Histochemical approach of cryobiopsy for glycogen distribution in living mouse livers under fasting and local circulation loss conditions

Saitoh, Yurika; Terada, Nobuo; Saitoh, Shu‐ichi; Ohno, Nobuhiko; Fujii, Yasuhisa; Ohno, Shinichi

doi:10.1007/s00418-009-0663-4

Cited by 29 publications

(17 citation statements)

References 24 publications

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“…In IVCT-FS samples of adult sciatic nerves, which preserve soluble proteins well in tissue sections (30), MPP6 immunoreactivity was detected in regions of noncompact myelin, including the SLI and the paranodes (Fig. 1A).…”

Section: Resultsmentioning

confidence: 99%

Essential Function of Protein 4.1G in Targeting of Membrane Protein Palmitoylated 6 into Schmidt-Lanterman Incisures in Myelinated Nerves

Terada

Saitoh

Ohno

et al. 2012

Molecular and Cellular Biology

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Protein 4.1G is a membrane skeletal protein found in specific subcellular structures in myelinated Schwann cells and seminiferous tubules. Here, we show that in the mouse sciatic nerve, protein 4.1G colocalized at Schmidt-Lanterman incisures (SLI) and the paranodes with a member of the membrane-associated guanylate kinase (MAGUK) family, membrane protein palmitoylated 6 (MPP6). Coimmunoprecipitation experiments revealed that MPP6 was interacting with protein 4.1G. In contrast to wild-type nerves, in 4.1G knockout mice, MPP6 was found largely in the cytoplasm near Schwann cell nuclei, indicating an abnormal protein transport. Although the SLI remained in the 4.1G knockout sciatic nerves, as confirmed by E-cadherin immunostaining, their shape was altered in aged 4.1G knockout nerves compared to their shape in wild-type nerves. In the seminiferous tubules, MPP6 was localized similarly to protein 4.1G along cell membranes of the spermatogonium and early spermatocytes. However, in contrast to myelinated peripheral nerves, the specific localization of MPP6 in the seminiferous tubules was unaltered in the absence of protein 4.1G. These results indicate that 4.1G has a specific role in the targeting of MPP6 to the SLI and the assembly of these subcellular structures. P rotein 4.1G (4.1G) is a member of the 4.1 family (27, 46), a group of membrane skeletal proteins that link various components to the spectrin-actin network (10). We have previously reported that 4.1G is present in rodent Schwann cells (7, 24) and mouse seminiferous tubules (40, 41). In peripheral nerves, 4.1G is found at the Schmidt-Lanterman incisures (SLI) and the paranodal loops of myelinated Schwann cells (24). The SLI are funnelshaped interruptions within the myelin sheath of nerve fibers. They contain high concentrations of actin and spectrin (35, 42), which forms a membrane skeleton that might contribute to the elasticity and stability of these structures.Membrane-associated guanylate kinase (MAGUK) family proteins contain PDZ (for postsynaptic density 95 [PSD-95]/Drosophila disks large [Dlg]/zonula occludens 1 [ZO-1]), GUK (guanylate kinase), and SH3 (src homology 3) domains, and they localize to specific domains at the plasma membranes (9, 11). In epithelial cells, for example, some MAGUKs, such as Dlg and ZO-1, are required for the formation of adherens and tight junctions, respectively. In addition to their function as membrane scaffolds (16, 21), several MAGUKs also control intracellular protein transport through their ability to bind motor proteins (45, 49). Interestingly, some MAGUKs contain specific domains that interact with 4.1 proteins (14,17,18). In the current study, we report the identification of membrane protein palmitoylated 6 (MPP6) (also known as PALS2, VAM1, and p55T [http://www .genenames.org/]) as a novel MAGUK molecule that interacts with 4.1G in peripheral nerves. We further demonstrate that the interaction between 4.1G and MPP6 is essential for the targeting of the latter into SLI. MATERIALS AND METHODS Animals and anesthes...

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Section: Resultsmentioning

confidence: 99%

Essential Function of Protein 4.1G in Targeting of Membrane Protein Palmitoylated 6 into Schmidt-Lanterman Incisures in Myelinated Nerves

Terada

Saitoh

Ohno

et al. 2012

Molecular and Cellular Biology

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“…For evaluation of hepatic collagen deposition, Sirius red staining was performed and a red color staining was considered where the collagen deposited. Glycogen storage was detected by periodic acid-Schiff (PAS) staining staining as described elsewhere (Saitoh et al 2010).…”

Section: Hematoxylin and Eosin Sirius Red And Periodic Acid-schiff mentioning

confidence: 99%

Metformin improves hepatic IRS2/PI3K/Akt signaling in insulin-resistant rats of NASH and cirrhosis

Hong

Zhou

Liu

et al. 2016

Journal of Endocrinology

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Nonalcoholic fatty liver disease and cirrhosis are strongly associated with insulin resistance and glucose intolerance. To date, the influence of metformin on glycogen synthesis in the liver is controversial. Limited studies have evaluated the effect of metformin on hepatic insulin signaling pathway in vivo. In this study, an insulin-resistant rat model of nonalcoholic steatohepatitis and cirrhosis was developed by high-fat and high-sucrose diet feeding in combination with subcutaneous injection of carbon tetrachloride. Liver tissues of the model rats were featured with severe steatosis and cirrhosis, accompanied by impaired liver function and antioxidant capacity. The glucose tolerance was impaired, and the index of insulin resistance was increased significantly compared with the control. The content of hepatic glycogen was dramatically decreased. The expression of insulin receptor β (IRβ); phosphorylations of IRβ, insulin receptor substrate 2 (IRS2), and Akt; and activities of phosphatidylinositol 3-kinase (PI3K) and glycogen synthase (GS) in the liver were significantly decreased, whereas the activities of glycogen synthase kinase 3α (GSK3α) and glycogen phosphorylase a (GPa) were increased. Metformin treatment remarkably improved liver function, alleviated lipid peroxidation and histological damages of the liver, and ameliorated glucose intolerance and insulin resistance. Metfromin also significantly upregulated the expression of IRβ; increased the phosphorylations of IRβ, IRS2, and Akt; increased the activities of PI3K and GS; and decreased GSK3α and GPa activities. In conclusion, our study suggests that metformin upregulates IRβ expression and the downstream IRS2/PI3K/Akt signaling transduction, therefore, to increase hepatic glycogen storage and improve insulin resistance. These actions may be attributed to the improved liver histological alterations by metformin.

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“…During PAS staining, the tissue is incubated with periodic acid to generate reactive aldehyde groups that further react with pararosaniline to form a Schiff base; this gives the tissue a characteristic pink color. In the case of liver tissue, it is necessary to remove glycogen by pretreatment with α‐amylase to avoid interference . There is strong PAS staining for FFPE and DST‐PE tissue prior to treatment with α‐amylase (Figure , left), whereas only DST‐PE tissue gave strong PAS staining after glycogen removal (Figure , right).…”

Section: Resultsmentioning

confidence: 99%

“…PAS staining was performed by paraffin removal from 4 μm tissue sections (Ultraclear 15 min, two times), tissue rehydration (100 % ethanol 2 min, 100 % ethanol 2 min, 96 % ethanol 2 min, 96 % ethanol 2 min, 70 % ethanol 2 min, water 2 min), treatment with 1 % ( w/v ) periodic acid in water for 20 min followed by reaction with the Schiff reagent for 20 min. The PAS staining protocol was also performed after removal of glycogen by incubation at 37 °C for 30 min with 1000 U mL −1 α‐amylase …”

Section: Methodsmentioning

confidence: 99%

Cleavable Crosslinkers as Tissue Fixation Reagents for Proteomic Analysis

et al. 2018

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Formaldehyde fixation is widely used for long-term maintenance of tissue. However, due to formaldehyde-induced crosslinks, fixed tissue proteins are difficult to extract, which hampers mass spectrometry (MS) proteomic analyses. Recent years have seen the use of different combinations of high temperature and solubilizing agents (usually derived from antigen retrieval techniques) to unravel formaldehyde-fixed paraffin-embedded tissue proteomes. However, to achieve protein extraction yields similar to those of fresh-frozen tissue, high-temperature heating is necessary. Such harsh extraction conditions can affect sensitive amino acids and post-translational modifications, resulting in the loss of important information, while still not resulting in protein yields comparable to those of fresh-frozen tissue. Herein, the objective is to evaluate cleavable protein crosslinkers as fixatives that allow tissue preservation and efficient protein extraction from fixed tissue for MS proteomics under mild conditions. With this goal in mind, disuccinimidyl tartrate (DST) and dithiobis(succinimidylpropionate) (DSP) are investigated as cleavable fixating reagents. These compounds crosslink proteins by reacting with amino groups, leading to amide bond formation, and can be cleaved with sodium metaperiodate (cis-diols, DST) or reducing agents (disulfide bonds, DSP), respectively. Results show that cleavable protein crosslinking with DST and DSP allows tissue fixation with morphology preservation comparable to that of formaldehyde. In addition, cleavage of DSP improves protein recovery from fixed tissue by a factor of 18 and increases the number of identified proteins by approximately 20 % under mild extraction conditions compared with those of formaldehyde-fixed paraffin-embedded tissue. A major advantage of DSP is the introduction of well-defined protein modifications that can be taken into account during database searching. In contrast to DSP fixation, DST fixation followed by cleavage with sodium metaperiodate, although effective, results in side reactions that prevent effective protein extraction and interfere with protein identification. Protein crosslinkers that can be cleaved under mild conditions and result in defined modifications, such as DSP, are thus viable alternatives to formaldehyde as tissue fixatives to facilitate protein analysis from paraffin-embedded, fixed tissue.

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Histochemical approach of cryobiopsy for glycogen distribution in living mouse livers under fasting and local circulation loss conditions

Cited by 29 publications

References 24 publications

Essential Function of Protein 4.1G in Targeting of Membrane Protein Palmitoylated 6 into Schmidt-Lanterman Incisures in Myelinated Nerves

Essential Function of Protein 4.1G in Targeting of Membrane Protein Palmitoylated 6 into Schmidt-Lanterman Incisures in Myelinated Nerves

Metformin improves hepatic IRS2/PI3K/Akt signaling in insulin-resistant rats of NASH and cirrhosis

Cleavable Crosslinkers as Tissue Fixation Reagents for Proteomic Analysis

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