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Circadian rhythms are central to optimal physiological function, as disruption contributes to the development of several chronic diseases. Alcohol (EtOH) intoxication disrupts circadian rhythms within liver, brain, and intestines, but it is unknown whether alcohol also disrupts components of the core clock in skeletal muscle. Female C57BL/6Hsd mice were randomized to receive either saline (control) or alcohol (EtOH) (5 g/kg) via intraperitoneal injection at the start of the dark cycle [ Zeitgeber time (ZT12) ], and gastrocnemius was collected every 4 h from control and EtOH-treated mice for the next 48 h following isoflurane anesthetization. In addition, metyrapone was administered before alcohol intoxication in separate mice to determine whether the alcohol-induced increase in serum corticosterone contributed to circadian gene regulation. Finally, synchronized C2C12 myotubes were treated with alcohol (100 mM) to assess the influence of centrally or peripherally mediated effects of alcohol on the muscle clock. Alcohol significantly disrupted mRNA expression of Bmal1 , Per1/2 , and Cry1/2 in addition to perturbing the circadian pattern of clock-controlled genes, Myod1 , Dbp , Tef , and Bhlhe40 ( P < 0.05), in muscle. Alcohol increased serum corticosterone levels and glucocorticoid target gene, Redd1 , in muscle. Metyrapone prevented the EtOH-mediated increase in serum corticosterone but did not normalize the EtOH-induced change in Per1 , Cry1 and Cry2 , and Myod1 mRNA expression. Core clock gene expression ( Bmal , Per1/2 , and Cry1/2 ) was not changed following 4, 8, or 12 h of alcohol treatment on synchronized C2C12 myotubes. Therefore, binge alcohol disrupted genes of the core molecular clock independently of elevated serum corticosterone or direct effects of EtOH on the muscle. NEW & NOTEWORTHY Alcohol is a myotoxin that impairs skeletal muscle metabolism and function following either chronic consumption or acute binge drinking; however, mechanisms underlying alcohol-related myotoxicity have not been fully elucidated. Herein, we demonstrate that alcohol acutely interrupts oscillation of skeletal muscle core clock genes, and this is neither a direct effect of ethanol on the skeletal muscle, nor an effect of elevated serum corticosterone, a major clock regulator.

A role for fat overfeeding in metabolic dysfunction in humans is commonly implied in the literature. Comparatively less is known about acute carbohydrate overfeeding (COF). We tested the hypothesis that COF predisposes to oxidative stress by channeling electrons away from antioxidants to support energy storage. In a study of 24 healthy human subjects with and without obesity, COF was simulated by oral administration of excess carbohydrates; a two-step hyperinsulinemic clamp was used to evaluate insulin action. The distribution of electrons between oxidative and reductive pathways was evaluated by the changes in the reduction potentials (Eh) of cytoplasmic (lactate, pyruvate) and mitochondrial (β-hydroxybutyrate, acetoacetate) redox couples. Antioxidant redox was measured by the ratio of reduced to oxidized glutathione. We used cross-correlation analysis to evaluate the relationships between the trajectories of Eh, insulin, glucose, and respiratory exchange during COF. DDIT3 and XBP1s/u mRNA were measured as markers of endoplasmic reticulum stress (ER stress) in adipose tissue before and after COF. Here, we show that acute COF is characterized by net transfer of electrons from mitochondria to cytoplasm. Circulating glutathione is oxidized in a manner that significantly cross-correlates with increasing insulin levels and precedes the decrease in cytoplasmic Eh. This effect is more pronounced in overweight individuals (OW). Markers of ER stress in subcutaneous fat are detectable in OW within 4 h. We conclude that acute COF contributes to metabolic dysfunction through insulin-dependent pathways that promote electron transfer to the cytoplasm and decrease antioxidant capacity. Characterization of redox during overfeeding is important for understanding the pathophysiology of obesity and type 2 diabetes. NEW & NOTEWORTHY Current principles assume that conversion of thermic energy to metabolically useful energy follows fixed rules. These principles ignore the possibility of variable proton uncoupling in mitochondria. Our study shows that the net balance of electron distribution between mitochondria and cytoplasm is influenced by insulin in a manner that reduces proton leakage during overfeeding. Characterization of the effects of insulin on redox balance is important for understanding obesity and insulin resistance.

During pregnancy, the uterus transitions from a quiescent state to an excitable, highly contractile state to deliver the fetus. Two important contributors essential for this transition are hormones and ion channels, both of which modulate myometrial smooth muscle cell (MSMC) excitability. Recently, the sodium (Na + ) leak channel, nonselective (NALCN), was shown to contribute to a Na + leak current in human MSMCs, and mice lacking NALCN in the uterus had dysfunctional labor. Microarray data suggested that the proquiescent hormone progesterone (P4) and the procontractile hormone estrogen (E2) regulated this channel. Here, we sought to determine whether P4 and E2 directly regulate NALCN. In human MSMCs, we found that NALCN mRNA expression decreased by 2.3-fold in the presence of E2 and increased by 5.6-fold in the presence of P4. Similarly, E2 treatment decreased, and P4 treatment restored NALCN protein expression. Additionally, E2 significantly inhibited, and P4 significantly enhanced an NALCN-dependent leak current in MSMCs. Finally, we identified estrogen response and progesterone response elements (EREs and PREs) in the NALCN promoter. With the use of luciferase assays, we showed that the PREs, but not the ERE, contributed to regulation of NALCN expression. Our findings reveal a new mechanism by which NALCN is regulated in the myometrium and suggest a novel role for NALCN in pregnancy.

Hepatic fat-specific protein 27 [cell death-inducing DNA fragmentation effector protein C ( Cidec )/ Fsp27 ] mRNA levels have been associated with hepatic lipid droplet extent under certain circumstances. To address its hepatic expression under different dietary conditions and in both sexes, apolipoprotein E ( Apoe ) - deficient mice were subjected to different experimental conditions for 11 wk to test the influence of cholesterol, Western diet, squalene, oleanolic acid, sex, and surgical castration on Cidec/Fsp27 mRNA expression. Dietary cholesterol increased hepatic Cidec/Fsp27β expression, an effect that was suppressed when cholesterol was combined with saturated fat as represented by Western diet feeding. Using the latter diet, neither oleanolic acid nor squalene modified its expression. Females showed lower levels of hepatic Cidec/Fsp27β expression than males when they were fed Western diets, a result that was translated into a lesser amount of CIDEC/FSP27 protein in lipid droplets and microsomes. This was also confirmed in low-density lipoprotein receptor ( Ldlr )-deficient mice. Incubation with estradiol resulted in decreased Cidec/Fsp27β expression in AML12 cells. Whereas male surgical castration did not modify the expression, ovariectomized females did show increased levels compared with control females. Females also showed increased expression of peroxisome proliferator-activated receptor-γ coactivator 1-α ( Pgc1a ), suppressed by ovariectomy, and the values were significantly and inversely associated with those of Cidec/Fsp27β . When Pgc1a -deficient mice were used, the sex differences in Cidec/Fsp27β expression disappeared. Therefore, hepatic Cidec/Fsp27β expression has a complex regulation influenced by diet and sex hormonal milieu. The mRNA sex differences are controlled by Pgc1a .

Exercise-induced increases in peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) and p53 protein content in the nucleus mediate the initial phase of exercise-induced mitochondrial biogenesis. Here, we investigated whether exercise-induced increases in these and other markers of mitochondrial biogenesis were altered after 40 sessions of twice-daily high-volume, high-intensity interval training (HVT) in human skeletal muscle. Vastus lateralis muscle biopsies were collected from 10 healthy recreationally active participants before, immediately postexercise, and 3 h after a session of high-intensity interval exercise (HIIE) performed at the same absolute exercise intensity before and after HVT (pre-HVT and post-HVT, respectively). The protein content of common markers of exercise-induced mitochondrial biogenesis was assessed in nuclear- and cytosolic-enriched fractions by immunoblotting; mRNA contents of key transcription factors and mitochondrial genes were assessed by qPCR. Despite exercise-induced increases in PGC-1α, p53, and plant homeodomain finger-containing protein 20 (PHF20) protein content, the phosphorylation of p53 and acetyl-CoA carboxylase (p-p53 Ser 15 and p-ACC Ser 79 , respectively), and PGC-1α mRNA Pre-HVT, no significant changes were observed post-HVT. Forty sessions of twice-daily high-intensity interval training blunted all of the measured exercise-induced molecular events associated with mitochondrial biogenesis that were observed pre-HVT. Future studies should determine whether this loss relates to the decrease in relative exercise intensity, habituation to the same exercise stimulus, or a combination of both.

Experimental data suggest that iron sucrose (FeS) injection, used either alone or in combination with other prooxidants, can induce "renal preconditioning," in part by upregulating cytoprotective ferritin levels. However, the rapidity, degree, composition (heavy vs. light chain), and renal ferritin changes after FeS administration in humans remain to be defined. To address these issues, healthy human volunteers ( n = 9) and patients with stage 3-4 chronic kidney disease( n = 9) were injected once with FeS (120, 240, or 360 mg). Plasma ferritin was measured from 0 to 8 days postinjection as an overall index of ferritin generation. Urinary ferritin served as a "biomarker" of renal ferritin production. FeS induced rapid (≤2 h), dose-dependent, plasma ferritin increases in all study participants, peaking at approximately three to five times baseline within 24-48 h. Significant urinary ferritin increases (~3 times), without dose-dependent increases in albuminuria, neutrophil gelatinase-associated lipocalin, or N -acetyl-β-d-glucosaminidase excretion, were observed. Western blot analysis with ferritin heavy chain (Fhc)- and light chain (Flc)-specific antibodies demonstrated that FeS raised plasma Flc but not Fhc levels. Conversely, FeS increased both Fhc and Flc in urine. To assess sites of FeS-induced ferritin generation, organs from FeS-treated mice were probed for Fhc, Flc, and their mRNAs. FeS predominantly raised hepatic Flc. Conversely, marked Fhc and Flc elevations developed in the kidney and spleen. No cardiopulmonary ferritin increases occurred. Ferritin mRNAs remained unchanged throughout, implying posttranscriptional ferritin production. We conclude that FeS induces rapid, dramatic, and differential Fhc and Flc upregulation in organs. Renal Fhc and Flc increases, in the absence of nephrotoxicity, suggest potential FeS utility as a clinical renal "preconditioning" agent.

Skeletal muscle satellite cells (SC) play an important role in muscle repair following injury. The regulation of SC activity is governed by myogenic regulatory factors (MRF), including MyoD, Myf5, myogenin, and MRF4. The mRNA expression of these MRF in humans following muscle damage has been predominately measured in whole muscle homogenates. Whether the temporal expression of MRF in a whole muscle homogenate reflects SC-specific expression of MRF remains largely unknown. Sixteen young men (23.1 ± 1.0 yr) performed 300 unilateral eccentric contractions (180°/s) of the knee extensors. Percutaneous muscle biopsies from the vastus lateralis were taken before (Pre) and 48 h postexercise. Fluorescence-activated cell sorting analysis was utilized to purify NCAM + muscle SC from the whole muscle homogenate. Forty-eight hours post-eccentric exercise, MyoD, Myf5, and myogenin mRNA expression were increased in the whole muscle homogenate (~1.4-, ~4.0-, ~1.7-fold, respectively, P < 0.05) and in isolated SC (~19.3-, ~17.5-, ~58.9-fold, respectively, P < 0.05). MRF4 mRNA expression was not increased 48 h postexercise in the whole muscle homogenate ( P > 0.05) or in isolated SC ( P > 0.05). In conclusion, our results suggest that the directional changes in mRNA expression of the MRF in a whole muscle homogenate in response to acute eccentric exercise reflects that observed in isolated muscle SC. NEW & NOTEWORTHY The myogenic program is controlled via transcription factors referred to as myogenic regulatory factors (MRF). Previous studies have derived MRF expression from whole muscle homogenates, but little work has examined whether the mRNA expression of these transcripts reflects the pattern of expression in the actual population of satellite cells (SC). We report that MRF expression from an enriched SC population reflects the directional pattern of expression from skeletal muscle biopsy samples following eccentric contractions.

Posthepatectomy liver failure (PHLF) may occur after extended partial hepatectomy (PH). If malignancy is widespread in the liver, the size of PH and hence the size of the future liver remnant (FLR) may limit curability. We aimed to characterize differences in protein expression between different sizes of FLRs and identify proteins specific to the regenerative process of minimal-size FLR (MSFLR), with special focus on postoperative day (POD) 1 when PHLF is present. A total of 104 male Wistar rats were subjected to 30, 70, or 90% PH (MSFLR in rats), sham operation, or no operation. Blood and liver tissue were harvested at POD1, 3, and 5 ( n = 8 per group). Protein expression was assessed by proteomic profiling by unsupervised two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) liquid chromatography tandem mass spectrometry (LC-MS/MS), followed by supervised selected reaction monitoring (SRM)-MS/MS. In all, 1,035 protein spots were detected, 54 of which were significantly differentially expressed between groups and identifiable. During PHLF after PH(90%) at POD1, urea cycle and related proteins showed significant perturbations, including the urea cycle flux-regulating enzyme of carbamoyl phosphate synthase-1, ornithine transcarbamylase, and arginase-1, as well as the ornithine aminotransferase and propionyl-CoA carboxylase alpha chain. Plasma-ammonia increased significantly at POD1 after PH(90%), followed by a prompt decrease. At the protein level, we found perturbations of urea cycle and related enzymes in the MSFLR during PHLF. Our results suggest that these perturbations may augment urea cycle function, which may be pivotal for increased ammonia elimination after extensive PHs and potential PHLF. NEW & NOTEWORTHY Posthepatectomy liver failure (PHLF) is associated with high mortality. In a rat model of 90% hepatectomy, PHLF is present. Our results on liver tissue proteomics suggest that the ability of the liver remnant to sufficiently eliminate ammonia may be brought about by perturbation related to urea cycle proteins and that enhancing the urea cycle capacity may play a key role in surviving PHLF.

Angiopoietin-like protein 4 (Angptl4)/fasting-induced adipose factor (Fiaf) expression levels are increased by exercise in skeletal muscle. We have previously shown that Angptl4 regulates food intake and energy expenditure via modulation of hypothalamic AMP-activated protein kinase (AMPK) activity. AMPK is an important signaling molecule that integrates skeletal muscle metabolism during exercise. Therefore, we investigated the involvement of Angptl4 in exercise-induced AMPK activation in skeletal muscle. Angptl4 protein and mRNA expression levels were significantly increased in the gastrocnemius and soleus muscles of mice following a 50-min running bout. Treatment of C2C12 myotubes with Angptl4 increased phosphorylation of AMPK and acetyl-CoA carboxylase (ACC), which were markers of AMPK activation, and the mitochondrial maximum respiratory capacity. Treadmill exercise increased AMPK and ACC phosphorylation in the gastrocnemius of normal mice; this phosphorylation increase was attenuated in mice lacking Angptl4. Endurance to swimming and hanging was also reduced in Angptl4 knockout mice. Taken together, our current data demonstrate that exercise-induced upregulation of skeletal muscle Angptl4 is critical for AMPK activation and exercise tolerance. These findings unveil a new role for skeletal muscle Angptl4 in exercise physiology. NEW & NOTEWORTHY 1) Angiopoietin-like protein 4 (Angptl4) treatment activates AMP-activated protein kinase (AMPK) signaling in skeletal muscle cells. 2) Angptl4 increases the maximum mitochondrial oxidative capacity through AMPK activation in skeletal muscle cells. 3) Lack of Angptl4 mitigates exercise-induced skeletal muscle AMPK activation. 4) Angptl4-deficient mice show a lower endurance to exercise.

Peroxisome proliferator-activated receptor-α coactivator-1α (PGC-1α) mRNA is increased with both exercise and exposure to cold temperature. However, transcriptional control has yet to be examined during exercise in the cold. Additionally, the need for environmental cold exposure after exercise may not be a practical recovery modality. The purpose of this study was to determine mitochondrial-related gene expression and transcriptional control of PGC-1α following exercise in a cold compared with room temperature environment. Eleven recreationally trained males completed two 1-h cycling bouts in a cold (7°C) or room temperature (20°C) environment, followed by 3 h of supine recovery in standard room conditions. Muscle biopsies were taken from the vastus lateralis preexercise, postexercise, and after a 3-h recovery. Gene expression and transcription factor binding to the PGC-1α promoter were analyzed. PGC-1α mRNA increased from preexercise to 3 h of recovery, but there was no difference between trials. Estrogen-related receptor-α (ERRα), myocyte enhancer factor-2 (MEF2A), and nuclear respiratory factor-1 (NRF-1) mRNA were lower in cold than at room temperature. Forkhead box class-O (FOXO1) and cAMP response element-binding protein (CREB) binding to the PGC-1α promoter were increased postexercise and at 3 h of recovery. MEF2A binding increased postexercise, and activating transcription factor 2 (ATF2) binding increased at 3 h of recovery. These data indicate no difference in PGC-1α mRNA or transcriptional control after exercise in cold versus room temperature and 3 h of recovery. However, the observed reductions in the mRNA of select transcription factors downstream of PGC-1α indicate a potential influence of exercise in the cold on the transcriptional response related to mitochondrial biogenesis.

Iron homeostasis is tightly regulated, and the peptide hormone hepcidin is considered to be a principal regulator of iron metabolism. Previous studies in a limited number of mouse strains found equivocal sex- and strain-dependent differences in mRNA and serum levels of hepcidin and reported conflicting data on the relationship between hepcidin ( Hamp1 ) mRNA levels and iron status. Our aim was to clarify the relationships between strain, sex, and hepcidin expression by examining multiple tissues and the effects of different dietary conditions in multiple inbred strains. Two studies were done: first, Hamp1 mRNA, liver iron, and plasma diferric transferrin levels were measured in 14 inbred strains on a control diet; and second, Hamp1 mRNA and plasma hepcidin levels in both sexes and iron levels in the heart, kidneys, liver, pancreas, and spleen in males were measured in nine inbred/recombinant inbred strains raised on an iron-sufficient or high-iron diet. Both sex and strain have a significant effect on both hepcidin mRNA (primarily a sex effect) and plasma hepcidin levels (primarily a strain effect). However, liver iron and diferric transferrin levels are not predictors of Hamp1 mRNA levels in mice fed iron-sufficient or high-iron diets, nor are the Hamp1 mRNA and plasma hepcidin levels good predictors of tissue iron levels, at least in males. We also measured plasma erythroferrone, performed RNA-sequencing analysis of liver samples from six inbred strains fed the iron-sufficient, low-iron, or high-iron diets, and explored differences in gene expression between the strains with the highest and lowest hepcidin levels. NEW & NOTEWORTHY Both sex and strain have a significant effect on both hepcidin mRNA (primarily a sex effect) and plasma hepcidin levels (primarily a strain effect). Liver iron and diferric transferrin levels are not predictors of Hamp1 mRNA levels in mice, nor are the Hamp1 mRNA and plasma hepcidin levels good predictors of tissue iron levels, at least in males., (Copyright © 2017 the American Physiological Society.)

Krüppel-like factor 5 (KLF5) is a member of the zinc finger family of transcription factors that regulates homeostasis of the intestinal epithelium. Previous studies suggested an indispensable role of KLF5 in maintaining intestinal barrier function. In the current study, we investigated the mechanisms by which KLF5 regulates colonic barrier function in vivo and in vitro. We used an inducible and a constitutive intestine-specific Klf5 knockout mouse models ( Villin-CreER T2 ;Klf5 fl/fl designated as Klf5 ΔIND and Villin-Cre;Klf5 fl/fl as Klf5 ΔIS ) and studied an inducible KLF5 knockdown in Caco-2 BBe cells using a lentiviral Tet-on system (Caco-2 BBe KLF5ΔIND ). Specific knockout of Klf5 in colonic tissues, either inducible or constitutive, resulted in increased intestinal permeability. The phenotype was accompanied by a significant reduction in Dsg2 , which encodes desmoglein-2, a desmosomal cadherin, at both mRNA and protein levels. Transmission electron microscopy showed alterations of desmosomal morphology in both KLF5 knockdown Caco-2 BBe cells and Klf5 knockout mouse colonic tissues. Inducible knockdown of KLF5 in Caco-2BBe cells grown on Transwell plates led to impaired barrier function as evidenced by decreased transepithelial electrical resistance and increased paracellular permeability to fluorescein isothiocyanate-4 kDa dextran. Furthermore, DSG2 was significantly decreased in KLF5 knockdown cells, and DSG2 overexpression partially rescued the impaired barrier function caused by KLF5 knockdown. Electron microscopy studies demonstrated altered desmosomal morphology after KLF5 knockdown. In combination with chromatin immunoprecipitation analysis and promoter study, our data show that KLF5 regulates intestinal barrier function by mediating the transcription of DSG2 , a gene encoding a major component of desmosome structures. NEW & NOTEWORTHY The study is original research on the direct function of a Krüppel-like factor on intestinal barrier function, which is commonly exerted by cell junctions, including tight junctions, adherens junctions, and desmosomes. Numerous previous studies were focused on tight junctions and adherens junctions. However, this study provided a new perspective on how the intestinal barrier function is regulated by KLF5 through DSG2, a component of desmosome complexes., (Copyright © 2017 the American Physiological Society.)

Endothelial cystathionine γ-lyase (CSEγ) contributes to cardiovascular homeostasis, mainly through production of H 2 S. However, the molecular mechanisms that control CSEγ gene expression in the endothelium during cardiovascular diseases are unclear. The aim of the current study is to determine the role of specific histone deacetylases (HDACs) in the regulation of endothelial CSEγ. Reduced CSEγ mRNA expression and protein abundance were observed in human aortic endothelial cells (HAEC) exposed to oxidized LDL (OxLDL) and in aortas from atherogenic apolipoprotein E knockout (ApoE -/- ) mice fed a high-fat diet compared with controls. Intact murine aortic rings exposed to OxLDL (50 μg/ml) for 24 h exhibited impaired endothelium-dependent vasorelaxation that was blocked by CSEγ overexpression or the H 2 S donor NaHS. CSEγ expression was upregulated by pan-HDAC inhibitors and by class II-specific HDAC inhibitors, but not by other class-specific inhibitors. The HDAC6 selective inhibitor tubacin and HDAC6-specific siRNA increased CSEγ expression and blocked OxLDL-mediated reductions in endothelial CSEγ expression and CSEγ promoter activity, indicating that HDAC6 is a specific regulator of CSEγ expression. Consistent with this finding, HDAC6 mRNA, protein expression, and activity were upregulated in OxLDL-exposed HAEC, but not in human aortic smooth muscle cells. HDAC6 protein levels in aortas from high-fat diet-fed ApoE -/- mice were comparable to those in controls, whereas HDAC6 activity was robustly upregulated. Together, our findings indicate that HDAC6 is upregulated by atherogenic stimuli via posttranslational modifications and is a critical regulator of CSEγ expression in vascular endothelium. Inhibition of HDAC6 activity may improve endothelial function and prevent or reverse the development of atherosclerosis. NEW & NOTEWORTHY Oxidative injury to endothelial cells by oxidized LDL reduced cystathionine γ-lyase (CSEγ) expression and H 2 S production, leading to endothelial dysfunction, which was prevented by histone deacetylase 6 (HDAC6) inhibition. Our data suggest HDAC6 as a novel therapeutic target to prevent the development of atherosclerosis., (Copyright © 2017 the American Physiological Society.)

Secondary hyperparathyroidism is a well-known complication of end-stage renal disease (ESRD). Both nodular and diffuse parathyroid hyperplasia occur in ESRD patients. However, their distinct molecular mechanisms remain poorly understood. Parathyroid tissue obtained from ESRD patients who had undergone parathyroidectomy was used for Illumina transcriptome screening and subsequently for discriminatory gene analysis, pathway mapping, and gene annotation enrichment analysis. Results were further validated using quantitative RT-PCR on the independent larger cohort. Microarray screening proved homogeneity of gene transcripts in hemodialysis patients compared with the transplant cohort and primary hyperparathyroidism; therefore, further experiments were performed in hemodialysis patients only. Enrichment analysis conducted on 485 differentially expressed genes between nodular and diffuse parathyroid hyperplasia revealed highly significant differences in Gene Ontology terms and the Kyoto Encyclopedia of Genes and Genomes database in ribosome structure (P = 3.70 × 10 -18 ). Next, quantitative RT-PCR validation of the top differently expressed genes from microarray analysis proved higher expression of RAN guanine nucleotide release factor (RANGRF; P < 0.001), calcyclin-binding protein (CACYBP; P < 0.05), and exocyst complex component 8 (EXOC8; P < 0.05) and lower expression of peptidylprolyl cis/trans-isomerase and NIMA-interacting 1 (PIN1; P < 0.01) mRNA in nodular hyperplasia. Multivariate analysis revealed higher RANGRF and lower PIN1 expression along with parathyroid weight to be associated with nodular hyperplasia. In conclusion, our study suggests the RANGRF transcript, which controls RNA metabolism, to be likely involved in pathways associated with the switch to nodular parathyroid growth. This transcript, along with PIN1 transcript, which influences parathyroid hormone secretion, may represent new therapeutical targets to cure secondary hyperparathyroidism., (Copyright © 2016 the American Physiological Society.)

In angiotensin II (ANG II)-dependent hypertension, there is an angiotensin type 1 receptor-dependent amplification mechanism enhancing intrarenal angiotensinogen (AGT) formation and secretion in the tubular fluid. To evaluate the role of increased arterial pressure, AGT mRNA, protein expression, and urinary AGT (uAGT) excretion and tissue injury were assessed in both kidneys of two-kidney, one-clip Sprague-Dawley hypertensive rats subjected to left renal arterial clipping (0.25-mm gap). By 18-21 days, systolic arterial pressure increased to 180 ± 3 mmHg, and uAGT increased. Water intake, body weights, 24-h urine volumes, and sodium excretion were similar. In separate measurements of renal function in anesthetized rats, renal plasma flow and glomerular filtration rate were similar in clipped and nonclipped kidneys and not different from those in sham rats, indicating that the perfusion pressure to the clipped kidneys remained within the autoregulatory range. The nonclipped kidneys exhibited increased urine flow and sodium excretion. The uAGT excretion was significantly greater in nonclipped kidneys compared with clipped and sham kidneys. AGT mRNA was 2.15-fold greater in the nonclipped kidneys compared with sham (1.0 ± 0.1) or clipped (0.98 ± 0.15) kidneys. AGT protein levels were also greater in the nonclipped kidneys. The nonclipped kidneys exhibited greater glomerular expansion and immune cell infiltration, medullary fibrosis, and cellular proliferation than the clipped kidneys. Because both kidneys have elevated ANG II levels, the greater tissue injury in the nonclipped kidneys indicates that an increased arterial pressure synergizes with increased intrarenal ANG II to stimulate AGT production and exert greater renal injury., (Copyright © 2016 the American Physiological Society.)

Hepatic glucose production (HGP) normally begins just prior to birth. Prolonged fetal hypoglycemia, intrauterine growth restriction, and acute hypoxemia produce an early activation of fetal HGP. To test the hypothesis that prolonged hypoxemia increases factors which regulate HGP, studies were performed in fetuses that were bled to anemic conditions (anemic: n = 11) for 8.9 ± 0.4 days and compared with control fetuses (n = 7). Fetal arterial hematocrit and oxygen content were 32% and 50% lower, respectively, in anemic vs. controls (P < 0.005). Arterial plasma glucose was 15% higher in the anemic group (P < 0.05). Hepatic mRNA expression of phosphonenolpyruvate carboxykinase (PCK1) was twofold higher in the anemic group (P < 0.05). Arterial plasma glucagon concentrations were 70% higher in anemic fetuses compared with controls (P < 0.05), and they were positively associated with hepatic PCK1 mRNA expression (P < 0.05). Arterial plasma cortisol concentrations increased 90% in the anemic fetuses (P < 0.05), but fetal cortisol concentrations were not correlated with hepatic PCK1 mRNA expression. Hepatic glycogen content was 30% lower in anemic vs. control fetuses (P < 0.05) and was inversely correlated with fetal arterial plasma glucagon concentrations. In isolated primary fetal sheep hepatocytes, incubation in low oxygen (3%) increased PCK1 mRNA threefold compared with incubation in normal oxygen (21%). Together, these results demonstrate that glucagon and PCK1 may potentiate fetal HGP during chronic fetal anemic hypoxemia., (Copyright © 2016 the American Physiological Society.)

Estradiol (E2) decreases both water and saline intakes by female rats. The ERα and ERβ subtypes are expressed in areas of the brain that control fluid intake; however, the role that these receptors play in E2's antidipsogenic and antinatriorexigenic effects have not been examined. Accordingly, we tested the hypothesis that activation of ERα and ERβ decreases water and saline intakes by female rats. We found a divergence in E2's inhibitory effect on intake: activation of ERα decreased water intake, whereas activation of ERβ decreased saline intake. E2 decreases expression of the angiotensin II type 1 receptor (AT1R), a receptor with known relevance to water and salt intakes, in multiple areas of the brain where ERα and ERβ are differentially expressed. Therefore, we tested for agonist-induced changes in AT1R mRNA expression by RT-PCR and protein expression by analyzing receptor binding to test the hypothesis that the divergent effects of these ER subtypes are mediated by region-specific changes in AT1R expression. Although we found no changes in AT1R mRNA or binding in areas of the brain known to control fluid intake associated with agonist treatment, the experimental results replicate and extend previous findings that body weight changes mediate alterations in AT1R expression in distinct brain regions. Together, the results reveal selective effects of ER subtypes on ingestive behaviors, advancing our understanding of E2's inhibitory role in the controls of fluid intake by female rats., (Copyright © 2016 the American Physiological Society.)

To investigate age-associated impairments in fluid homeostasis, 4-mo (young) and 32-mo (old) Fischer 344/BN male rats were studied before and after a dietary sodium load. Transferring young rats from a low-sodium (LS) to a high-sodium (HS) diet increased water intake and urine volume by 1.9- and 3.0-fold, respectively, while urine osmolality and plasma aldosterone decreased by 33 and 98%. Concomitantly, adrenocortical angiotensin type 1 receptor (AT1R) density decreased by 35%, and AT1bR mRNA decreased by 39%; no changes were observed in AT1aR mRNA. In contrast, the increase in water intake (1.4-fold) was lower in the old rats, and there was no effect of the HS diet on urine volume or urine osmolality. AT1R densities were 29% less in the old rats before transferring to the HS diet, and AT1R densities were not reduced as rapidly in response to a HS diet compared with the young animals. After 6 days on the HS diet, plasma potassium was lowered by 26% in the old rats, whereas no change was detected in the young rats. Furthermore, while plasma aldosterone was substantially decreased after 2 days on the HS diet in both young and old rats, plasma aldosterone was significantly lower in the old compared with the young animals after 2 wk on the LS diet. These findings suggest that aging attenuates the responsiveness of the adrenocortical AT1R to a sodium load through impaired regulation of AT1bR mRNA, and that this dysregulation contributes to the defects in water and electrolyte homeostasis observed in aging., (Copyright © 2016 the American Physiological Society.)

Recent research suggest that anoxia-tolerant fish transfer extracellular nitrite into the tissues, where it is used for nitric oxide (NO) generation, iron-nitrosylation, and S-nitrosation of proteins, as part of the cytoprotective response toward prolonged hypoxia and subsequent reoxygenation. We hypothesized that crucian carp take up ambient nitrite and use it as a source of cellular NO availability during hypoxia. Fish were exposed for 1 day to normoxia (Po2 > 140 mmHg) and deep hypoxia (1 < Po2 < 3 mmHg) at both low (< 0.2 μM) and moderately elevated (10 μM) ambient [nitrite] to decipher NO metabolites in plasma and several tissues. We also compared NO metabolite changes during acute (10 min) and chronic (1 day) exposures to three different O2 levels. Plasma [nitrite] decreased with decreasing [O2], while the cellular concentrations of nitrite and nitros(yl)ated compounds either increased or stayed constant, depending on O2 level and tissue type. Nitrite was notably increased in the heart during deep hypoxia, and the increase was amplified by elevated ambient [nitrite]. Raised nitrite also increased gill [nitrite] and decreased mRNA expression of an inducible nitric oxide synthase-2 gene variant. The data support that ambient nitrite is taken up across the gills to be distributed via the blood to the tissues, particularly the heart, where it assists in cytoprotection and other functions. Cardiac nitrite was not elevated in acutely exposed fish, revealing that the response requires time. NO metabolite levels were higher during acute than chronic exposures, possibly caused by increased swimming activity and stress in acutely exposed fish., (Copyright © 2016 the American Physiological Society.)

Prolonged food deprivation in mammals typically reduces glucose, insulin, and thyroid hormone (TH) concentrations, as well as tissue deiodinase (DI) content and activity, which, collectively, suppress metabolism. However, in elephant seal pups, prolonged fasting does not suppress TH levels; it is associated with upregulation of adipose TH-mediated cellular mechanisms and adipose-specific insulin resistance. The functional relevance of this apparent paradox and the effects of glucose and insulin on TH-mediated signaling in an insulin-resistant tissue are not well defined. To address our hypothesis that insulin increases adipose TH signaling in pups during extended fasting, we assessed the changes in TH-associated genes in response to an insulin infusion in early- and late-fasted pups. In late fasting, insulin increased DI1, DI2, and THrβ-1 mRNA expression by 566%, 44%, and 267% at 60 min postinfusion, respectively, with levels decreasing by 120 min. Additionally, we performed a glucose challenge in late-fasted pups to differentiate between insulin- and glucose-mediated effects on TH signaling. In contrast to the insulin-induced effects, glucose infusion did not increase the expressions of DI1, DI2, and THrβ-1 until 120 min, suggesting that glucose delays the onset of the insulin-induced effects. The data also suggest that fasting duration increases the sensitivity of adipose TH-mediated mechanisms to insulin, some of which may be mediated by increased glucose. These responses appear to be unique among mammals and to have evolved in elephant seals to facilitate their adaptation to tolerate an extreme physiological condition., (Copyright © 2016 the American Physiological Society.)

Adropin, a recently described peptide hormone produced in the brain and liver, has been reported to have physiologically relevant actions on glucose homeostasis and lipogenesis, and to exert significant effect on endothelial function. We describe a central nervous system action of adropin to inhibit water drinking and identify a potential adropin receptor, the orphan G protein-coupled receptor, GPR19. Reduction in GPR19 mRNA levels in medial basal hypothalamus of male rats resulted in the loss of the inhibitory effect of adropin on water deprivation-induced thirst. The identification of a novel brain action of adropin and a candidate receptor for the peptide should extend and accelerate the study of the potential therapeutic value of adropin or its mimetics for the treatment of metabolic disorders., (Copyright © 2016 the American Physiological Society.)

Muscle-restricted coiled-coil protein (MURC), also referred to as cavin-4, is a member of the cavin family that works cooperatively with caveolins in caveola formation and function. Cavins are cytoplasmic proteins with coiled-coil domains and form heteromeric complexes, which are recruited to caveolae in cells expressing caveolins. Among caveolins, caveolin-3 (Cav3) is exclusively expressed in muscle cells, similar to MURC/cavin-4. In the heart, Cav3 overexpression contributes to cardiac protection, and its deficiency leads to progressive cardiomyopathy. Mutations in the MURC/cavin-4 gene have been identified in patients with dilated cardiomyopathy. In the present study, we show the role of MURC/cavin-4 as a caveolar component in the heart. In H9c2 cells, MURC/cavin-4 was localized at the plasma membrane, whereas a MURC/cavin-4 mutant lacking the coiled-coil domain (ΔCC) was primarily localized to the cytoplasm. ΔCC bound to Cav3 and impaired membrane localization of Cav3 in cardiomyocytes. Additionally, although ΔCC did not alter Cav3 mRNA expression, ΔCC decreased the Cav3 protein level. MURC/cavin-4 and ΔCC similarly induced cardiomyocyte hypertrophy; however, ΔCC showed higher hypertrophy-related fetal gene expression than MURC/cavin-4. ΔCC induced ERK activation in cardiomyocytes. Transgenic mice expressing ΔCC in the heart (ΔCC-Tg mice) showed impaired cardiac function accompanied by cardiomyocyte hypertrophy and marked interstitial fibrosis. Hearts from ΔCC-Tg mice showed a reduction of the Cav3 protein level and activation of ERK. These results suggest that MURC/cavin-4 requires its coiled-coil domain to target the plasma membrane and to stabilize Cav3 at the plasma membrane of cardiomyocytes and that MURC/cavin-4 functions as a crucial caveolar component to regulate cardiac function., (Copyright © 2015 the American Physiological Society.)

Obesity is associated with increased fibrinogen production and fibrin formation, which produces fibrin degradation products (FDP-E and FDP-D). Fibrin and FDPs both contribute to inflammation, which would be expected to suppress glucose uptake and insulin signaling in adipose tissue, yet the effect of FDP-E and FDP-D on adipocyte function and glucose disposal is completely unknown. We tested the effects of FDPs on inflammation in 3T3-L1 adipocytes and primary macrophages and adipocyte glucose uptake in vitro. High-fat-fed mice increased hepatic fibrinogen mRNA expression ninefold over chow-fed mice, with concomitant increases in plasma fibrinogen protein levels. Obese mice also displayed increased fibrinogen content of epididymal fat pads. We treated cultured 3T3-L1 adipocytes and primary macrophages with FDP-E, FDP-D, or fibrinogen degradation products (FgnDP-E). FDP-D and FgnDP-E had no effect on inflammation or glucose uptake. Cytokine mRNA expression in RAW264.7 macrophage-like cells and 3T3-L1 adipocytes treated with FDP-E induced inflammation with maximal effects at 100 nM and 6 h. Insulin-stimulated 2-deoxy-d-[(3)H]glucose uptake was reduced by 71% in adipocytes treated with FDP-E. FDP-E, but not FDP-D or FgnDP-E, induces inflammation in macrophages and adipocytes and decreases glucose uptake in vitro. FDP-E may contribute toward obesity-associated acute inflammation and glucose intolerance, although its chronic role in obesity remains to be elucidated., (Copyright © 2015 the American Physiological Society.)

We tested the hypothesis that ingestion of sodium bicarbonate (NaHCO3) prior to an acute session of high-intensity interval training (HIIT) would augment signaling cascades and gene expression linked to mitochondrial biogenesis in human skeletal muscle. On two occasions separated by ∼1 wk, nine men (mean ± SD: age 22 ± 2 yr, weight 78 ± 13 kg, V̇O(2 peak) 48 ± 8 ml·kg(-1)·min(-1)) performed 10 × 60-s cycling efforts at an intensity eliciting ∼90% of maximal heart rate (263 ± 40 W), interspersed with 60 s of recovery. In a double-blind, crossover manner, subjects ingested a total of 0.4 g/kg body weight NaHCO3 before exercise (BICARB) or an equimolar amount of a placebo, sodium chloride (PLAC). Venous blood bicarbonate and pH were elevated at all time points after ingestion (P < 0.05) in BICARB vs. PLAC. During exercise, muscle glycogen utilization (126 ± 47 vs. 53 ± 38 mmol/kg dry weight, P < 0.05) and blood lactate accumulation (12.8 ± 2.6 vs. 10.5 ± 2.8 mmol/liter, P < 0.05) were greater in BICARB vs. PLAC. The acute exercise-induced increase in the phosphorylation of acetyl-CoA carboxylase, a downstream marker of AMP-activated protein kinase activity, and p38 mitogen-activated protein kinase were similar between treatments (P > 0.05). However, the increase in PGC-1α mRNA expression after 3 h of recovery was higher in BICARB vs. PLAC (approximately sevenfold vs. fivefold compared with rest, P < 0.05). We conclude that NaHCO3 before HIIT alters the mRNA expression of this key regulatory protein associated with mitochondrial biogenesis. The elevated PGC-1α mRNA response provides a putative mechanism to explain the enhanced mitochondrial adaptation observed after chronic HIIT supplemented with NaHCO3 in rats., (Copyright © 2015 the American Physiological Society.)

Transgenic mouse models are designed to study the role of specific proteins. To increase transgene expression the human growth hormone (hGH) minigene, including introns, has been included in many transgenic constructs. Until recently, it was thought that the hGH gene was not spliced, transcribed, and translated to produce functional hGH protein. We generated a transgenic mouse with the transcription factor Forkhead box M1 (FoxM1) followed by the hGH minigene, under control of the mouse insulin promoter (MIP) to target expression specifically in the pancreatic β-cell. Expression of FoxM1 in isolated pancreatic islets in vitro stimulates β-cell proliferation. We aimed to investigate the effect of FoxM1 on β-cell mass in a mouse model for diabetes mellitus. However, we found inadvertent coexpression of hGH protein from a spliced, bicistronic mRNA. MIP-FoxM1-hGH mice had lower blood glucose and higher pancreatic insulin content, due to increased β-cell proliferation. hGH signals through the murine prolactin receptor, and expression of its downstream targets tryptophan hydroxylase-1 (Tph1), tryptophan hydroxylase-2 (Tph2), and cytokine-inducible SH2 containing protein (Cish) was increased. Conversely, transcriptional targets of FoxM1 were not upregulated. Our data suggest that the phenotype of MIP-FoxM1-hGH mice is due primarily to hGH activity and that the FoxM1 protein remains largely inactive. Over the past decades, multiple transgenic mouse strains were generated that make use of the hGH minigene to increase transgene expression. Our work suggests that each will need to be carefully screened for inadvertent hGH production and critically evaluated for the use of proper controls.

Limited data exist on the efficacy of low-load blood flow-restricted strength training (BFR), as compared directly to heavy-load strength training (HST). Here, we show that 12 wk of twice-a-week unilateral BFR [30% of one repetition maximum (1RM) to exhaustion] and HST (6-10RM) of knee extensors provide similar increases in 1RM knee extension and cross-sectional area of distal parts of musculus quadriceps femoris in nine untrained women (age 22 ± 1 yr). The two protocols resulted in similar acute increases in serum levels of human growth hormone. On the cellular level, 12 wk of BFR and HST resulted in similar shifts in muscle fiber composition in musculus vastus lateralis, evident as increased MyHC2A proportions and decreased MyHC2X proportions. They also resulted in similar changes of the expression of 29 genes involved in skeletal muscle function, measured both in a rested state following 12 wk of training and subsequent to singular training sessions. Training had no effect on myonuclei proportions. Of particular interest, 1) gross adaptations to BFR and HST were greater in individuals with higher proportions of type 2 fibers, 2) both BFR and HST resulted in approximately four-fold increases in the expression of the novel exercise-responsive gene Syndecan-4, and 3) BFR provided lesser hypertrophy than HST in the proximal half of musculus quadriceps femoris and also in CSApeak, potentially being a consequence of pressure from the tourniquet utilized to achieve blood flow restriction. In conclusion, BFR and HST of knee extensors resulted in similar adaptations in functional, physiological, and cell biological parameters in untrained women., (Copyright © 2015 the American Physiological Society.)

Siberian hamsters (Phodopus sungorus) show spontaneous daily torpor only after ∼2 mo in winter-like short photoperiods (SP). Although some SP-induced hormonal changes have been demonstrated to be necessary for the occurrence of seasonal torpor, the whole set of preconditions is still unknown. Recent findings provide evidence that the hypothalamic pituitary growth axis is involved in endocrine responses to SP exposure in the photoperiodic hamsters. To examine whether suppression of growth hormone (GH) and insulin-like growth factor-1 (IGF-1) secretion affects the incidence of daily torpor, we used two somatostatin receptor agonists, pasireotide (SOM230) and octreotide, with different affinity profiles for receptor subtypes. Pasireotide strikingly increased the torpor frequency in male hamsters compared with sham-treated controls, and torpor duration was often increased, which in some cases exceeded 12 h. In contrast, administration of octreotide reduced the body weight of SP hamsters but had only a marginal effect on torpor frequency in males and no effect in females. Together with measured concentrations of circulating IGF-1, the present results strongly suggest that reduced activity of the GH/IGF-1 axis is not critical for stimulation of torpor expression but activation of specific somatostatin receptors is critical. This putative role for certain somatostatin receptor subtypes in torpor induction provides a promising new approach to unravel the endocrine mechanisms of torpor regulation., (Copyright © 2015 the American Physiological Society.)

Butyrate is a major metabolite in colonic lumen. It is produced from bacterial fermentation of dietary fiber. Butyrate has been shown to stimulate electroneutral sodium absorption through its regulation on sodium/hydrogen exchanger 3 (NHE3). Although NHE8, the newest addition of intestinal NHE family, is involved in sodium absorption in the intestinal tract, whether butyrate modulates NHE8 expression in the intestinal epithelial cells is not known. In the current study, we showed that butyrate treatment strongly induced NHE8 protein and NHE8 mRNA expression in human intestinal epithelial cells. Transfection with the human NHE8 promoter reporter constructs showed that butyrate treatment stimulated reporter gene expression at an amount comparable with its stimulation of NHE8 mRNA expression. Interestingly, a similar result was also observed in human NHE8 promoter transfected cells after trichostatin (TSA) treatment. Gel mobility shift assay identified an enhanced Sp3 protein binding on the human NHE8 basal promoter region upon butyrate stimulation. Furthermore, Sp3 acetylation modification is involved in butyrate-mediated NHE8 activation in Caco-2 cells. Our findings suggest that the mechanism of butyrate action on NHE8 expression involves enhanced Sp3 interaction at the basal promoter region of the human NHE8 gene promoter to activate NHE8 gene transcription. Thus butyrate is involved in intestinal regulation of NHE8 resulting enhanced sodium absorption., (Copyright © 2015 the American Physiological Society.)

Cytokines drive many inflammatory diseases, including asthma. Understanding the molecular mechanisms responsible for cytokine secretion will allow us to develop novel strategies to repress inflammation in the future. Harnessing the power of endogenous anti-inflammatory proteins is one such strategy. In this study, we investigate the p38 MAPK-mediated regulatory interaction of two anti-inflammatory proteins, mitogen-activated protein kinase phosphatase 1 (MKP-1) and tristetraprolin (TTP), in the context of asthmatic inflammation. Using primary cultures of airway smooth muscle cells in vitro, we explored the temporal regulation of IL-6 cytokine mRNA expression upon stimulation with TNF-α. Intriguingly, the temporal profile of mRNA expression was biphasic. This was not due to COX-2-derived prostanoid upregulation, increased expression of NLRP3 inflammasome components, or upregulation of the cognate receptor for TNF-α-TNFR1. Rather, the biphasic nature of TNF-α-induced IL-6 mRNA expression was regulated temporally by the RNA-destabilizing molecule, TTP. Importantly, TTP function is controlled by p38 MAPK, and our study reveals that its expression in airway smooth muscle cells is p38 MAPK-dependent and its anti-inflammatory activity is also controlled by p38 MAPK-mediated phosphorylation. MKP-1 is a MAPK deactivator; thus, by controlling p38 MAPK phosphorylation status in a temporally distinct manner, MKP-1 ensures that TTP is expressed and made functional at precisely the correct time to repress cytokine expression. Together, p38 MAPK, MKP-1, and TTP may form a regulatory network that exerts significant control on cytokine secretion in proasthmatic inflammation through precise temporal signaling., (Copyright © 2015 the American Physiological Society.)

Supplemental oxygen, used to treat hypoxia in preterm and term neonates, increases the risk of neonatal lung diseases, such as bronchopulmonary dysplasia (BPD) and asthma. There is a known sex predilection for BPD, but the underlying mechanisms are not clear. We tested the hypothesis that altered, local estradiol following hyperoxia contributes to pathophysiological changes observed in immature lung. In human fetal airway smooth muscle (fASM) cells exposed to normoxia or hyperoxia, we measured the expression of proteins involved in estrogen metabolism and cell proliferation responses to estradiol. In fASM cells, CYP1a1 expression was increased by hyperoxia, whereas hyperoxia-induced enhancement of cell proliferation was blunted by estradiol. Pharmacological studies indicated that these effects were attributable to upregulation of CYP1a1 and subsequent increased metabolism of estradiol to a downstream intermediate 2-methoxyestradiol. Microarray analysis of mouse lung exposed to 14 days of hyperoxia showed the most significant alteration in CYP1a1 expression, with minimal changes in expression of five other genes related to estrogen receptors, synthesis, and metabolism. Our novel results on estradiol metabolism in fetal and early postnatal lung in the context of hyperoxia indicate CYP1a1 as a potential mechanism for the protective effect of estradiol in hyperoxia-exposed immature lung, which may help explain the sex difference in neonatal lung diseases., (Copyright © 2015 the American Physiological Society.)

Surfactant protein A (SP-A), a molecule with roles in lung innate immunity and surfactant-related functions, is encoded by two genes in humans: SFTPA1 (SP-A1) and SFTPA2 (SP-A2). The mRNAs from these genes differ in their 5'-untranslated regions (5'-UTR) due to differential splicing. The 5'-UTR variant ACD' is exclusively found in transcripts of SP-A1, but not in those of SP-A2. Its unique exon C contains two upstream AUG codons (uAUGs) that may affect SP-A1 translation efficiency. The first uAUG (u1) is in frame with the primary start codon (p), but the second one (u2) is not. The purpose of this study was to assess the impact of uAUGs on SP-A1 expression. We employed RT-qPCR to determine the presence of exon C-containing SP-A1 transcripts in human RNA samples. We also used in vitro techniques including mutagenesis, reporter assays, and toeprinting analysis, as well as in silico analyses to determine the role of uAUGs. Exon C-containing mRNA is present in most human lung tissue samples and its expression can, under certain conditions, be regulated by factors such as dexamethasone or endotoxin. Mutating uAUGs resulted in increased luciferase activity. The mature protein size was not affected by the uAUGs, as shown by a combination of toeprint and in silico analysis for Kozak sequence, secondary structure, and signal peptide and in vitro translation in the presence of microsomes. In conclusion, alternative splicing may introduce uAUGs in SP-A1 transcripts, which in turn negatively affect SP-A1 translation, possibly affecting SP-A1/SP-A2 ratio, with potential for clinical implication., (Copyright © 2015 the American Physiological Society.)

REDD1 (regulated in development and DNA damage response 1) has been proposed to inhibit the mechanistic target of rapamycin complex 1 (mTORC1) during in vitro hypoxia. REDD1 expression is low under basal conditions but is highly increased in response to several catabolic stresses, like hypoxia and glucocorticoids. However, REDD1 function seems to be tissue and stress dependent, and its role in skeletal muscle in vivo has been poorly characterized. Here, we investigated the effect of REDD1 deletion on skeletal muscle mass, protein synthesis, proteolysis, and mTORC1 signaling pathway under basal conditions and after glucocorticoid administration. Whereas skeletal muscle mass and typology were unchanged between wild-type (WT) and REDD1-null mice, oral gavage with dexamethasone (DEX) for 7 days reduced tibialis anterior and gastrocnemius muscle weights as well as tibialis anterior fiber size only in WT. Similarly, REDD1 deletion prevented the inhibition of protein synthesis and mTORC1 activity (assessed by S6, 4E-BP1, and ULK1 phosphorylation) observed in gastrocnemius muscle of WT mice following single DEX administration for 5 h. However, our results suggest that REDD1-mediated inhibition of mTORC1 in skeletal muscle is not related to the modulation of the binding between TSC2 and 14-3-3. In contrast, our data highlight a new mechanism involved in mTORC1 inhibition linking REDD1, Akt, and PRAS40. Altogether, these results demonstrated in vivo that REDD1 is required for glucocorticoid-induced inhibition of protein synthesis via mTORC1 downregulation. Inhibition of REDD1 may thus be a strategy to limit muscle loss in glucocorticoid-mediated atrophy., (Copyright © 2014 the American Physiological Society.)

Aberrant expression of master phenotype regulators or alterations in their downstream pathways in lung fibroblasts may play a central role in idiopathic pulmonary fibrosis (IPF). Interrogating IPF fibroblast transcriptome datasets, we identified Forkhead Box F1 (FOXF1), a DNA-binding protein required for lung development, as a candidate actor in IPF. Thus we determined FOXF1 expression levels in fibroblasts cultured from normal or IPF lungs in vitro, and explored FOXF1 functions in these cells using transient and stable loss-of-function and gain-of-function models. FOXF1 mRNA and protein were expressed at higher levels in IPF fibroblasts compared with normal fibroblasts (mRNA: +44%, protein: +77%). Immunohistochemistry showed FOXF1 expression in nuclei of bronchial smooth muscle cells, endothelial cells, and lung fibroblasts including fibroblastic foci of IPF lungs. In normal lung fibroblasts, FOXF1 repressed cell growth and expression of collagen-1 (COL1) and actin-related protein 2/3 complex, subunit 2 (ARPC2). ARPC2 knockdown inhibited cell growth and COL1 expression, consistent with FOXF1 acting in part through ARPC2 repression. In IPF fibroblasts, COL1 and ARPC2 repression by FOXF1 was blunted, and FOXF1 did not repress growth. FOXF1 expression was induced by the antifibrotic mediator prostaglandin E2 and repressed by the profibrotic cytokine transforming growth factor-β1 in both normal and IPF lung fibroblasts. Ex vivo, FOXF1 knockdown conferred CCL-210 lung fibroblasts the ability to implant in uninjured mouse lungs. In conclusion, FOXF1 functions and regulation were consistent with participation in antifibrotic pathways. Alterations of pathways downstream of FOXF1 may participate to fibrogenesis in IPF fibroblasts., (Copyright © 2014 the American Physiological Society.)

Lung alveolar epithelial cells are exposed to hypoxia under a variety of physiological and pathological conditions. It has been shown recently that miR-143, which can directly target the key glycolytic enzyme hexokinase II (HK2), may be regulated by miR-155. We investigated whether microRNAs contribute to the cellular glycolysis in response to hypoxia. Using the A549 cells, we found that the expression of Dicer is decreased under hypoxia. When Dicer was knocked down with small-interfering RNA (siRNA), pre-miR143 was increased and mature miR-143 was decreased as that in hypoxia, indicating that reduction of Dicer is responsible for the change of miR-143 under hypoxia. Interestingly, both hypoxia and knockdown of Dicer resulted in miR-155 and pre-miR-155 expression increases. We also examined the expression of HK2 and glucose metabolism in the cells. Both HK2 mRNA and protein were increased under hypoxia, which is accompanied by an increase of glucose uptake and production of lactate. The same alterations were found with siRNA Dicer knockdown. Moreover, transfection with anti-miR-143 also led to a HK2 production and an increase of glucose uptake and lactate production, whereas anti-miR-155 had opposite effects. The miR-143 and anti-miR-155 transfection resulted in a significant cell apoptosis. The expression of Dicer was decreased with HK2 accumulating in mouse lung tissues under hypoxia identified by immunohistochemistry. The changes of miR-143 and miR-155 were similar to those in A549 cells. Our data demonstrate that Dicer regulation of miRNAs promotes HK2 activation and glycolysis, which might protect the cell from hypoxic damage and enter into an adaptive process., (Copyright © 2014 the American Physiological Society.)

Muscle-specific RING finger-1 (MuRF-1), a ubiquitin ligase and key regulator of proteasome-dependent protein degradation, is highly expressed during skeletal muscle atrophy. The transcription factor forkhead box O3 (FoxO3) induces MuRF-1 expression, but the direct role of other major atrophy-related transcription factors, such as SMAD3, is largely unknown. The goal of this study was to determine whether SMAD3 individually regulates, or with FoxO3 coordinately regulates, MuRF-1 expression. In cultured myotubes or human embryonic kidney cells, MuRF-1 mRNA content and promoter activity were increased by FoxO3 but not by SMAD3 overexpression. However, FoxO3 and SMAD3 coexpression synergistically increased MuRF-1 mRNA and promoter activity. Mutation of the SMAD-binding element (SBE) in the proximal MuRF-1 promoter or overexpression of a SMAD3 DNA-binding mutant attenuated FoxO3-dependent MuRF-1 promoter activation, showing that SMAD binding to DNA is required for optimal activation of FoxO3-induced transcription of MuRF-1. Using chromatin immunoprecipitation, SMAD3 DNA binding increased FoxO3 abundance and SBE mutation reduced FoxO3 abundance on the MuRF-1 promoter. Furthermore, SMAD3 overexpression dose-dependently increased FoxO3 protein content, and coexpression of FoxO3 and SMAD3 synergistically increased FoxO-dependent gene transcription [assessed with a FoxO response element (FRE)-driven reporter]. Collectively, these results show that SMAD3 regulates transcription of MuRF-1 by increasing FoxO3 binding at a conserved FRE-SBE motif within the proximal promoter region, and by increasing FoxO3 protein content and transcriptional activity. These data are the first to indicate that two major transcription factors regulating protein degradation, FoxO3 and SMAD3, converge to coordinately and directly regulate transcription of MuRF-1., (Copyright © 2014 the American Physiological Society.)

Our knowledge of the molecular mechanisms underlying human embryonic stem cell (hESC) self-renewal and differentiation is incomplete. The level of octamer-binding transcription factor 4 (Oct4), a critical regulator of pluripotency, is precisely controlled in mouse embryonic stem cells. However, studies of human OCT4 are often confounded by the presence of three isoforms and six expressed pseudogenes, which has complicated the interpretation of results. Using an inducible lentiviral overexpression and knockdown system to manipulate OCT4A above or below physiological levels, we specifically examine the functional role of the OCT4A isoform in hESC. (We also designed and generated a comparable series of vectors, which were not functional, for the overexpression and knockdown of OCT4B.) We show that specific knockdown of OCT4A results in hESC differentiation, as indicated by morphology changes, cell surface antigen expression, and upregulation of ectodermal genes. In contrast, inducible overexpression of OCT4A in hESC leads to a transient instability of the hESC phenotype, as indicated by changes in morphology, cell surface antigen expression, and transcriptional profile, that returns to baseline within 5 days. Interestingly, sustained expression of OCT4A past 5 days enhances hESC cloning efficiency, suggesting that higher levels of OCT4A can support self-renewal. Overall, our results indicate that high levels of OCT4A increase hESC cloning efficiency and do not induce differentiation (whereas OCT4B expression cannot be induced in hESC), highlighting the importance of isoform-specific studies in a stable and inducible expression system for human OCT4. Additionally, we demonstrate the utility of an efficient method for conditional gene expression in hESC., (Copyright © 2014 the American Physiological Society.)

CCK and leptin are anorectic hormones produced in the small intestine and white adipose tissue, respectively. Investigating how these hormones act together as an integrated anorectic signal is important for elucidating the mechanisms by which energy balance is maintained. We found here that coadministration of subthreshold CCK and leptin, which individually have no effect on feeding, dramatically reduced food intake in rats. Phosphorylation of AMP-activated protein kinase (AMPK) in the hypothalamus significantly decreased after coinjection of CCK and leptin. In addition, coadministration of these hormones significantly increased mRNA levels of anorectic cocaine- and amphetamine-regulated transcript (CART) and thyrotropin-releasing hormone (TRH) in the hypothalamus. The interactive effect of CCK and leptin on food intake was abolished by intracerebroventricular preadministration of the AMPK activator AICAR or anti-CART/anti-TRH antibodies. These findings indicate that coinjection of CCK and leptin reduces food intake via reduced AMPK phosphorylation and increased CART/TRH in the hypothalamus. Furthermore, by using midbrain-transected rats, we investigated the role of the neural pathway from the hindbrain to the hypothalamus in the interaction of CCK and leptin to reduce food intake. Food intake reduction induced by coinjection of CCK and leptin was blocked in midbrain-transected rats. Therefore, the neural pathway from hindbrain to hypothalamus plays an important role in transmitting the anorectic signals provided by coinjection of CCK and leptin. Our findings give further insight into the mechanisms of feeding and energy balance., (Copyright © 2014 the American Physiological Society.)

A loss of glucose effectiveness to suppress hepatic glucose production as well as increase hepatic glucose uptake and storage as glycogen is associated with a defective increase in glucose phosphorylation catalyzed by glucokinase (GK) in Zucker diabetic fatty (ZDF) rats. We extended these observations by investigating the role of persistent hyperglycemia (glucotoxicity) in the development of impaired hepatic GK activity in ZDF rats. We measured expression and localization of GK and GK regulatory protein (GKRP), translocation of GK, and hepatic glucose flux in response to a gastric mixed meal load (MMT) and hyperglycemic hyperinsulinemic clamp after 1 or 6 wk of treatment with the sodium-glucose transporter 2 inhibitor (canaglifrozin) that was used to correct the persistent hyperglycemia of ZDF rats. Defective augmentation of glucose phosphorylation in response to a rise in plasma glucose in ZDF rats was associated with the coresidency of GKRP with GK in the cytoplasm in the midstage of diabetes, which was followed by a decrease in GK protein levels due to impaired posttranscriptional processing in the late stage of diabetes. Correcting hyperglycemia from the middle diabetic stage normalized the rate of glucose phosphorylation by maintaining GK protein levels, restoring normal nuclear residency of GK and GKRP under basal conditions and normalizing translocation of GK from the nucleus to the cytoplasm, with GKRP remaining in the nucleus in response to a rise in plasma glucose. This improved the liver's metabolic ability to respond to hyperglycemic hyperinsulinemia. Glucotoxicity is responsible for loss of glucose effectiveness and is associated with altered GK regulation in the ZDF rat., (Copyright © 2014 the American Physiological Society.)

Hyperoxic acute lung injury (HALI) is characterized by inflammation and epithelial cell death. CLOCK genes are master regulators of circadian rhythm also implicated in inflammation and lung diseases. However, the relationship of CLOCK genes in hyperoxia-induced lung injury has not been studied. This study will determine if HALI alters CLOCK gene expression. To test this, wild-type and NALP3(-/-) mice were exposed to room air or hyperoxia for 24, 48, or 72 h. In addition, mice were exposed to different concentrations of hyperoxia (50, 75, or 100% O2) or room air for 72 h. The mRNA and protein levels of lung CLOCK genes, based on quantitative PCR and Western blot analysis, respectively, and their target genes are significantly elevated in mice exposed to hyperoxia compared with controls. Alterations in CLOCK genes are associated with increased inflammatory markers in bronchoalveolar lavage fluid of hyperoxic mice compared with controls. Histological examination of mice lungs exposed to hyperoxia show increased inflammation and alveolar congestion compared with controls. Our results indicate sequential increase in CLOCK gene expression in lungs of mice exposed to hyperoxia compared with controls. Additionally, data suggest a dose-dependent increase in CLOCK gene expression with increased oxygen concentrations. To validate if the expression changes related to CLOCK genes are indeed associated with inflammation, NALP3(-/-) was introduced to analyze loss of function in inflammation. Western blot analysis showed significant CLOCK gene downregulation in NALP3(-/-) mice compared with wild-type controls. Together, our results demonstrate that hyperoxia-mediated lung inflammation is associated with alterations in CLOCK gene expression., (Copyright © 2014 the American Physiological Society.)

In diabetes, when glucose uptake and oxidation are impaired, the heart is compelled to use fatty acid (FA) almost exclusively for ATP. The vascular content of lipoprotein lipase (LPL), the rate-limiting enzyme that determines circulating triglyceride clearance, is largely responsible for this FA delivery and increases following diabetes. Glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein [GPIHBP1; a protein expressed abundantly in the heart in endothelial cells (EC)] collects LPL from the interstitial space and transfers it across ECs onto the luminal binding sites of these cells, where the enzyme is functional. We tested whether ECs respond to hyperglycemia by increasing GPIHBP1. Streptozotocin diabetes increased cardiac LPL activity and GPIHBP1 gene and protein expression. The increased LPL and GPIHBP1 were located at the capillary lumen. In vitro, passaging EC caused a loss of GPIHBP1, which could be induced on exposure to increasing concentrations of glucose. The high-glucose-induced GPIHBP1 increased LPL shuttling across EC monolayers. GPIHBP1 expression was linked to the EC content of heparanase. Moreover, active heparanase increased GPIHBP1 gene and protein expression. Both ECs and myocyte heparan sulfate proteoglycan-bound platelet-derived growth factor (PDGF) released by heparanase caused augmentation of GPIHBP1. Overall, our data suggest that this protein "ensemble" (heparanase-PDGF-GPIHBP1) cooperates in the diabetic heart to regulate FA delivery and utilization by the cardiomyocytes. Interrupting this axis may be a novel therapeutic strategy to restore metabolic equilibrium, curb lipotoxicity, and help prevent or delay heart dysfunction that is characteristic of diabetes., (Copyright © 2014 the American Physiological Society.)

Skeletal muscle atrophy is prevalent in chronic diseases, and microRNAs (miRs) may play a key role in the wasting process. miR-23a was previously shown to inhibit the expression of atrogin-1 and muscle RING-finger protein-1 (MuRF1) in muscle. It also was reported to be regulated by cytoplasmic nuclear factor of activated T cells 3 (NFATc3) in cardiomyocytes. The objective of this study was to determine if miR-23a is regulated during muscle atrophy and to evaluate the relationship between calcineurin (Cn)/NFAT signaling and miR-23a expression in skeletal muscle cells during atrophy. miR-23a was decreased in the gastrocnemius of rats with acute streptozotocin-induced diabetes, a condition known to increase atrogin-1 and MuRF1 expression and cause atrophy. Treatment of C2C12 myotubes with dexamethasone (Dex) for 48 h also reduced miR-23a as well as RCAN1.4 mRNA, which is transcriptionally regulated by NFAT. NFATc3 nuclear localization and the amount of miR-23a decreased rapidly within 1 h of Dex administration, suggesting a link between Cn signaling and miR-23a. The level of miR-23a was lower in primary myotubes from mice lacking the α- or β-isoform of the CnA catalytic subunit than wild-type mice. Dex did not further suppress miR-23a in myotubes from Cn-deficient mice. Overexpression of CnAβ in C2C12 myotubes prevented Dex-induced suppression of miR-23a. Finally, miR-23a was present in exosomes isolated from the media of C2C12 myotubes, and Dex increased its exosomal abundance. Dex did not alter the number of exosomes released into the media. We conclude that atrophy-inducing conditions downregulate miR-23a in muscle by mechanisms involving attenuated Cn/NFAT signaling and selective packaging into exosomes.

Oxidant injury contributes to acute lung injury (ALI). We previously reported that activation of protein kinase GI (PKGI) posttranscriptionally increased the key antioxidant enzymes catalase and glutathione peroxidase 1 (Gpx-1) and attenuated oxidant-induced cytotoxicity in mouse lung microvascular endothelial cells (MLMVEC). The present studies tested the hypothesis that the antioxidant effect of PKGI is mediated via inhibition of the c-Abl tyrosine kinase. We found that activation of PKGI with the cGMP analog 8pCPT-cGMP inhibited c-Abl activity and decreased c-Abl expression in wild-type but not PKGI(-/-) MLMVEC. Treatment of wild-type MLMVEC with atrial natriuretic peptide also inhibited c-Abl activation. Moreover, treatment of MLMVEC with the c-Abl inhibitor imatinib increased catalase and GPx-1 protein in a posttranscriptional fashion. In imatinib-treated MLMVEC, there was no additional effect of 8pCPT-cGMP on catalase or GPx-1. The imatinib-induced increase in antioxidant proteins was associated with an increase in extracellular H2O2 scavenging by MLMVEC, attenuation of oxidant-induced endothelial barrier dysfunction, and prevention of oxidant-induced endothelial cell death. Finally, in the isolated perfused lung, imatinib prevented oxidant-induced endothelial toxicity. We conclude that cGMP, through activation of PKGI, inhibits c-Abl, leading to increased key antioxidant enzymes and resistance to lung endothelial oxidant injury. Inhibition of c-Abl by active PKGI may be the downstream mechanism underlying PKGI-mediated antioxidant signaling. Tyrosine kinase inhibitors may represent a novel therapeutic approach in oxidant-induced ALI.

Cardiac fibrosis, a known risk factor for heart disease, is typically caused by uncontrolled proliferation of fibroblasts and excessive deposition of extracellular matrix proteins in the myocardium. Cyclin-dependent kinase 1 (CDK1) is involved in the control of G2/M transit phase of the cell cycle. Here, we showed that isoproterenol (ISO)-induced cardiac fibrosis is associated with increased levels of CDK1 exclusively in fibroblasts in the adult mouse heart. Treatment of primary embryonic ventricular cell cultures with ISO (a nonselective β-adrenergic receptor agonist) increased CDK1 protein expression in fibroblasts and promoted their cell cycle activity. Quantitative PCR analysis confirmed that ISO increases CDK1 transcription in a transient manner. Further, the ISO-responsive element was mapped to the proximal -100-bp sequence of the CDK1 promoter region using various 5'-flanking sequence deletion constructs. Sequence analysis of the -100-bp CDK1 minimal promoter region revealed two putative nuclear factor-Y (NF-Y) binding elements. Overexpression of the NF-YA subunit in primary ventricular cultures significantly increased the basal activation of the -100-bp CDK1 promoter construct but not the ISO-induced transcription of the minimal promoter construct. In contrast, dominant negative NF-YA expression decreased the basal activity of the minimal promoter construct and ISO treatment fully rescued the dominant negative effects. Furthermore, site-directed mutagenesis of the distal NF-Y binding site in the -100-bp CDK1 promoter region completely abolished both basal and ISO-induced promoter activation of the CDK1 gene. Collectively, our results raise an exciting possibility that targeting CDK1 or NF-Y in the diseased heart may inhibit fibrosis and subsequently confer cardioprotection.

Adiponectin is an adipose-derived hormone with anti-inflammatory activity. Following subacute ozone exposure (0.3 ppm for 24-72 h), neutrophilic inflammation and IL-6 are augmented in adiponectin-deficient (Adipo(-/-)) mice. The IL-17/granulocyte colony-stimulating factor (G-CSF) axis is required for this increased neutrophilia. We hypothesized that elevated IL-6 in Adipo(-/-) mice contributes to their augmented responses to ozone via effects on IL-17A expression. Therefore, we generated mice deficient in both adiponectin and IL-6 (Adipo(-/-)/IL-6(-/-)) and exposed them to ozone or air. In ozone-exposed mice, bronchoalveolar lavage (BAL) neutrophils, IL-6, and G-CSF, and pulmonary Il17a mRNA expression were greater in Adipo(-/-) vs. wild-type mice, but reduced in Adipo(-/-)/IL-6(-/-) vs. Adipo(-/-) mice. IL-17A(+) F4/80(+) cells and IL-17A(+) γδ T cells were also reduced in Adipo(-/-)/IL-6(-/-) vs. Adipo(-/-) mice exposed to ozone. Only BAL neutrophils were reduced in IL-6(-/-) vs. wild-type mice. In wild-type mice, IL-6 was expressed in Gr-1(+)F4/80(-)CD11c(-) cells, whereas in Adipo(-/-) mice F4/80(+)CD11c(+) cells also expressed IL-6, suggesting that IL-6 is regulated by adiponectin in these alveolar macrophages. Transcriptomic analysis identified serum amyloid A3 (Saa3), which promotes IL-17A expression, as the gene most differentially augmented by ozone in Adipo(-/-) vs. wild-type mice. After ozone, Saa3 mRNA expression was markedly greater in Adipo(-/-) vs. wild-type mice but reduced in Adipo(-/-)/IL-6(-/-) vs. Adipo(-/-) mice. In conclusion, our data support a pivotal role of IL-6 in the hyperinflammatory condition observed in Adipo(-/-) mice after ozone exposure and suggest that this role of IL-6 involves its ability to induce Saa3, IL-17A, and G-CSF.

Cytosolic phospholipase A2 (cPLA2) plays a pivotal role in mediating agonist-induced arachidonic acid release for prostaglandin (PG) synthesis during inflammation triggered by tumor necrosis factor-α (TNF-α). However, the mechanisms underlying TNF-α-induced cPLA2 expression in human lung epithelial cells (HPAEpiCs) were not completely understood. Here, we demonstrated that TNF-α induced cPLA2 mRNA and protein expression, promoter activity, and PGE2 secretion in HPAEpiCs. These responses induced by TNF-α were inhibited by pretreatment with the inhibitor of Jak2 (AG490), platelet-derived growth factor receptor (PDGFR) (AG1296), phosphoinositide 3 kinase (PI3K) (LY294002), or MEK1/2 (PD98059) and transfection with siRNA of Jak2, PDGFR, Akt, or p42. We showed that TNF-α markedly stimulated Jak2, PDGFR, Akt, and p42/p44 MAPK phosphorylation, which were attenuated by their respective inhibitors. Moreover, TNF-α stimulated Akt activation via a Jak2/PDGFR pathway in HPAEpiCs. In addition, TNF-α-induced p42/p44 MAPK phosphorylation was reduced by AG1296 or LY294002. On the other hand, TNF-α could induce Akt and p42/p44 MAPK translocation from the cytosol into the nucleus, which was inhibited by AG490, AG1296, or LY294002. Finally, we showed that TNF-α stimulated Elk-1 phosphorylation, which was reduced by LY294002 or PD98059. We also observed that TNF-α time dependently induced p300/Elk-1 and p300/Akt complex formation in HPAEpiCs, which was reduced by AG490, AG1296, or LY294002. The activity of cPLA2 protein upregulated by TNF-α was reflected on the PGE2 release, which was reduced by AG490, AG1296, LY294002, or PD98059. Taken together, these results demonstrated that TNF-α-induced cPLA2 expression and PGE2 release were mediated through a Jak2/PDGFR/PI3K/Akt/p42/p44 MAPK/Elk-1 pathway in HPAEpiCs.

K(+) channels in mammary secretory (MS) cells are believed to play a role in transcellular electrolyte transport and thus determining ionic composition of the aqueous phase of milk. However, direct evidence for specific K(+) channel activity in native MS cells is lacking at the single-cell level. Here, we show for the first time that an inwardly rectifying K(+) (Kir) channel is functionally expressed in fully differentiated MS cells that were freshly isolated from the mammary gland of lactating mice. Using the standard whole cell patch-clamp technique, we found that mouse MS cells consistently displayed a K(+) current, whose electrophysiological properties are similar to those previously reported for Kir2.x channels, particularly Kir2.1: 1) current-voltage relationship with strong inward rectification, 2) slope conductance approximately proportional to the square root of external K(+) concentration, 3) voltage- and time-dependent and high-affinity block by external Ba(2+), and 4) voltage-dependent inhibition by external Cs(+). Accordingly, RT-PCR analysis revealed the gene expression of Kir2.1, but not Kir2.2, Kir2.3, and Kir2.4, in lactating mouse mammary gland, and immunohistochemical staining showed Kir2.1 protein expression in the secretory cells. Cell-attached patch recordings from MS cells revealed that a 31-pS K(+) channel with strong inward rectification was likely active at the resting membrane potential. Collectively, the present work demonstrates that a functional Kir2.1-like channel is expressed in lactating mouse MS cells. We propose that the channel might be involved, at least in part, in secretion and/or preservation of ionic components of milk stored into the lumen of these cells.

Increase in matrix protein content in the kidney is a cardinal feature of diabetic kidney disease. While renal matrix protein content is increased by chronic hyperglycemia, whether it is regulated by acute elevation of glucose and insulin has not been addressed. In this study, we aimed to evaluate whether short duration of combined hyperglycemia and hyperinsulinemia, mimicking the metabolic environment of prediabetes and early type 2 diabetes, induces kidney injury. Normal rats were subjected to either saline infusion (control, n = 4) or 7 h of combined hyperglycemic-hyperinsulinemic clamp (HG+HI clamp; n = 6). During the clamp, plasma glucose and plasma insulin were maintained at about 350 mg/dl and 16 ng/ml, respectively. HG+HI clamp increased the expression of renal cortical transforming growth factor-β (TGF-β) and renal matrix proteins, laminin and fibronectin. This was associated with the activation of SMAD3, Akt, mammalian target of rapamycin (mTOR) complexes, and ERK signaling pathways and their downstream target events in the initiation and elongation phases of mRNA translation, an important step in protein synthesis. Additionally, HG+HI clamp provoked renal inflammation as shown by the activation of Toll-like receptor 4 (TLR4) and infiltration of CD68-positive monocytes. Urinary F2t isoprostane excretion, an index of renal oxidant stress, was increased in the HG+HI clamp rats. We conclude that even a short duration of hyperglycemia and hyperinsulinemia contributes to activation of pathways that regulate matrix protein synthesis, inflammation, and oxidative stress in the kidney. This finding could have implications for the control of short-term rises in blood glucose in diabetic individuals at risk of developing kidney disease.

An acute bout of exercise activates downstream signaling cascades that ultimately result in mitochondrial biogenesis. In addition to inducing mitochondrial synthesis, exercise triggers the removal of damaged cellular material via autophagy and of dysfunctional mitochondria through mitophagy. Here, we investigated the necessity of p53 to the changes that transpire within the muscle upon an imposed metabolic and physiological challenge, such as a bout of endurance exercise. We randomly assigned wild-type (WT) and p53 knockout (KO) mice to control, acute exercise (AE; 90 min at 15 m/min), and AE + 3 h recovery (AER) groups and measured downstream alterations in markers of mitochondrial biogenesis, autophagy, and mitophagy. In the absence of p53, activation of p38 MAPK upon exercise was abolished, whereas CaMKII and AMP-activated protein kinase only displayed an attenuated enhancement in the AER group compared with WT mice. The translocation of peroxisome proliferator-activated receptor-γ coactivator-1 α to the nucleus was diminished and only observed in the AER group, and the subsequent increase in messenger RNA transcripts related to mitochondrial biogenesis with exercise and recovery was absent in the p53 KO animals. Whole-muscle autophagic and lysosomal markers did not respond to exercise, irrespective of the genotype of the exercised mice, with the exception of increased ubiquitination observed in KO mice with exercise. Markers of mitophagy were elevated in response to AE and AER conditions in both WT and p53 KO runners. The data suggest that p53 is important for the exercise-induced activation of mitochondrial synthesis and is integral in regulating autophagy during control conditions but not in response to exercise.

Riboflavin, also known as vitamin B2, is transported across the biological membrane into various organs by transport systems. Riboflavin transporter RFVT3 is expressed in the small intestine and has been suggested to localize in the apical membranes of the intestinal epithelial cells. In this study, we investigated the functional involvement of RFVT3 in riboflavin absorption using intestinal epithelial T84 cells and mouse small intestine. T84 cells expressed RFVT3 and conserved unidirectional riboflavin transport corresponding to intestinal absorption. Apical [(3)H]riboflavin uptake was pH-dependent in T84 cells. This uptake was not affected by Na(+) depletion at apical pH 6.0, although it was significantly decreased at apical pH 7.4. The [(3)H]riboflavin uptake from the apical side of T84 cells was prominently inhibited by the RFVT3 selective inhibitor methylene blue and significantly decreased by transfection of RFVT3-small-interfering RNA. In the gastrointestinal tract, RFVT3 was expressed in the jejunum and ileum. Mouse jejunal and ileal permeabilities of [(3)H]riboflavin were measured by the in situ closed-loop method and were significantly reduced by methylene blue. These results strongly suggest that RFVT3 would functionally be involved in riboflavin absorption in the apical membranes of intestinal epithelial cells.

Sodium/potassium/chloride cotransporter (NKCC1) proteins play important roles in Na(+) and K(+) concentrations in key physiological systems, including cardiac, vascular, renal, nervous, and sensory systems. NKCC1 levels and functionality are altered in certain disease states, and tend to decline with age. A sensitive, effective way of regulating NKCC1 protein expression has significant biotherapeutic possibilities. The purpose of the present investigation was to determine if the naturally occurring hormone aldosterone (ALD) could regulate NKCC1 protein expression. Application of ALD to a human cell line (HT-29) revealed that ALD can regulate NKCC1 protein expression, quite sensitively and rapidly, independent of mRNA expression changes. Utilization of a specific inhibitor of mineralocorticoid receptors, eplerenone, implicated these receptors as part of the ALD mechanism of action. Further experiments with cycloheximide (protein synthesis inhibitor) and MG132 (proteasome inhibitor) revealed that ALD can upregulate NKCC1 by increasing protein stability, i.e., reducing ubiquitination of NKCC1. Having a procedure for controlling NKCC1 protein expression opens the doors for therapeutic interventions for diseases involving the mis-regulation or depletion of NKCC1 proteins, for example during aging.