American journal of physiology. Cell physiology最新文献

Glutamine is a critical amino acid that serves as an energy source, building block, and signaling molecule for the heart tissue and the immune system. However, the role of glutamine metabolism in regulating cardiac remodeling following myocardial infarction (MI) is unknown. In this study, we show in adult male mice that glutamine metabolism is altered both in the remote (contractile) area and in infiltrating macrophages in the infarct area after permanent left anterior descending artery occlusion. We found that metabolites related to glutamine metabolism were differentially altered in macrophages at days 1, 3, and 7 after MI using untargeted metabolomics. Glutamine metabolism in live cells was increased after MI relative to no MI controls. Gene expression in the remote area of the heart indicated a loss of glutamine metabolism. Glutamine administration improved left ventricle (LV) function at days 1, 3, and 7 after MI, which was associated with improved contractile and metabolic gene expression. Conversely, administration of BPTES, a pharmacological inhibitor of glutaminase-1, worsened LV function after MI. Neither glutamine nor BPTES administration impacted gene expression or bioenergetics of macrophages isolated from the infarct area. Our results indicate that glutamine metabolism plays a critical role in maintaining LV contractile function following MI and that glutamine administration improves LV function. Glutamine metabolism may also play a role in regulating macrophage function, but macrophages are not responsive to exogenous pharmacological manipulation of glutamine metabolism.NEW & NOTEWORTHY Glutamine metabolism is altered in both infarct macrophages and the remote left ventricle (LV) following myocardial infarction (MI). Supplemental glutamine improves LV function following MI while inhibiting glutamine metabolism with BPTES worsens LV function. Supplemental glutamine or BPTES does not impact macrophage immunometabolic phenotypes after MI.

ATP and benzoylbenzoyl-ATP (BzATP) increase free cytosolic Ca²⁺ concentration ([Ca²⁺]_i) in conjunctival goblet cells (CGCs) resulting in mucin secretion. The purpose of this study was to investigate the source of the Ca²⁺_i mobilized by ATP and BzATP. First-passage cultured rat CGCs were incubated with Fura-2/AM, and [Ca²⁺]_i was measured under several conditions with ATP and BzATP stimulation. The following conditions were used: 1) preincubation with the Ca²⁺ chelator EGTA, 2) preincubation with the SERCA inhibitor thapsigargin (10^-6 M), which depletes ER Ca²⁺ stores, 3) preincubation with phospholipase C (PLC) or protein kinase A (PKA) inhibitor, or 4) preincubation with the voltage-gated calcium channel antagonist nifedipine (10^-5 M) and the ryanodine receptor (RyR) antagonist dantrolene (10^-5 M). Immunofluorescence microscopy (IF) and quantitative reverse transcription polymerase chain reaction (RT-qPCR) were used to investigate RyR presence in rat and human CGCs. ATP-stimulated peak [Ca²⁺]_i was significantly lower after chelating Ca²⁺_i with 2 mM EGTA in Ca²⁺-free buffer. The peak [Ca²⁺]_i increase in CGCs preincubated with thapsigargin, the PKA inhibitor H89, nifedipine, and dantrolene, but not the PLC inhibitor, was reduced for ATP at 10^-5 M and BzATP at 10^-4 M. Incubating CGCs with dantrolene alone decreased [Ca²⁺]_i and induced CGC cell death at a high concentration. RyR3 was detected in rat and human CGCs with IF and RT-qPCR. We conclude that ATP- and BzATP-induced Ca²⁺_i increases originate from the ER and that RyR3 may be an essential regulator of CGC [Ca²⁺]_i. This study contributes to the understanding of diseases arising from defective Ca²⁺ signaling in nonexcitable cells.NEW & NOTEWORTHY ATP and benzoylbenzoyl-ATP (BzATP) induce mucin secretion through an increase in free cytosolic calcium concentration ([Ca²⁺]_i) in conjunctival goblet cells (CGCs). The mechanisms through which ATP and BzATP increase [Ca²⁺]_i in CGCs are unclear. Ryanodine receptors (RyRs) are fundamental in [Ca²⁺]_i regulation in excitable cells. Herein, we find that ATP and BzATP increase [Ca²⁺]_i through the activation of protein kinase A, voltage-gated calcium channels, and RyRs, and that RyRs are crucial for nonexcitable CGCs' Ca²⁺_i homeostasis.

In this issue, Burke et al. discuss the utility of the rodent synergist ablation (SA) model for examining mechanisms associated with skeletal muscle hypertrophy. In this invited perspective, we aim to complement their original perspective by discussing limitations to the model along with alternative mechanical overload models that have strengths and limitations.

A thin film of pulmonary surfactant lines the surface of the airways and alveoli, where it lowers the surface tension in the peripheral lungs, preventing collapse of the bronchioles and alveoli and reducing the work of breathing. It also possesses a barrier function for maintaining the blood-gas interface of the lungs and plays an important role in innate immunity. The surfactant film covers the epithelium lining both large and small airways, forming the first line of defense between toxic airborne particles/pathogens and the lungs. Furthermore, surfactant has been shown to relax airway smooth muscle (ASM) after exposure to ASM agonists, suggesting a more subtle function. Whether surfactant masks irritant sensory receptors or interacts with one of them is not known. The relaxant effect of surfactant on ASM is absent in bronchial tissues denuded of an epithelial layer. Blocking of prostanoid synthesis inhibits the relaxant function of surfactant, indicating that prostanoids might be involved. Another possibility for surfactant to be active, namely through ATP-dependent potassium channels and the cAMP-regulated epithelial chloride channels [cystic fibrosis transmembrane conductance regulators (CFTRs)], was tested but could not be confirmed. Hence, this review discusses the mechanisms of known and potential relaxant effects of pulmonary surfactant on ASM. This review summarizes what is known about the role of surfactant in smooth muscle physiology and explores the scientific questions and studies needed to fully understand how surfactant helps maintain the delicate balance between relaxant and constrictor needs.

The mitochondrial citrate shuttle, which relies on the solute carrier family 25 member 1 (SLC25A1), plays a pivotal role in transporting citrate from the mitochondria to the cytoplasm. This shuttle supports glycolysis, lipid biosynthesis, and protein acetylation. Previous research has primarily focused on SLC25A1 in pathological models, particularly high-fat diet (HFD)-induced obesity. However, the impact of SLC25A1 inhibition on nutrient metabolism under HFD remains unclear. To address this gap, we used zebrafish (Danio rerio) and Nile tilapia (Oreochromis niloticus) to evaluate the effects of inhibiting Slc25a1. In zebrafish, we administered Slc25a1-specific inhibitors (CTPI-2) for 4 wk, whereas Nile tilapia received intraperitoneal injections of dsRNA to knock down slc25a1b for 7 days. Inhibition of the mitochondrial citrate shuttle effectively protected zebrafish from HFD-induced obesity, hepatic steatosis, and insulin resistance. Of note, glucose tolerance was unaffected. Inhibition of Slc25a1 altered hepatic protein acetylation patterns, with decreased cytoplasmic acetylation and increased mitochondrial acetylation. Under HFD conditions, Slc25a1 inhibition promoted fatty acid oxidation and reduced hepatic triglyceride (TAG) accumulation by deacetylating carnitine palmitoyltransferase 1a (Cpt1a). In addition, Slc25a1 inhibition triggered acetylation-induced inactivation of Pdhe1α, leading to a reduction in glucose oxidative catabolism. This was accompanied by enhanced glucose uptake and storage in zebrafish livers. Furthermore, Slc25a1 inhibition under HFD conditions activated the SIRT1/PGC1α pathway, promoting mitochondrial proliferation and enhancing oxidative phosphorylation for energy production. Our findings provide new insights into the role of nonhistone protein acetylation via the mitochondrial citrate shuttle in the development of hepatic lipid deposition and hyperglycemia caused by HFD.NEW & NOTEWORTHY The mitochondrial citrate shuttle is a crucial physiological process for maintaining metabolic homeostasis. In the present study, we found that inhibition of mitochondrial citrate shuttle (Slc25a1) could alleviate metabolic syndromes induced by high-fat diet (HFD) through remodeling hepatic protein acetylation modification. Briefly, Slc25a1 inhibition reduces hepatic triglyceride deposition by deacetylating Cpt1a and reduces glucose oxidative catabolism by acetylating Pdhe1α. Our study provides new insights into the treatment of diet-induced metabolic syndromes.

Within the tetramerization domain (T1) of most voltage-gated potassium channels (Kv) are highly conserved charged residues that line the T1-T1 interface. We investigated the Kv1.1 residue R86 located at the narrowest region of the T1 interface. A Kv1.1 R86Q mutation was reported in a child diagnosed with lower limb dyskinesia (Set KK, Ghosh D, Huq AHM, Luat AF. Mov Disord Clin Pract 4: 784-786, 2017). The child did not present with episodic ataxia 1 (EA1) symptoms typically associated with Kv1.1 loss-of-function mutations. We characterized the electrophysiological outcome of the R86Q substitution by expressing Kv1.1 in Xenopus laevis oocytes. Mutated α-subunits were able to form functional channels that pass delayed rectifier currents. Oocytes that expressed only mutated α-subunits produced a significant reduction in Kv1.1 current and showed a positive shift in voltage dependence of activation. In addition, there was substantially slower activation and faster deactivation implying a reduction in the time the channel is in its open state. Oocytes co-injected with both mutated and wild-type cRNA in equal amounts, to mimic the heterozygous condition of the disease, showed a decrease in current amplitude at -10 mV, a positive shift in activation voltage-dependence and faster deactivation kinetics when compared with the wild-type channel. These findings indicate that T1 plays a role in Kv1.1's voltage-dependent activation and in its kinetics of activation and deactivation.NEW & NOTEWORTHY This is the first Kv1.1 study to characterize the electrophysiological and structural phenotype of a tetramerization (T1) domain mutation. Surprisingly, the mutated α-subunits were able to tetramerize, albeit with different gating kinetics and voltage dependence. This novel finding points to a clear role of T1 in the channel's voltage dependence and gating. Mimicking the heterozygous condition resulted in milder alterations in channel function when compared with previously reported mutations. This is in agreement with the child's milder symptoms.

In cell biology, ribosomal RNA (rRNA) 2'O-methyl (2'-O-Me) is the most prevalent posttranscriptional chemical modification contributing to ribosome heterogeneity. The modification involves a family of small nucleolar RNAs (snoRNAs) and is specified by box C/D snoRNAs (SNORDs). Given the importance of ribosome biogenesis for skeletal muscle growth, we asked if rRNA 2'-O-Me in nascent ribosomes synthesized in response to a growth stimulus is an unrecognized mode of ribosome heterogeneity in muscle. To determine the pattern and dynamics of 2'-O-Me rRNA, we used a sequencing-based profiling method called RiboMeth-seq (RMS). We applied this method to tissue-derived rRNA of skeletal muscle and rRNA specifically from the muscle fiber using an inducible myofiber-specific RiboTag mouse in sedentary and mechanically overloaded conditions. These analyses were complemented by myonuclear-specific small RNA sequencing to profile SNORDs and link the rRNA epitranscriptome to known regulatory elements generated within the muscle fiber. We demonstrate for the first time that mechanical overload of skeletal muscle 1) induces decreased 2'-O-Me at a subset of skeletal muscle rRNA and 2) alters the SNORD profile in isolated myonuclei. These findings point to a transient diversification of the ribosome pool via 2'-O-Me during growth and adaptation in skeletal muscle. These findings suggest changes in ribosome heterogeneity at the 2'-O-Me level during muscle hypertrophy and lay the foundation for studies investigating the functional implications of these newly identified "growth-induced" ribosomes.NEW & NOTEWORTHY Ribosomal RNAs (rRNAs) are posttranscriptionally modified by 2'O-methyl (2'-O-Me). This study applied RiboMeth-seq (RMS) to detect changes in 2'-O-Me levels during skeletal muscle hypertrophy, uncovering transient diversification of the ribosome pool in skeletal muscle fibers. This work implies a role for ribosome heterogeneity in skeletal muscle growth and adaptation.

Cancer cachexia, the unintentional loss of lean mass, contributes to functional dependency, poor treatment outcomes, and decreased survival. Although its pathogenicity is multifactorial, metabolic dysfunction remains a hallmark of cachexia. However, significant knowledge gaps exist in understanding the role of skeletal muscle lipid metabolism and dynamics in this condition. We examined skeletal muscle metabolic dysfunction, intramyocellular lipid droplet (LD) content, LD morphology and subcellular distribution, and LD-mitochondrial interactions using the Lewis lung carcinoma (LLC) murine model of cachexia. C57/BL6 male mice (n = 20) were implanted with LLC cells (10⁶) in the right flank or underwent PBS sham injections. Skeletal muscle was excised for transmission electron microscopy (TEM; soleus), oil red O/lipid staining [tibialis anterior (TA)], and protein (gastrocnemius). LLC mice had a greater number (232%; P = 0.006) and size (130%; P = 0.023) of intramyocellular LDs further supported by increased oil-red O positive (87%; P = 0.0109) and "very high" oil-red O positive (178%; P = 0.0002) fibers compared with controls and this was inversely correlated with fiber size (R² = 0.5294; P < 0.0001). Morphological analyses of LDs show increased elongation and complexity [aspect ratio: intermyofibrillar (IMF) = 9%, P = 0.046) with decreases in circularity [circularity: subsarcolemmal (SS) = 6%, P = 0.042] or roundness (roundness: whole = 10%, P = 0.033; IMF = 8%, P = 0.038) as well as decreased LD-mitochondria touch (-15%; P = 0.006), contact length (-38%; P = 0.036), and relative contact (86%; P = 0.004). Furthermore, dysregulation in lipid metabolism (adiponectin, CPT1b) and LD-associated proteins, perilipin-2 and perilipin-5, in cachectic muscle (P < 0.05) were observed. Collectively, we provide evidence that skeletal muscle myosteatosis, altered LD morphology, and decreased LD-mitochondrial interactions occur in a preclinical model of cancer cachexia.NEW & NOTEWORTHY We sought to advance our understanding of skeletal muscle lipid metabolism and dynamics in cancer cachexia. Cachexia increased the number and size of intramyocellular lipid droplets (LDs). Furthermore, decreases in LD-mitochondrial touch, contact length, and relative contact along with increased LD shape complexity with decreases in circularity and roundness. Dysregulation in lipid metabolism and LD-associated proteins was also documented. Collectively, we show that myosteatosis, altered LD morphology, and decreased LD-mitochondrial interactions occur in cancer cachexia.

Lower oxidative capacity in skeletal muscles (SKMs) is a prevailing cause of metabolic diseases. Exercise not only enhances the fatty acid oxidation (FAO) capacity of SKMs but also increases lactate levels. Given that lactate may contribute to tricarboxylic acid cycle (TCA) flux and impact monocarboxylate transporter 1 in the SKMs, we hypothesize that lactate can influence glucose and fatty acid (FA) metabolism. To test this hypothesis, we investigated the mechanism underlying lactate-driven FAO regulation in the SKM of mice with diet-induced obesity (DIO). Lactate was administered to DIO mice immediately after exercise for over 3 wk. We found that increased lactate levels enhanced energy expenditure mediated by fat metabolism during exercise recovery and decreased triglyceride levels in DIO mice SKMs. To determine the lactate-specific effects without exercise, we administered lactate to mice on a high-fat diet (HFD) for 8 wk. Similar to our exercise conditions, lactate increased FAO, TCA cycle activity, and mitochondrial respiration in the SKMs of HFD-fed mice. In addition, under sufficient FA conditions, lactate increased uncoupling protein-3 abundance via the NADH-NAD⁺ shuttle. Conversely, ATP synthase abundance decreased in the SKMs of HFD mice. Taken together, our results suggest that lactate amplifies the adaptive increase in FAO capacity mediated by the TCA cycle and mitochondrial respiration in SKMs under sufficient FA abundance.NEW & NOTEWORTHY Lactate administration post-exercise promotes triglyceride content loss in skeletal muscles (SKMs) and reduced body weight. Lactate enhances fatty acid oxidation in the SKMs of high-fat diet (HFD)-fed mice due to enhanced mitochondrial oxygen consumption. In addition, lactate restores the malate-aspartate shuttle, which is reduced by a HFD, and activates the tricarboxylic acid cycle (TCA) cycle in SKMs. Interestingly, supraphysiological lactate facilitates uncoupling protein-3 expression through NADH/NAD⁺, which is enhanced under high-fat levels in SKMs.

Euryhaline fish experience variable osmotic environments requiring physiological adjustments to tolerate elevated salinity. Mozambique tilapia (Oreochromis mossambicus) possess one of the highest salinity tolerance limits of any fish. In tilapia and other euryhaline fish species, the myo-inositol biosynthesis (MIB) pathway enzymes, myo-inositol phosphate synthase (MIPS) and inositol monophosphatase 1 (IMPA1.1), are among the most upregulated mRNAs and proteins indicating the high importance of this pathway for hyperosmotic (HO) stress tolerance. These abundance changes must be precluded by HO perception and signaling mechanism activation to regulate the expression of MIPS and IMPA1.1 genes. In previous work using a O. mossambicus cell line (OmB), a reoccurring osmosensitive enhancer element (OSRE1) in both MIPS and IMPA1.1 was shown to transcriptionally upregulate these enzymes in response to HO stress. The OSRE1 core consensus (5'-GGAAA-3') matches the core binding sequence of the predominant mammalian HO response transcription factor, nuclear factor of activated T-cells (NFAT5). HO-challenged OmB cells showed an increase in NFAT5 mRNA suggesting NFAT5 may contribute to MIB pathway regulation in euryhaline fish. Ectopic expression of wild-type NFAT5 induced an IMPA1.1 promoter-driven reporter by 5.1-fold (P < 0.01). Moreover, expression of dominant negative NFAT5 in HO media resulted in a 47% suppression of the reporter signal (P < 0.005). Furthermore, reductions of IMPA1.1 (37-49%) and MIPS (6-37%) mRNA abundance were observed in HO-challenged NFAT5 knockout cells relative to control cells. Collectively, these multiple lines of experimental evidence establish NFAT5 as a tilapia transcription factor contributing to HO-induced activation of the MIB pathway.NEW & NOTEWORTHY In our study, we use a multi-pronged synthetic biology approach to demonstrate that the fish homolog of the predominant mammalian osmotic stress transcription factor nuclear factor of activated T-cells (NFAT5) also contributes to the activation of hyperosmolality inducible genes in cells of extremely euryhaline fish. However, in addition to NFAT5 the presence of other strong osmotically inducible signaling mechanisms is required for full activation of osmoregulated tilapia genes.