, but then entered a phase of inactivity, which lasted until Lights Out, when they became active again. The highest dose of NES induced a state of almost complete inactivity, which lasted beyond Lights Out. These results indicate that the rise in MAP could reflect the initial behavioral activity. Furthermore, the anorexigenic effect of NES may also be secondary to its action to reduce spontaneous locomotor activity.
Hypothalamic a‐MSH is increased in male rats fed a high soy protein diet
Authors: Doane Dolores Fernandez , Barnes Meredith Blythe , Heppner Kristy , Vallabh Anuja , Beverly J. Lee
Published: June 2021

The hypothalamus is actively involved in energy homeostasis, including food intake regulation. Increased activity of the melanocortin system within the mediobasal hypothalamus (MBH) may facilitate reduced food intake. The effectiveness of high protein diets in weight loss may be through an increase in satiety. The objective of this study was to characterize changes in the hypothalamic melanocortin system in the MBH of male Sprague‐Dawley rats fed a high protein diet (HP; 50% protein: 34% CHO: 16% fat) versus an adequate protein diet (C; 15:69:16). The concentration of a‐melanocyte stimulating hormone (a‐MSH), a known anorexigenic neuropeptide in the MBH, was determined following 10 days on the high protein diet. The whole brain was removed, snap‐frozen in ice‐cold isopentane and stored for analysis. The MBH was dissected out, homogenized and a‐MSH levels measured by RIA. Animals on the HP diet consumed an average of 77.9 ± 2.5 kcal/d, 12.4 ± 3.8 kcal/d less than the control (p < 0.01) and α‐MSH was 15.2 ± 1.0 pg/mg protein in animals on the HP diet, 3.0 ± 1.3 pg/mg higher than control (p < 0.05). Increased levels of a‐MSH in the MBH of rats chronically fed the HP diet are consistent with increased satiety and subsequent reduced food intakes as compared to their low‐protein‐fed counterparts. Supported by APS Porter Physiology Fellowship Award.
Sympathetically mediated energy expenditure in subjects who have had gastric bypass surgery
Authors: Curry Timothy Brian , Somaraju Madhuri , Vaa Brianna E. , Roberts Shelly K. , McKenzie Jeffery A , Eisenach John H. , Miles John M. , Joyner Michael J.
Published: June 2021

The physiological changes that occur after gastric bypass surgery (GBS) are not well understood but changes in activation of the sympathetic nervous system may be involved. We hypothesized that GBS would be associated with greater sympathetically mediated resting energy expenditure as measured by a greater decrease in REE after beta‐blockade (beta‐REE). Subjects who had undergone GBS (average reduction in BMI of 31.6% from peak, N=7) along with lean (BMI<25 kg/m2, N=5) and obese (BMI=35–45 kg/m2, N=4) subjects underwent measurements of REE before and after IV propranolol (0.25 mg/kg bolus followed by an infusion of 0.004 mg/kg/min). The decrease in REE (mean±SD) after beta‐blockade tended to be greater in lean (102±58 kcal) compared with obese (−22±100 kcal) and GBS (26±55 kcal) subjects (P=0.06). The decrease in REE after beta‐blockade negatively correlated with the % body fat (r=0.54, P<0.05) and VO2max (r=0.67, P=0.01) of the subjects, but not with resting catecholamine or leptin levels. We did not find that subjects who had undergone GBS had a significant difference in the amount of their REE that is dependent on sympathetic stimulation. Beta‐REE appears to be related to fitness and also to fatness and the fact that GBS subjects had high body fat even after significant weight loss may explain our results. Support: NIH HL46493, NIH CTSA UL1‐RR24150, Department of Anesthesiology.
Kv1.3 channels in postganglionic sympathetic neurons: Modulation by preganglionic and vascular target interactions
Authors: Damon Deborah Heins , Doczi Megan A. , Morielli Anthony D.
Published: June 2021

The sympathetic nervous system is an important modulator of cardiovascular function. We have shown that Kv1.3 channels, affect neurotransmitter release from postganglionic sympathetic neurons. The present study tests the hypothesis that preganglionic and target interactions modulate the function of Kv1.3 channels in postganglionic sympathetic neurons. Electrophysiological function (whole cell patch clamp recordings) and neurotransmitter release (nicotinic agonist‐induced tritiated norepinephrine release) were assessed in the presence and absence of margatoxin (MgTX; 100 μM), a specific inhibitor of Kv1.3. Electrophysiological analyses indicate MgTX decreased tetraethylammonium‐insensitive voltage‐dependent outward currents in the absence (p < 0.05; unpaired t‐test; n = 12) but not in the presence of bethanechol, a muscarinic acetylcholine receptor agonist (100 μM). Neurotransmitter release assays indicate that MgTX enhanced neurotransmitter release in the absence (p < 0.05; unpaired t‐test; n = 10) but not in the presence (n = 6) of vascular target interactions. These data indicate that both preganglionic and target interactions modulate the function of Kv1.3 channels in postganglionic sympathetic neurons and suggest novel mechanisms for modulating the activity of the sympathetic nervous system. HL076774 and NS050623.