bluuebunny/crossref_metadata_2025 huggingface dataset:
The MBL‐complex is necessary for FeCl3‐mediated thrombosis
Authors: Pavlov Vasile I. , La Bonte Laura , Takahashi Kazue , Stahl Gregory L.
Published: June 2021

Occlusive thrombosis, resulting from atherosclerotic plaque rupture or restenosis plays an important role in the onset of two major causes of death in developed nations: myocardial infarction and ischemic stroke. It is well‐known that the complement and coagulation systems interact, however no studies have investigated the specific role of the lectin complement pathway in thrombus formation. Thus, the purpose of this study was to examine the role of the complement system, specifically the mannose‐binding lectin (MBL) complex, in a mouse model of ferric‐chloride (FeCl3)‐induced thrombosis. For this model, 2 strips of Whatman paper were soaked in 3.5% FeCl3 and applied to either side of the exposed right carotid artery for 3min then removed. Blood flow was measured at baseline, during FeCl3 administration, and for 30min following FeCl3 exposure using a Doppler flow probe positioned distal to the FeCl3 application. Using wild‐type (WT) and C2/fB−/− mice, we observed occlusive thrombosis of the carotid artery 10min after FeCl3 administration. In contrast, MBL−/− mice had significantly decreased FeCl3‐induced thrombosis formation compared to WT or C2/fB−/− mice (P<0.05). In conclusion, our study provides evidence for a significant role for the MBL‐complex in FeCl3‐induced coagulation and may lead to the development of improved anti‐thrombotic therapies. Supported by HL56086, HL92469 and HL99130.
The role of β‐carotene and its cleavage enzyme β‐carotene‐ 15,15′‐oxygenase (CMO1) during mammalian embryonic development
Authors: Kim Youn‐Kyung , Wassef Lesley , Chung Stacey , Wyss Adrian , Blaner William S.
Published: June 2021

The mammalian developing fetus relies on maternal circulating retinoids (vitamin A and its derivatives) to support its elevated vitamin A requirements. Β‐carotene is the major precursor of vitamin A in the human diet, and the central cleavage enzyme, β‐ carotene‐15,15′‐oxygenase (CMO1) has been suggested as the main enzyme involved in the production of vitamin A from β‐ carotene in vivo. To study the role of β‐carotene as vitamin A precursor during development we performed loss of CMO1 function studies in mice lacking retinol‐binding protein (RBP), an established model of embryonic vitamin A deficiency. Embryos from dams lacking both CMO1 and RBP (CMO1−/−RBP−/−) bred on a vitamin A‐deficient diet were malformed, but their defects were more severe than those of RBP−/ − mice. Since no differences were observed in serum retinoids levels between CMO1−/ −RBP−/ − and RBP−/ − dams, the embryonic malformations were likely due to the lack of CMO1 in the developing tissues. Upon maternal administration of β‐carotene, defects were almost fully rescued in CMO1+/−RBP−/ − embryos, while only partially improved in CMO1−/ −RBP−/ − embryos. These data suggest that CMO1 is a key enzyme in the conversion of β‐carotene to vitamin A in the developing tissues. However, they do not rule out a contribution of the β‐carotene asymmetric cleavage enzyme, β‐carotene‐9,10′‐ oxygenase (CMO2), to this process.

Grant Funding Source: NIH 5R01HD57493
Nephrolithiasis in the Wpk/+ Rats
Authors: Gattone Vincent , Williams James , Bledsoe Sharon , Eggleston‐Gulyas Tracy , Miller Caroline , Moe Sharon , Evan Andrew
Published: June 2021

Nephrolithiasis affects over 10% of Americans and 10–15% of stones are calcium phosphate (CaP). CaP stone disease is poorly understood. Male Brown Norway Wpk/+ rats (12–18 months old, heterozygote carriers of a mutation for Meckel Syndrome) develop renal calcium deposits and pelvic stones. We evaluated kidney and stone morphologies from old BN‐Wpk/+ rats; kidneys were evaluated by light microscopy, microCT and infrared spectroscopy (FT‐IR). Renal calcification was commonly associated with hypertrophic papillary epithelium, and associated with the apical surface, not classic Randall's plaques. Additionally, kidney stones were found in pelvis and pelvic fornix in most rats, some of which were adherent to the papillae while others were free floating. Micro CT analysis of adherent kidney stones identified the attachment site surrounded by multiple laminations, suggesting slow growth over time—similar to human kidney stones. FT‐IR showed amorphous CaP as the primary mineral component. Rodent models of spontaneous kidney stone disease are few; this model is linked to a gene mutation and therefore could provide insight into the pathogenesis of CaP nephrolithiasis.
Microfluidic Platform for Reproducing Blood Vessel Microenvironment
Authors: Song Jonathan W. , Munn Lance L.
Published: June 2021

The critical determinants of blood vessel physiology – cell‐cell interactions, cell‐matrix interactions and blood flow – are difficult to control or monitor in vivo. We have developed a microfluidic platform for studying blood vessels in vitro featuring a unique combination of: (i) controllable flow rates, both tangential and transverse to the endothelial monolayer, (ii) 3‐D extracellular matrix localized to a defined region of the system, and (iii) spatiotemporally defined growth factors, cytokines or inhibitors. Consisting of endothelial‐lined channels adjacent to collagen gel, our capillary analogs support fluid flow and allow assessment of how endothelial cells collectively determine vessel physiology and morphogenesis. Research support: T32CA073479, R21CA12676‐02, and R56HL064240‐09A2.
Antidiabetic activities of Abutilon indicum Sweet mediated by enhancement PPARγ and activation of the GLUT1 promoter
Authors: Krisanapun Chutwadee , Lee Seong‐Ho , Peungvicha Penchom , Temsiririrkkul Rungravi , Baek Seung Joon
Published: June 2021

Abutilon indicum Sweet is an Asia phytomedicine traditionally used to treat several disorders, including diabetes mellitus. However, molecular mechanisms supporting the antidiabetic effect of A. indicum Sweet remains unknown. Since a tremendous increase in the number of individuals with type 2 diabetes mellitus worldwide and insulin resistance is a major pathogenic factor for type 2 diabetes. The aim of this study was to evaluate whether extract of A. indicum Sweet improves insulin sensitivity. First, we observed the antidiabetic activity of aqueous extract of A. indicum on postprandial plasma glucose in diabetic rats. The subsequent experiments revealed that butanol fractions of the extract bind to PPARγ and activate 3T3‐L1 differentiation. To measure glucose uptake enhanced by insulin‐like activity, we used rat diaphragm incubated with various concentrations of the extract and found that the extract enhances glucose consumption in the incubated solution. Our data also indicate that the extract and the fractions did not affect the activity of kinases involved in Akt and GSK‐3β pathways; however, the reporter assay showed that the crude extract could activate glucose transporter 1 (GLUT1) promoter activity. These results suggest that the extract from A. indicum Sweet may be beneficial for reducing insulin resistance through its potency in regulating adipocyte differentiation through PPARγ agonist activity and increasing glucose utilization via GLUT1.
L‐4F Improves Metabolic Syndrome Phenotype in HO‐2 Null Mice by Decreasing NFkB Activity & Increasing Adiponectin Levels
Authors: Palladino Leah Marie , Burgess Angela , Vanella Luca , Kim Dong Hyun , Sodhi Komal , Peterson Stephen J. , Abraham Nader G. , Mensah‐Osman Edith
Published: June 2021

We hypothesized that L‐4F, an apoA‐I mimetic peptide, may reduce adiposity and oxidative stress in mice lacking heme oxygenase 2 (HO‐2), which display the metabolic syndrome, leading to the amelioration of insulin resistance. Mice were divided into four groups: Wild‐type (WT) vehicle treated, WT treated with L‐4F (I.P., 2mg/kg daily), HO‐2 null vehicle treated, HO‐2 null treated with L‐4F. In null mice there was limited weight gain, decreased visceral and subcutaneous fat content, decreased plasma TNFα and IL‐6 levels, and increased insulin sensitivity compared to WT controls. We further examined the effect of L‐4F on inflammation by measuring levels of NFκB activity through measurement of p65 and IκBα. The levels of p65 remained the same in both the control and HO‐2 null mice, but there were increased levels of phosphorylated p65 in the nucleus which was reversed in the mice treated with L‐4F. IκBα levels were decreased in HO‐2 knockout mice, but levels were restored with L‐4F treatment implying decreased NFκB activity. In addition, L‐4F treatment increased aortic HO activity and decreased aortic dysfunction. The decrease in adiposity by L‐4F treatment was associated with an increase in serum adiponectin levels. The action of L‐4F decreasing adiposity and increasing insulin sensitivity in an oxidative stress model of metabolic syndrome suggests that L‐4F offers promise in the management of the metabolic syndrome.
Modulation of inflammatory pathway in insulin sensitive tissues of normo and hyperphagic obese animals.
Authors: Quadros Caren Dal'Mora , Alves Eduardo Silva , Haidar Andre Abou , Morgan Daniela , Rocha Marlene Santos , Carpinelli Angelo Rafael , Hirata Aparecida Emiko
Published: June 2021

Obesity is characterized by the activation of an inflammatory process in metabolically active sites such as adipose tissue and liver. The consequence of this response is an increase proinflammatory markers. This activation response in obesity is mediated by some specific signaling pathways including kinase families such as JNK, MAPK and IKKb. It is known that these molecules can lead to impairment of insulin signaling and result in the development of insulin resistance. The aim of this study was to evaluate the expression of these molecules in different models of obesity. Liver, muscle and adipose tissue from MSG‐obese rats (normophagic) and High‐fat induced obesity‐HF (hyperphagic) were evaluated. MSG‐obese rats showed significant increase in phosphorylation of ERK1/2 in liver and adipose tissue while HF‐rats showed a decreased in phosphorylation of this kinase in adipose tissue. Preliminary data also showed a differential modulation of p38MAPK, JNK and IKKb of these models. It is possible that the different energy load condition may have an important role in the signaling pathways of these molecules associated with the development of obesity‐induced inflammation.
Vascular ACE and eNOS Expression in Syrian Cardiomyopathic Hamsters
Authors: Cruz Nildris , Quidgley Jose , Escobales Nelson , Miranda Jorge , Altieri Pablo I , Crespo Maria J
Published: June 2021

Angiotensin Converting Enzyme (ACE) and endothelial Nitric Oxide Synthase (eNOS) have been implicated in the pathophysiology of heart failure. We evaluated the time‐course expression of ACE and eNOS in aortic tissue from 1‐ to 4‐month‐old Syrian cardiomyopathic hamsters (SCH) to correlate these variables with vascular status. The expression of both genes in 2‐month‐old SCH after one month of treatment with the antioxidant N‐acetylcysteine (NAC, 1g/kg/d) and the β‐blocker‐antioxidant carvedilol (1mg/kg/d) was also evaluated. Age‐matched golden hamsters were used as controls (CT). Total RNA was extracted and gene expression was quantified by Real‐Time RT‐PCR. Our results showed up‐regulation in ACE (1.5 fold) and eNOS (4‐fold) expression in 2‐month‐old SCH when compared to 1‐, 3‐ and 4‐month‐old SCH and to CT. In 2‐month‐old SCH, carvedilol down‐regulated eNOS and ACE gene expressions, whereas NAC down‐regulated eNOS expression without modifying ACE expression. These results suggest that the improvements in CV function observed previously in SCH after early administration of NAC or carvedilol may involve modifications in the expression of ACE and eNOS genes. Supported by NIH‐SCORE S06‐RR 08224.
Identification of glucocorticoid response elements in the rat angiotensin II receptor (AT2R) promoter
Authors: Dasgupta Chiranjib , Xue Qin , Zhang Lubo
Published: June 2021

Objective

Identification of molecular mechanism responsible for glucocorticoid induced down‐regulation of angiotensin II receptor (AT2R) gene expression.

Methods

Transcription of AT2R is repressed by glucocorticoids. To understand the molecular basis of glucocorticoid effect on AT2R, we amplified a 2130 base pair promoter region of rat AT2R, and cloned it in‐frame to pGL3‐Luciferase repoter (wild‐type AT2R‐pGL3‐Luc) into KpnI/XhoI sites. In silico analyses of this region of AT2R promoter identified several putative glucocorticoid response elements (GRE). Several constructs were created out of the wild‐type AT2R‐pGL3‐Luc, by deleting one putative GRE at a time.

Results

Reporter gene assay in H9C2 rat cardiomyocytes using above mentioned constructs identified that deletion of eight, (but not all of the presumptive GREs) augmented Luciferase activity. Authenticity of these GREs was confirmed by Electrophoretic Mobility Shift Assay (EMSA), and by competition experiments with a rat consensus and AT1a‐R GRE oligos. Immunological identity of the protein causing gel‐retardation was shown to be glucocorticoid receptor (GR) in supershift analysis using anti‐GR antibody.

Conclusions

Our study confirmed presence of eight GRE on AT2R promoter. Further studies are underway to identify physiological processes that use these GREs for regulation of AT2R expression.
Chronic angiotensin II (Ang II) increases renal oxidative stress in male spontanously hypertensive rats (SHR), but not in female SHR
Authors: Brinson Krystal N. , Sullivan Jennifer C.
Published: June 2021

Male SHR are more sensitive to Ang II‐induced increases in blood pressure than females. Ang II‐induced oxidative stress contributes to hypertension in males; less is known regarding Ang II‐induced oxidative stress in females. We hypothesize female SHR have less Ang II‐induced oxidative stress compared to males. 13 week old control and Ang II infused (200 ng/kg/min, 14 day minipump) male and female SHR were studied (n=3–4). The renal cortex was isolated to measure O2·− levels via lucigenin. Males had higher O2·− levels than females (see figure, * indicates p<0.05). Ang II increased O2·− levels in males(+ indicates p<0.05) yet levels were similar in females and female + Ang. Renal cortical NADPH oxidase NOX4 and p22phox subunits, SOD and catalase protein expression were assessed. p22phox expression was comparable in males (4.0±0.2 rdu) and male + Ang (4.0±0.2 rdu), however, Ang II tended to decrease p22phox in females (female: 4.0±0.3 rdu; female + Ang: 3.4±0.1 rdu, p=0.07). Ang II decreased ECSOD expression in males (male: 24±1; male + Ang: 20±1 rdu, p<0.05), with no effect in females (female: 20±2; female + Ang: 22±1 rdu). NOX4, CuZn SOD, MnSOD, and catalase expression were comparable among groups. Ang II infusion increased oxidative stress in male, but not female SHR. We hypothesize that this is related to increased antioxidant potential and lowered NADPH oxidase activity in female SHR with Ang II infusion. Funded by NIH.
Sex differences in estrogen receptor mediation of vasopressin synthesis and release in rats chronically exposed to alcohol
Authors: Andaya January May , Hernandez Claudia A. , Sato Aileen K. , Uyehara Catherine F.T.
Published: June 2021

We tested the hypothesis that estrogen receptor (ER) mediation of vasopressin (VP) synthesis or release from pituitary stores can be altered by alcohol exposure. Thus, pituitary VP levels and VP mRNA expression in alcohol‐exposed (7% ethanol in 80 ml/day liquid diet for 4 weeks) rats were compared with that in control rats. In control females (n=10), pituitary VP content and hypothalamic VP mRNA correlated with ERα (r=0.73, p≤0.05) and ERβ (r=0.86, r=0.82, p≤0.01). These relationships seem to be disrupted in alcohol fed females (n=10). Also, pituitary VP content and VP mRNA were not linked in all females indicating that there were adequate VP pituitary stores in both alcohol fed and control rats. Control males (n=8) did not exhibit significant relationships between ERα or ERβ and pituitary VP or VP mRNA. However, a negative relationship between pituitary VP and VP mRNA (r=0.93, p≤0.01) was evident in alcohol fed males (n=6), which perhaps corresponds to their greater need for increased VP release to keep up with increased renal water losses. Also, in alcohol fed males, it appeared that the influence of ERβ became unmasked with a tendency for pituitary VP to be negatively linked with ERβ only when VP synthesis was tightly coupled to pituitary VP stores. Overall, results suggest that sex differences in ER mediation of VP regulation may be involved with alcohol‐induced water balance differences in males and females.
Progesterone administration antagonizes the effect of estradiol on endothelium‐dependent vasodilation in young healthy women
Authors: Miner Jennifer Ann , Smith Michael M , Martini Emily R , Brunt Vienna E , Kaplan Paul F , Halliwill John R , Minson Christopher T
Published: June 2021

Purpose

To examine the acute effects of progesterone and estrogen on endothelium‐dependent vasodilation in healthy reproductive‐aged women.

Methods

We suppressed endogenous estrogens and progesterone in 16 premenopausal women for 10 days using a gonadotropin‐releasing hormone antagonist (GnRHa). On day 4, subjects were tested and then supplemented either 0.1 mg estradiol (GnRHA+E2; N=8) transdermally or 200 mg progesterone (GnRHa+P4; N=8) orally per day. On day 7 subjects were tested and began supplementation with both hormones (GnRHa+P4+E2), and were tested again on day 10. Flow‐mediated vasodilation (FMD) of the brachial artery was assessed using B‐mode arterial ultrasound, combined with synchronized Doppler analysis.

Results

Significant differences were observed when comparing FMD in GnRHa (7.85 ± 2.77%) and GnRHa+E2 conditions (10.14 ± 1.40%; p<0.05). The E2 increase was abolished when progesterone was also supplemented (6.27 ± 2.41%). In contrast, GnRHa+P4 (6.66 ± 2.56%) did not significantly alter FMD from GnRHa (7.80 ± 2.76%) and GnRHa+P4+E2 (7.40 ± 3.02%). However, a multi‐level prediction model demonstrates progesterone (p<0.02) and estradiol (p<0.01) predict FMD within subjects.

Conclusion

These data suggest that acute progesterone administration antagonizes the effect of estrogen on endothelium‐dependent vasodilation.

Supported by NIH Grant HL081671.
Role of Transient Receptor Potential (TRP) Channels in Cyclic ADP‐Ribose (cADPR)‐Mediated Sex‐Differences in Agonist‐Induced Contraction of Rat Aorta
Authors: Guedes Alonso G.P. , Stallone John N.
Published: June 2021

cADPR signaling is emerging as a significant mechanism underlying sex differences in agonist‐induced vasoconstriction. Here we investigated the role of TRP‐channels in cADPR‐mediated sex differences in vasopressin (VP)‐ and phenylephrine (PE)‐induced contraction of male (M) and female (F) rat aorta. Isometric tension was measured in endothelium‐denuded aortic rings from age‐matched, sex intact, M and F SD rats (n=6–10/group). Maximal responses (mean ± SE) to VP in M (3.3 ± 0.2 g/mg dry tissue) and F (4.1 ± 0.2), respectively, decreased by 53% (1.5 ± 0.3) and 0.1% (4.0 ± 0.3) with cADPR antagonist 8‐Br‐cADPR (8‐Br; 30 μM); by 56% (1.4 ± 0.1) and 39% (2.4 ± 0.4) with TRP‐channel antagonist SKF‐96365 (SKF; 15 μM); and by 64% (1.2 ± 0.3) and 53% (1.7 ± 0.3) with both 8‐Br and SKF. Maximal responses to PE in M (4.2 ± 0.2) and F (3.9 ± 0.2), respectively, decreased by 16% (3.5 ± 0.1) and 26% (2.9 ± 0.3) with 8‐Br; by 38% (2.6 ± 0.2) and 33% (2.6 ± 0.2) with SKF; and by 59% (1.7 ± 0.3) and 51% (1.9 ± 0.4) with both 8‐Br and SKF. Thus, the cADPR contribution to VP‐induced contraction in M is markedly TRP channel‐dependent. VP‐induced contraction of F aorta is noticeably less dependent on TRP‐channels and almost entirely cADPR‐independent. cADPR contribution to PE‐induced contraction appears to be independent of TRP channels in both M and F. Hence, the role of TRP‐channels in cADPR‐dependent contraction of rat aorta is both agonist‐ and sex‐specific.
Laboratory evaluation of the accuracy of a triaxial accelerometer embedded into a cell phone platform for measuring physical activity
Authors: MANOHAR CHINMAY , CRADY SHELLY MC , Fujiki Yuichi , Pavlidis Ioannis , Levine James
Published: June 2021

Background

Physical activity is important in obesity, metabolic and cardiovascular diseases and cancer prevention. Several cell phone platforms integrate an accelerometer onto the motherboard. We wrote a software application to access the on board accelerometer from the iPhone (Apple Computer, Cupertino, CA) to measure and quantify physical activity. Here we tested the validity of the cell phone accelerometer to assess physical activity in a controlled laboratory setting.

Methods

31 subjects wore the cell phone along with the validated Physical Activity Monitoring System (PAMS) with different body postures and during graded walking at 7 velocities. Energy expenditure was measured using indirect calorimetry. Data were also collected from 7346 users who downloaded the “Walk n’ Play” application from the internet.

Results

In all the 31 subjects, the cell phone accelerometer distinguished sedentary and walking activity reliably even with ½ mph increments in walking and was accurate and precise compared to the PAMS, with an intra‐class correlation coefficient (r2> 0.98). The cell phone accelerometer showed excellent sequential increases with increased in walking velocity and energy expenditure (r2>0.9). The data from the 7346 free‐living people demonstrated that most daily activity detected with the cell phone accelerometer occurs in the low intensity.

Conclusions

An accelerometer embedded into a cell phone was shown to be accurate and reliable in measuring and quantifying physical activity in the laboratory setting. Before a comprehensive integrated physical activity promoting and weight loss platform can be deployed, free‐living evaluation is necessary.
AMPKα2 deletion leads to marked alterations in SIRT1 activity, CPT1 expression and basal FA muscle metabolism in high‐fat‐fed mice
Authors: Abbott Marcia J , Turcotte Lorraine P
Published: June 2021

Because AMPKα2 is a critical signal in the regulation of muscle metabolism, we sought to determine if muscle AMPKα2 deletion would exacerbate the loss of insulin sensitivity, induced by high fat diet (HFD), as it pertains to the regulation of fatty acid (FA) metabolism. Male C57BL/6 mice (8–12 wks), wild‐type (WT) or dominant negative (DN) AMPKα2 in heart and skeletal muscle, were fed control diet (CD; 12%fat) or HFD (60%fat). After 6 wks, hindlimbs of CD (n=10) and HFD (n=10) mice were perfused (550μM palmitate, 6mM glucose, [1‐14C]palmitate) ±450μU insulin (IS). CD (n=8) and HFD (n=8) muscles were used for basal protein expression. In CD, AMPKα2 deletion did not affect basal FA uptake (FAU), but it increased basal FA oxidation (FAO) by 81% and α2 deletion prevented the IS increase in FAU and decrease in FAO. HFD abolished the IS increase in FAU and decrease in FAO in WT. In HFD, α2 deletion increased basal FAU by 139% (P<0.05) but did not affect IS FAU nor FAO (P>0.05). IS SIRT1 activity was higher in HFD‐DN than HFD‐WT (P<0.05). Neither DN nor HFD affected PGC1α content. HFD increased (P<0.05) PTP1b, SIRT1, and CD36 content and α2 deletion prevented the HFD‐induced increases in PTP1b and SIRT1. In HFD, α2 deletion decreased (P<0.05) CPT1 content. These data show that AMPKα2 may be more critical for the regulation of FAU and FAO under basal than IS conditions and that it affects CPT1, PTP1b, and SIRT1 content and IS SIRT1 activity with HFD.
Effects of acute exercise and epigallocatechin gallate (EGCG) incubation on mouse skeletal muscle Nampt levels
Authors: Urcuyo Dawnette Massiel , McMahon Kristen Eileen , Brandauer Josef
Published: June 2021

Activating AMP‐activated protein kinase (AMPK) in skeletal muscle may increase expression of nicotinamide phosphoribosyltransferase (Nampt), a protein that has been implicated in the regulation of the aging process. We investigated whether incubation with the green‐tea compound epigallocatechin gallate (EGCG) directly activates AMPK in mouse skeletal muscle, and whether a single bout of acute exercise increases skeletal muscle Nampt concentrations in mice. The effect of various physiological EGCG concentrations on AMPK phosphorylation was determined in vitro in extensor digitorum longus (EDL) muscle. For exercise, mice ran on a treadmill for 1 hour (21.5 m/min, 10% grade). Liver and skeletal muscle tissues were removed immediately after exercise, and Western blots were performed. EGCG incubation did not alter AMPK phosphorylation in EDL muscle (p > 0.05). Total Nampt levels were higher in sedentary soleus than gastrocnemius muscle (mean ± S.E.M., 1.00 ± 0.08 vs. 2.07 ± 0.18 arbitrary units; p < 0.001). Acute exercise failed to increase Nampt levels in skeletal muscle and liver tissues (p > 0.05) despite increased activation of the AMPK target acetyl‐CoA carboxylase in soleus muscle (≈ 32 %; p < 0.05). Neither acute exercise nor EGCG treatment had direct or immediate effects on Nampt concentrations in the tested tissues. Support: American Physiological Society (D.M.U.), Mellon Foundation (K.E.M).
Dominant anabolic effects of low‐dose corticosteroid in C2C12 cells
Authors: Zhu Ercheng , Fang Liwei , Caiozzo Vincent J , Sassoon Catherine S.H.
Published: June 2021

High‐dose corticosteroid induces diaphragm muscle dysfunction, whereas low‐dose corticosteroid preserves function. To elucidate the mechanisms of the protective effects of low‐dose corticosteroid, in C2C12 cell cultures we tested the hypothesis that low‐dose methylprednisolone (MP) increases protein synthesis via IGF/PI3K/Akt/mTOR, and decreases proteolysis via IGF/PI3K/Akt/Foxo pathways.

Methods

Myotubes of normal control (NC) and dominant negative MAF‐box (shRNA MAF‐box‐targeted interference) were treated with 0, 10 (low‐dose), and 100 (high‐dose) μMol MP (MP0, MP10, and MP100, respectively) for 3 days in the presence of rhIGF‐1 (50 ng/ml), the former with or without of rapamycin (RP) (25 ng/ml). We measured total, cytoplasmic, and nuclear protein pools, MAF‐box protein expressions, and MAF‐box mRNA.

Results

Both mTOR‐mediated protein synthesis increased and MAF‐box‐mediated proteolysis decreased following MP10 administration (Figure). MP10 treatment did not increase MAF‐box protein despite enhanced MAF‐box mRNA levels. MP100 overexpressed both MAF‐box protein and mRNA levels.

Conclusion

Low‐dose methylprednisolone promotes protein synthesis and reduces proteolysis via Akt/mTOR and Akt/Foxo pathways, respectively. The reduced proteolysis is likely related to MAF‐box RNA stabilization.

Funded By: VA Medical Research, NIH AR‐46856
Up regulation of skeletal muscle IL‐6 mRNA in response to hyperthermia
Authors: Welc Steven Stanley , Oca‐Cossio Jose , Payne Anthony M , Clanton Thomas L
Published: June 2021

Circulating interleukin‐6 (IL‐6) is thought to play a protective function in whole body hyperthermia. We hypothesized that an important source of IL‐6 in such conditions might be skeletal muscles. Whole mouse soleus (sol) muscle was hung vertically in muscle baths containing physiological saline, stimulated (≈.017Hz) and exposed to temperatures of 37°C (CON) or 40–41°C (HEAT) for 1 hour (hr), followed by 37°C recovery for 1 hr. In parallel experiments, C2C12 cells were grown in 6 well plates with each well representing a different treatment group. mRNA was isolated from each sample, reverse transcribed into cDNA, and quantified using real‐time PCR. We observed significant up regulation of both IL‐6 and heat shock protein 72 (HSP 72) mRNA in sol HEAT. Additionally, IL‐6 mRNA levels were significantly elevated in C2C12 cells in response to 1 hr exposure to 42°C, but not during the heat exposure period itself. These results demonstrate that hyperthermia is a functional stimulus for IL‐6 mRNA transcription in skeletal muscle. We speculate that it may contribute to the suppression of the whole body hyperthermic inflammatory responses.
Reversible Modification of Thick‐ and Thin‐filament Proteins in Oxidant‐induced Changes to Skeletal Muscle Function
Authors: Gross Sean M , Lehman Steven L
Published: June 2021

Exposure of skeletal muscle to reactive oxygen species (ROS) has been shown to modify the calcium sensitivity of force production.

Calcium sensitivity changes were largely reversed by the reducing agent DTT, indicating modification of protein thiol groups. To determine which proteins are modified we used permeabilized rabbit psoas in which the intracellular and redox conditions can be tightly controlled. Oxidants hydrogen peroxide and dithiodypridine (DTDP) and reductants DTT and glutathione were used to modify thiols. We identified myofilament proteins with modified thiols using 3 gel electrophoresis techniques: 1d SDS page, 2d IEF/SDS page, and diagonal SDS page electrophoresis. To relate modifications of myofilament protein thiols to altered muscle mechanics, force‐pCa curves were measured. Exposure to oxidants caused increased actin and myosin oxidation. Treatment with glutathione produced gluathionylation of myofilament proteins including myosin. 2d diagonal gels showed that several myofilament proteins formed intermolecular disulfide bonds and that the regulatory myosin light chain could form an intramolecular disulfide bond. Force‐pCa curves confirmed that treatments that modified free thiol groups also modified calcium sensitivity. Results indicate that the reversible modification of both thick and thin filament proteins play a role in the oxidant‐induced changes to muscle function.
AMPKα2 is not necessary to increase the activation state of ERK1/2, CaMKI, or AS160 during moderate intensity muscle contraction in perfused mouse muscle
Authors: Abbott Marcia J , Turcotte Lorraine P.
Published: June 2021

AMPK has been implicated in the regulation of multiple metabolic signaling pathways and has been deemed an energy sensor during muscle contraction. The purpose of this study was to determine if AMPKα2 is required to measure alterations in the activation state of signaling molecules during caffeine treatment (C) and moderate intensity muscle contraction (MIMC). Male C57BL/6 mice were either wild type (WT) or skeletal and heart muscle specific dominant negative (DN) AMPKα2. Hindquarters were perfused with 550μM palmitate and 6mM glucose at rest (R; WT n=5 and DN n=6), during C (3mM; WT n=5 and DN n=5) and during MIMC (WT n=6 and DN n=5). Hindlimb muscles were freeze‐clamped in situ and used for analysis of protein expression and phosphorylation state. ACC~p increased (P<0.05) 32 and 64% with C and MC. In the DN mice, ACC~pwas 11, 24, and 35% lower (P<0.05) in the R, C, and MIMC groups when compared to similarly‐treated WT groups. ERK1/2~p increased (P<0.05) 38% with MIMC. CaMKI~p increased (P<0.05) 150 and 103% with C and MIMC. AS160~p increased (P<0.05) 18 and 43% with C and MIMC. No genome‐induced differences were detected. Our results provide evidence for C‐ and MIMC‐induced activation of ACC, ERK1/2, CaMKI, and AS160 in a physiologically relevant muscle model and further suggest that AMPKα2 is not necessary to activate ERK1/2, CaMKI, or AS160 during C and MIMC in perfused mouse hindlimb muscle.
The role of calcium handling in the improved contractile function associated with high‐fat feeding in heart failure
Authors: CHENG YI‐HSIN , McElfresh Tracy A. , Chen Xiaoqin , Li Wei , Yu Xin , Van Wagoner David , Chandler Margaret P.
Published: June 2021

Heart failure (HF) is characterized by deteriorating left ventricular (LV) function. High saturated fat diets (SAT) are associated with increased risks for lipotoxicity & contractile dysfunction. However, HF animals fed SAT exhibit improved in vivo LV contractility versus HF animals fed normal chow (NC) as assessed by LV +dP/dt max. Ca2+ handling properties are known to be altered & contribute to HF. Therefore, we hypothesize that LV contractile dysfunction associated with alterations in Ca2+ handling properties in HF are improved with SAT. Male rats underwent coronary ligation or sham surgery (SH) & fed NC or SAT for 8 weeks. Cardiomyocytes were isolated, followed by measurements of cell shortening, Ca2+ transients, sarcoplasmic reticulum (SR) Ca2+ content, & L‐type Ca2+ current. Myocytes from both HF groups were hypertrophied & their Ca2+ decay (τ1/2) prolonged versus SH. HF+NC myocytes exhibited decreased cell shortening at 1Hz but increased Ca2+ transients despite decreased SR Ca2+ content versus SH+NC. HF+NC myocytes had depressed L‐type Ca2+ currents & a diminished response to β‐adrenergic stimulation versus SH+NC which may contribute to the decrease in cell shortening. However, cell shortening, L‐type ca2+ currents at baseline and in response to β‐adrenergic stimulation were not decreased in HF animals fed high fat and thus, may account for in vivo differences in contractility. (Grant: NHLBI HL‐081857)
Phosphatidylinositol 3,5 bisphosphate increases contractility and intracellular calcium in cardiac muscle by directly activating the cardiac ryanodine receptor.
Authors: Touchberry Chad D , Bales Ian K. , Brotto Marco , Valdivia Hector H. , Wacker Michael J
Published: June 2021

We have previously reported that treatment of left ventricular cardiac muscle strips with phosphatidylinositol 3,5 bisphosphate (PI(3,5)P2) during field stimulation enhanced the magnitude of isometric force, the rate of force development, and the area associated with the contractile waveforms. In addition, PI(3,5)P2 caused an increase in intracellular calcium (Ca) in cardiac myocytes. These data suggested that PI(3,5)P2 increased contractile force by promoting extracellular Ca entry or by facilitating intracellular Ca release. To elucidate the mechanism responsible for the effects of PI(3,5) on intracellular Ca, we have performed ratiometric Ca imaging in isolated adult mouse cardiac myocytes. We have found that PI(3,5)P2 induced a dramatic increase in intracellular Ca levels, which was inhibited by preincubation with ryanodine. To confirm that PI(3,5)P2 was acting on RyR2 we performed electrophysiological and biochemical studies. PI(3,5)P2 increased [3H]ryanodine binding and this effect was prevented by preincubation with an antibody against PI(3,5)P2. Furthermore, single RyR2 channels reconstituted in lipid bilayers were activated by PI(3,5)P2 in a dose‐dependent manner, indicating that PI(3,5)P2 binds directly to RyR2. We provide inaugural evidence that PI(3,5)P2 regulates cardiac contractility via modulation of intracellular Ca by direct binding and activation of the RyR2 channel.
Diet‐induced obesity differentially affects baroreflex‐mediated sympathetic and parasympathetic outflow
Authors: McCully Belinda Houghton , Brooks Virginia L , Andresen Michael C
Published: June 2021

Diet‐induced obesity (DIO) impairs the baroreflex control of heart rate (HR), which may arise from differential influences of the sympathetic (SNS) and parasympathetic (PNS) nervous systems. To test this hypothesis, Δ mean arterial pressure (MAP) and Δ HR to 30‐sec maximal stimulation of the aortic baroreceptors (aortic depressor nerve; 20V, 100Hz) were recorded before and after muscarinic receptor blockade (MB; methscopolamine, iv bolus 500ug/kg) in inactin‐anesthetized male SD rats. Rats were fed a normal (N, 13.5% kcal fat) or high fat diet (HFD, 33% kcal fat) for 4–5 weeks. Rats fed the HFD were separated into obesity prone (OP) or resistant (OR) groups, based on the top and bottom tertile of weight gain, respectively. Baseline values did not differ between groups.

As shown in the table above, Δ HR and Δ MAP were blunted in OP rats compared to N and OR rats (†p<0.05 vs OP). Removal of PNS influence with MB attenuated Δ HR and Δ MAP in N and OR rats (*p<0.05 control vs MB), which were similar to the OP rat control responses. MB had no effect on the OP group. These findings suggest that DIO impairs aortic baroreceptor activation of the PNS, but may minimally impact the SNS. Funded by AHA (Brooks) and NIH (McCully NS045553, Brooks HL088552, Andresen HL41119).

Δ HR (bpm)
Δ MAP (mmHg)

Control
MB
Control
MB

N (n=6)
−53±7†
−28±4*
−57±6†
−47±4*

OR (n=6)
−45±6†
−31±3*
−60±5†
−48±4*

OP (n=6)
−30±6
−28±6
−41±8
−47±4
MODULATION BY P2X RECEPTORS OF THE GLUTAMATERGIC SYNAPTIC TRANSMISSION IN RVLM‐PROJECTING NTS NEURONS OF RATS
Authors: Accorsi‐Mendonca Daniela , Bonagamba Leni GH , Leao Ricardo Mauricio , Machado Benedito H
Published: June 2021

The peripheral chemoreflex activation by hypoxia produces autonomic and respiratory adjustments. The NTS is the primary site of peripheral chemoreceptor afferents in the CNS and RVLM‐projecting NTS neurons are related to the sympathoexcitatory component of this reflex. In a previous study we demonstrated that ATP modulates the spontaneous and evoked glutamate release onto RVLM‐projecting NTS neurons. Herein, using a pharmacological approach, we evaluated the subtypes of purinergic receptors involved with this modulation. Adults Wistar rats received DiI tracer into RVLM and 24 hours later the labeled RVLM‐projecting NTS neurons were identified in transversal brainstem slices. Spontaneous and TS evoked excitatory pos‐synaptic currents (sEPSCs and TS‐eEPSCs) were recorded using whole cell patch clamp. PPADS (100 μM, P2X1/2/3/5/7 receptor antagonist) decreased the frequency of sEPSCs (control: 3.39 ± 0.1 vs PPADS: 1.21 ± 0.1 Hz, n=7), but did not change the amplitude or half‐width of events. TNP‐ATP (1 μM, P2X1/2/3 receptor antagonist) also decreased the frequency of currents (control: 4.3 ± 1.1 Hz vs TNP‐ATP: 1.6 ± 0.5 Hz, n=8) and did not alter the amplitude or half‐width of events. PPADS and TNP‐ATP also decreased the amplitude of TS‐eEPSC (−23% and −32% respectively). These data suggest that P2X1, P2X2 or P2X3 receptors are involved in the purinergic modulation of glutamate release onto RVLM‐projecting NTS neurons.
Enhanced peripheral chemoreflex function in conscious rats with ligation‐induced heart failure
Authors: Tang Yuhong , Ding Yanfeng , Li Yulong , Schultz Harold D.
Published: June 2021

Our previous studies showed peripheral chemoreflex sensitivity is enhanced in conscious rabbits with pacing‐induced chronic heart failure (CHF), which is mediated by an elevated Angiotensin II (Ang II) effect on the carotid body (CB). Here we investigated whether CB chemoreflex function and CB Ang II are similarly altered in a rat model of 8 week ligation‐induced CHF. Minute ventilation (VE) and renal sympathetic nerve activity (RSNA) in response to stimulation of peripheral chemoreceptor by isocapnic/hypoxic gases were measured in conscious state. The degree of CHF was assessed by echocardiography. It was found that the baselines VE and RSNA at normoxia and graded hypoxia were elevated in CHF rats (> 30% reduction of FS) than those in sham rats. In addition, the concentration of tissue Ang II measured by radioimmunoassay was higher in CHF CBs compared to sham CBs (51.44 ± 0.16 vs. 36.20 ± 1.29 pg/mg protein, p<0.05). Furthermore, protein expression of Ang II type 1 receptor and angiotensin converting enzyme 1 in CHF CBs were increased compared to those in sham CBs. These results suggest that, similar to the pacing model in rabbits, increased Ang II in the CBs may contribute to the enhanced peripheral chemoreflex function in the coronary ligation model of CHF in rats. Supported by NIH grant PO‐1 HL 62222.
Role of exercise in the regulation of NF‐kappaB‐dependent gene expression in Alzheimer's Disease‐affected brain
Authors: Lloyd Jesse William , Beaman‐Hall Carol , Heckstall Ebony Monique , Vallano Mary‐Lou , Keslacy Stefan
Published: June 2021

Introduction

A defining feature of Alzheimer's disease (AD) is plaque deposition induced by accumulation of Amyloid beta peptides (Aβ) that trigger chronic inflammation in the brain. Aβ‐related neurodegenerative changes are improved by chronic exercise. NF‐κB pathway is a “master controller” of the inflammatory process. Hypothesis: We hypothesize that exercise will diminish the AD‐associated NF‐κB activation in the brain. Aβ‐related model of AD APP/PS1 and wild‐type (WT) mice were exposed to voluntary running for 1 month (EX) and compared to sedentary (SED).

Results

EX demonstrated a decrease in plaque count. Using a qRT‐PCR array, we observed that exercise induced a downregulation of inflammatory genes, (IL‐1α, IL‐1β, Ltbr Tlr1/8/9/, Trfrs10b, Tnfrsf1a and Traf3) and an upregulation of anti‐inflammatory genes (Ccl2, IFNγ, Cd27, Card10 and IFNγ) associated with NF‐κB in hippocampus (Hip) of SED APP/PS1 mice. AD‐induced expression and phosphorylation of NF‐κB/P65 were diminished in Hip, cerebellum (Cb) and cortex (Cx) of EX. Phospho‐IKKα/β levels were reduced in Hip of EX compare to SED. P65, physically bound to IκB, was sequestrated into the cytoplasm following exercise.

Conclusion

The molecular mechanisms underlying the beneficial effect of exercise in AD‐affected brain may rely on i) P65/IκB physical interaction and ii) regulation of the immune system through IFNγ signaling.
Prolonged fasting suppresses cellular insulin‐dependent activity in adipose tissue of the northern elephant seal
Authors: Viscarra Jose Abraham , Vazquez‐Medina Jose Pablo , Minas Jacqueline , Crocker Daniel E. , Ortiz Rudy M.
Published: June 2021

Throughout its life, the northern elephant seal (Mirounga angustirostris) experiences periods of prolonged fasting during which plasma glucose reaches hyperglycemic levels. As the fast progresses, the levels of circulating glucose decrease, despite relatively low or decreasing plasma insulin levels. Cellular glucose uptake is dependent on an insulin‐activated signaling cascade resulting in the translocation of the glucose transport protein, Glut4, into the plasma membrane. In order to understand how this pathway is affected by prolonged fasting PI3K, AKT and VAMP2 content, and plasma insulin were examined. Blubber biopsies as well as plasma samples were obtained from two groups of northern elephant seal pups (n = 10/group) sampled during their post weaning fast: early (2 weeks) and late (6 to 8 weeks). Tissue content of PI3K, AKT, and VAMP2 proteins was quantified by western blot. Plasma insulin did not change (1.5 ± 0.4 vs. 1.2 ± 0.2 uU/mL) between early and late fasting. While PI3K and VAMP2 also did not change, AKT decreased 29% with the fast. We have previously shown that fasting is associated with a decrease in insulin receptor (IR), but an increase in Glut 4. Because insulin stimulates PI3K and AKT, the decreases in insulin and IR may explain the lack of change in PI3K and the decrease in AKT suggesting that fasting suppresses cellular insulin‐dependent activity. Funded by NIH HL91767 and UC Startup funds.
A complete profile of carbohydrate metabolism during prolonged fasting in the northern elephant seal
Authors: Champagne Cory , Houser Dorian , Fowler Melinda , Costa Dan , Crocker Dan
Published: June 2021

Glucose production is typically suppressed while fasting to facilitate protein sparing by avoiding the commitment of amino acids to gluconeogenesis. Previous work in fasting northern elephant seals, however, has found elevated rates of glucose production. To explore carbohydrate metabolism during prolonged fasting, we used a tracer technique utilizing D2O, [U‐13C]propionate, and [1,6‐13C]glucose. Nuclear magnetic resonance (NMR) was used to measure the abundance of each resulting positional isotope of glucose. We used the relative peak heights from the NMR spectra to quantify the rate of glucose production, the contribution of major gluconeogenic precursors to glucose production, the flux through the tricarboxylic‐acid cycle, and the activity of the phosphoenolpyruvate (PEP) cycle. Rates of glucose production closely agreed with previously measured values in this species. The combined contribution of glycogen and glycerol to gluconeogenesis was low, less than 10% of glucose production. The balance of glucose flux, greater than 90%, derived from PEP. The significant contribution of PEP to glucose production is consistent with high rates of carbon recycling through three‐carbon intermediates such as lactate. This is the first use of this positional isotope technique in a free‐ranging animal and provides the first comprehensive carbohydrate metabolism profile of an animal undertaking a prolonged fast.
Effects of β‐hydroxy‐β‐methylbutyrate (HMB) on Myofiber Dimension and Myogenic Response in Old Sprague‐Dawley Female Rats during 10‐Week Resistance Training
Authors: Kim Jeong‐Su , Park Young‐Min , Lee Sang‐Rok , Wilson Jacob M , Henning Paul C , Masad Ihssan S , Ugrinowitsch Carlos , Arjmandi Bahram H , Grant Samuel C
Published: June 2021

Aging mammalian skeletal muscles exhibit sarcopenia. We investigated the effects of HMB intake during intense resistance training (RT) on lean body mass (LBM) and myofiber dimensions (MD) of aged rats. Sixteen 19‐month‐old Sprague‐Dawley female rats were divided randomly into 3 groups: CON (control), HMB (0.46 g/kg/d) and Non‐HMB. CON was sacrificed for baseline; HMB and Non‐HMB underwent intense resistance training (weighted ladder climbing) every 3 days for 10 weeks. Dual energy X‐ray absorptiometry assessed pre‐ and post‐RT LBM. In the soleus, diffusion tensor imaging determined water diffusion in myofibers by calculating the fractional anisotropy (FA) and eigenvalues while myogenin mRNA levels were assessed with PCR. Overall, RT improved LBM (+21%, p<0.05) in both HMB and Non‐HMB. The longitudinal eigenvalue λ1 exhibited no change while λ2 (+17%) and λ3 (+20%) increased (p<0.01) in both groups after RT, indicating no change in diffusion along the myofiber length but increased fiber cross‐sectional area (CSA). Decreased FA and increased apparent diffusion coefficient appear to indicate hypertrophic fibers with RT. Myogenin mRNA expression increased (+69%, p<0.05) in both groups. No between group differences were identified for all variables. Our findings suggest that high intensity RT in aged individuals increases LBM, myofiber CSA and myogenic response; however, HMB may not facilitate the changes.
Evidence of crosstalk between A1 and A2A adenosine receptor in the regulation of renal O2 consumption
Authors: Di Sole Francesca , Babich Victor , Moe Orson W
Published: June 2021

Limited O2 availability leads to high level of adenosine which protects the kidney via both A1 and A2A adenosine receptor (A1R and A2AR). Relative contribution of A1R and A2AR is not clear. We evaluated the effect of acute A1R and A2AR activation on the rate of O2 consumption (VO2) in renal cortex which has high O2 requirement. A1R agonist, CPA, and A2A agonist, CGS, both inhibited VO2 in a dose and time dependent mode. Reduction in VO2 was Na+ dependent for A1R and Na+ independent for A2A, suggesting the two receptors act differently to reduce VO2. To eliminate endogenous adenosine, we metabolized it by incubation with adenosine deaminase (ADA). We found a strong reduction in VO2 that was Na+ dependent. Likewise, A1R antagonist, CPX, also reduced VO2. This data suggests that in contradistinction to exogenously added A1R agonist, endogenous adenosine increases VO2 via A1R. Furthermore, in ADA pretreated tissue, we found that: 1. A1R has dual mode of action. Low dose of CPA activates and high dose inhibits VO2 and both effects were Na+ dependent. 2. CGS has no effect on the reduction in VO2 induced by ADA but converted the Na+ dependent A1R mediated effect on VO2 to a mainly Na+ independent effect. In summary, A1R affects VO2 via Na+ dependent, while A2AR via Na+ independent mechanisms. If both A1R and A2AR are activated Na+ independent mechanisms prevails on the Na+ dependent mechanisms suggesting crosstalk between A1R and A2AR. These findings indicate that increasing level of adenosine decreases VO2 via both Na+ dependent and independent mechanisms depending on the receptor subtype activated and the two mechanisms are interdependent.