Fish Protein Promotes Skeletal Muscle Hypertrophy via the Akt/mTOR Signaling Pathways.

Skeletal muscle is the largest organ in the body and has a broad range of plasticity, undergoing atrophy in response to aging or disease and hypertrophy in response to nutritional supplements or exercise. Loss of skeletal muscle mass and force increases the risk of falls, impairs mobility, and leads to reduced quality of life. In a previous study, we demonstrated that taking in Alaska pollock protein (APP) for only 7 d increased the gastrocnemius muscle mass in rats. This study was conducted to identify hypertrophic myofibers and analyze how hypertrophy occurs within them. Twenty male rats were randomly divided into two groups and administered a diet of casein or APP for 7 d. The expression of each myosin heavy chain (MyHC) isoform in a cross-sectional area was then measured. MyHC IIb and IIx isoforms exhibited hypertrophic features in the gastrocnemius muscles of the APP-fed rats. Furthermore, comprehensive proteomic analyses were conducted to identify changes in protein expression due to muscle hypertrophy. Our results, evaluated by pathway analyses, indicated that the activity of the growth factor signaling pathway was significantly impacted by APP consumption. Moreover, APP could promote protein synthesis by activating the protein kinase B/mechanistic target of the rapamycin signaling pathway, which is also promoted by exercise.

Lipid Dynamics due to Muscle Atrophy Induced by Immobilization.

Muscle atrophy refers to skeletal muscle loss and dysfunction that affects glucose and lipid metabolism. Moreover, muscle atrophy is manifested in cancer, diabetes, and obesity. In this study, we focused on lipid metabolism during muscle atrophy. We observed that the gastrocnemius muscle was associated with significant atrophy with 8 days of immobilization of hind limb joints and that muscle atrophy occurred regardless of the muscle fiber type. Further, we performed lipid analyses using thin layer chromatography, liquid chromatography-mass spectrometry, and mass spectrometry imaging. Total amounts of triacylglycerol, phosphatidylserine, and sphingomyelin were found to be increased in the immobilized muscle. Additionally, we found that specific molecular species of phosphatidylserine, phosphatidylcholine, and sphingomyelin were increased by immobilization. Furthermore, the expression of adipose triglyceride lipase and the activity of cyclooxygenase-2 were significantly reduced by atrophy. From these results, it was revealed that lipid accumulation and metabolic changes in specific fatty acids occur during disuse muscle atrophy. The present study holds implications in validating preventive treatment strategies for muscle atrophy.

Effect of antioxidant supplementation on skeletal muscle and metabolic profile in aging mice.

Aging induces drastic changes in muscle mass and function (sarcopenia); however, the detailed mechanisms underlying sarcopenia remain poorly understood. Recent studies suggested that age-related increases in oxidative stress induce muscle atrophy. In this study, we investigated the effect of 6-month supplementation of antioxidants, specifically piceatannol (PIC) and enzymatically modified isoquercitrin (EMIQ), on age-related physiological changes, including skeletal muscle weight and quality, in 25-month-old (OLD) mice, compared to in 4-month-old (young, YNG) C57BL/6J mice. Muscle weight corrected by body weight significantly declined in OLD mice, compared to in YNG mice. The control OLD mice also showed changes in the expression of genes related to muscle fiber type, reduced locomotor activity, and increased oxidative stress markers in blood. Consistent with the muscle weight and quality changes, whole-body fat oxidation during sedentary conditions and exercise periods in control OLD mice was significantly lower than that in YNG mice. Interestingly, compared to the control OLD mice, the PIC- or EMIQ-fed OLD mice showed higher fat oxidation. Furthermore, EMIQ, but not PIC, increased locomotor activity, the expression of genes encoding antioxidant enzymes, and suppressed the carbonylated protein in the skeletal muscle of OLD mice. These results suggested that chronic antioxidant intake could alleviate aging-related muscle function changes.

Immunohistochemical expression analysis of leucine-rich PPR-motif-containing protein (LRPPRC), a candidate colorectal cancer biomarker identified by shotgun proteomics using iTRAQ.

BACKGROUND: Colorectal cancer (CRC) is the fourth most frequent cause of cancer deaths in the world. Novel biomarkers for the diagnosis, prognosis, and treatment of CRC are required to improve the clinical strategy. METHODS: We applied shotgun proteomics using isobaric tags for relative and absolute quantitation (iTRAQ) to identify novel biomarkers of CRC, and then we detected leucine-rich PPR-motif-containing protein (LRPPRC) expression in 83 normal colorectal tissues and 133 CRC tissues by immunohistochemistry. RESULTS: A total of 570 proteins were identified using iTRAQ. We validated the expression of LRPPRC protein by immunohistochemical analysis of the 77 proteins that showed expression changes in the cancer tissues >1.5-fold the levels in the normal tissues. The expression levels of LRPPRC were significantly higher in CRC tissues than those in normal colorectal tissues, and the expression levels were related with tumor differentiation and especially high in moderately differentiated CRC tissues. CONCLUSION: We identified a novel, differentially expressed protein, LRPPRC, which has the potential to serve as a molecular target for diagnosis and/or prognosis of CRC.

Imaging Mass Spectrometry Reveals a Unique Distribution of Triglycerides in the Abdominal Aortic Aneurysmal Wall.

The pathophysiology underlying abdominal aortic aneurysms (AAAs) remains unknown. In this study, we applied imaging mass spectrometry (IMS) to analyze the pathophysiology of the aneurysmal wall. Comparisons were performed between the tissue samples from the neck and the sac of the AAA, at a single time point, in 30 patients who underwent elective surgery of their AAAs. The localization of each lipid molecule in the aortic wall was assessed by IMS. Histopathological examination and IMS revealed a characteristic distribution of triglycerides (TGs) specifically in the aneurismal adventitia of the sac. This characteristic TG distribution was derived from an ectopic appearance of adipocytes in the adventitia. Furthermore, ectopic adipocyte accumulation in the aortic wall leads to the loss of the collagen fiber network subsequent to the wall rupture. The underlying mechanism of adipocyte accumulation involves the presence of adipose-derived stem cells (ADSCs) in the aneurismal adventitia and the expression of peroxisome proliferator-activated receptor gamma 2, a master regulator of adipocyte differentiation by some ADSCs. This study reveals new, previously overlooked aspects of AAA pathology.

Imaging mass spectrometry reveals fiber-specific distribution of acetylcarnitine and contraction-induced carnitine dynamics in rat skeletal muscles.

Carnitine is well recognized as a key regulator of long-chain fatty acyl group translocation into the mitochondria. In addition, carnitine, as acetylcarnitine, acts as an acceptor of excess acetyl-CoA, a potent inhibitor of pyruvate dehydrogenase. Here, we provide a new methodology for accurate quantification of acetylcarnitine content and determination of its localization in skeletal muscles. We used matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) to visualize acetylcarnitine distribution in rat skeletal muscles. MALDI-IMS and immunohistochemistry of serial cross-sections showed that acetylcarnitine was enriched in the slow-type muscle fibers. The concentration of ATP was lower in muscle regions with abundant acetylcarnitine, suggesting a relationship between acetylcarnitine and metabolic activity. Using our novel method, we detected an increase in acetylcarnitine content after muscle contraction. Importantly, this increase was not detected using traditional biochemical assays of homogenized muscles. We also demonstrated that acetylation of carnitine during muscle contraction was concomitant with glycogen depletion. Our methodology would be useful for the quantification of acetylcarnitine and its contraction-induced kinetics in skeletal muscles.

Lysophosphatidylcholine acyltransferase 1 altered phospholipid composition and regulated hepatoma progression.

BACKGROUND & AIMS: Several lipid synthesis pathways play important roles in the development and progression of hepatocellular carcinoma (HCC), although the precise molecular mechanisms remain to be elucidated. Here, we show the relationship between HCC progression and alteration of phospholipid composition regulated by lysophosphatidylcholine acyltransferase (LPCAT). METHODS: Molecular lipidomic screening was performed by imaging mass spectrometry (IMS) in 37 resected HCC specimens. RT-PCR and Western blotting were carried out to examine the mRNA and protein levels of LPCATs, which catalyze the conversion of lysophosphatidylcholine (LPC) into phosphatidylcholine (PC) and have substrate specificity for some kinds of fatty acids. We examined the effect of LPCAT1 overexpression or knockdown on cell proliferation, migration, and invasion in HCC cell lines. RESULTS: IMS revealed the increase of PC species with palmitoleic acid or oleic acid at the sn-2-position and the reduction of LPC with palmitic acid at the sn-1-position in HCC tissues. mRNA and protein of LPCAT1, responsible for LPC to PC conversion, were more abundant in HCCs than in the surrounding parenchyma. In cell line experiments, LPCAT1 overexpression enriched PCs observed in IMS and promoted cell proliferation, migration, and invasion. LPCAT1 knockdown did viceversa. CONCLUSIONS: Enrichment or depletion of some specific PCs, was found in HCC by IMS. Alteration of phospholipid composition in HCC would affect tumor character. LPCAT1 modulates phospholipid composition to create favorable conditions to HCC cells. LPCAT1 is a potent target molecule to inhibit HCC progression.

GPHR-dependent functions of the Golgi apparatus are essential for the formation of lamellar granules and the skin barrier.

The lumen of the Golgi apparatus is regulated to be weakly acidic, which is critical for its functions. The Golgi pH regulator (GPHR) is an anion channel essential for normal acidification of the Golgi apparatus, and is therefore required for its functions. The Golgi apparatus has been thought to be the origin of lamellar granules in the skin. To study the functional role(s) of GPHR in the skin, we established keratinocyte-specific GPHR-knockout mice using the Cre-loxP system. These mutant mice exhibited hypopigmented skin, hair loss, and scaliness. Histological examination of GPHR-knockout mice showed ballooning of the basal cells and follicular dysplasia. In addition, inflammatory cells were seen in the dermis. The expression of trans-Golgi network 46, a marker for lamellar bodies, and kallikrein 7, a protein within lamellar bodies, is diminished in GPHR-knockout mouse skin. Examination by electron microscopy revealed that keratinocytes produced aberrant lamellar bodies. The transepidermal water loss of these knockout mice was increased compared with wild-type mice. Moreover, expression of cathelicidin-related antimicrobial peptide (CRAMP) in the skin was diminished. These results suggest that GPHR is essential for the homeostasis of the epidermis including the formation of lamellar bodies and for the barrier function.

Imaging mass spectrometry-based histopathologic examination of atherosclerotic lesions.

AIMS: Imaging mass spectrometry (IMS) enables the visualization of individual molecules present on tissue sections. We attempted to identify and visualize specific markers for aortic atherosclerotic lesions. METHODS AND RESULTS: Atherosclerotic lesions were obtained from aortic roots of apolipoprotein E (ApoE)-deficient mice at 60 weeks of age and from femoral arteries of humans with peripheral artery occlusive disease. IMS was performed with a matrix-assisted laser desorption/ionization mass spectrometry time-of-flight (TOF)/TOF-type instrument. The molecular ions at m/z 671.6 and 673.6 were found to be specific molecules in the mouse and human lipid-rich regions. These molecules were assigned as cholesterol linoleate (CE 18:2) and cholesterol oleate (CE 18:1). In the case of the human samples, triacylglycerol was also localized in the lipid-rich regions. The distributions of the molecular ions at m/z 804.5 and 832.5 were the same as the distribution of both the mouse and the human SMCs. These molecules were assigned as phosphatidylcholine (PC) (diacyl 16:0/20:4) and PC (diacyl 18:0/20:4). The molecular ion at m/z 566.9 was localized in the mouse calcified regions, and the molecular ions at m/z 539.0 were localized in the human calcified regions. CONCLUSIONS: The IMS-based histopathologic examination (IbHE) revealed the characteristic peaks of lipid-rich regions, SMCs, and calcified regions in the atherosclerotic lesions. In addition, IbHE revealed the characteristic distribution of lipids in human atherosclerotic lesions. These data indicate that an IMS-based pathologic approach is of considerable value as a new histopathologic examination.

Microarray analysis identifies versican and CD9 as potent prognostic markers in gastric gastrointestinal stromal tumors.

Although the main cause of gastrointestinal stromal tumor (GIST) is gain-of-function mutations in the c-kit gene in the interstitial cells of Cajal, concomitant genetic or epigenetic changes other than c-kit appear to occur in the development of metastasis. We sought to identify the genes involved in the metastatic process of gastric GIST. Microarray analysis was performed to compare gene expressions between three gastric GIST and four metastatic liver GIST. Expression levels were higher for 165 genes and lower for 146 genes in metastatic liver GIST. The upregulation of five oncogenes and downregulation of four tumor suppressor genes including versican and CD9 were confirmed by quantitative reverse transcriptional PCR. Immunohistochemistry in 117 GIST revealed that protein levels of versican and CD9 were higher and lower, respectively, in metastatic GIST. High expression of versican and low expression of CD9 in 104 primary gastric GIST correlated with poor disease-free survival (P = 0.0078 and P = 0.0018). In addition to the c-kit gene mutation, genetic or epigenetic changes other than c-kit play important roles in the metastatic process. In particular, versican and CD9 are potential prognostic markers in gastric GIST.

Imaging mass spectrometry of gastric carcinoma in formalin-fixed paraffin-embedded tissue microarray.

The popularity of imaging mass spectrometry (IMS) of tissue samples, which enables the direct scanning of tissue sections within a short time-period, has been considerably increasing in cancer proteomics. Most pathological specimens stored in medical institutes are formalin-fixed; thus, they had been regarded to be unsuitable for proteomic analyses, including IMS, until recently. Here, we report an easy-to-use screening method that enables the analysis of multiple samples in one experiment without extractions and purifications of proteins. We scanned, with an IMS technique, a tissue microarray (TMA) of formalin-fixed paraffin-embedded (FFPE) specimens. We detected a large amount of signals from trypsin-treated FFPE-TMA samples of gastric carcinoma tissues of different histological types. Of the signals detected, 54 were classified as signals specific to cancer with statistically significant differences between adenocarcinomas and normal tissues. We detected a total of 14 of the 54 signals as histological type-specific with the support of statistical analyses. Tandem MS revealed that a signal specific to poorly differentiated cancer tissue corresponded to histone H4. Finally, we verified the IMS-based finding by immunohistochemical analysis of more than 300 specimens spotted on TMAs; the immunoreactivity of histone H4 was remarkably strong in poorly differentiated cancer tissues. Thus, the application of IMS to FFPE-TMA can enable high-throughput analysis in cancer proteomics to aid in the understanding of molecular mechanisms underlying carcinogenesis, invasiveness, metastasis, and prognosis. Further, results obtained from the IMS of FFPE-TMA can be readily confirmed by commonly used immunohistochemical analyses.

Convenient structural analysis of glycosphingolipids using MALDI-QIT-TOF mass spectrometry with increased laser power and cooling gas flow.

Matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight mass spectrometry (MALDI-QIT-TOF MS) was applied to the structural characterization of neutral glycosphingolipids. Lithium adduct ions of glycosphingolipids were analyzed using MALDI-QIT-TOF MS under strong conditions of increased laser power and cooling gas flow. The relative intensities of fragment ions were increased under the strong conditions, and the resulting spectra revealed the presence of oligosaccharide ions fragmented from the glycosphingolipids. Consequently, the oligosaccharide sequences of the glycosphingolipids were readily obtained. To obtain more detailed structural information, MS/MS (MS2) and MS/MS/MS (MS3) analyses were performed with selection of the lactosylceramide and ceramide ions, respectively. The resulting data were sufficient to determine the structures of both the oligosaccharide and the ceramide moiety of each glycosphingolipid. The fragmentation patterns of MS2 and MS3 for Forssman glycolipid under the strong conditions were comparable to those of MS3 and MS4 obtained under standard conditions, respectively. Thus, MALDI-QIT-TOF MS with increased laser power and cooling gas flow is a convenient method for glycosphingolipid analysis.