Role of caveolae in the pathogenesis of cholesterol-induced gallbladder muscle hypomotility

Muscle cells from human gallbladders (GB) with cholesterol stones (ChS) exhibit a defective contraction, excess cholesterol (Ch) in the plasma membrane, and lower binding of CCK-1 receptors. These abnormalities improved after muscle cells were incubated with Ch-free liposomes that remove the excess Ch from the plasma membrane. The present studies were designed to investigate the role of caveolin-3 proteins (Cav-3) in the pathogenesis of these abnormalities. Muscle cells from GB with ChS exhibit higher Ch levels in the plasma membrane that were mostly localized in caveolae and associated with parallel increases in the expression of Cav-3 in the caveolae compared with that in GB with pigment stones (PS). The overall number of CCK-1 receptors in the plasma membrane was not different between muscle cells from GB with ChS and PS, but they were increased in the caveolae in muscle cells from GB with ChS. Treatment of muscle cells from GB with ChS with a Gαi3 protein fragment increased the total binding of CCK-1 receptors (from 8.3 to 11.2%) and muscle contraction induced by CCK-8 (from 11.2 to 17.3% shortening). However, Gαq/11 protein fragment had no such effect. Moreover, neither fragment had any effect on muscle cells from GB with PS. We conclude that the defective contraction of muscle cells with excessive Ch levels in the plasma membrane is due to an increased expression of Cav-3 that results in the sequestration of CCK-1 receptors in the caveolae, probably by inhibiting the functions of Gαi3 proteins.

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The role of protein-mediated transport in regulating mitochondrial long-chain fatty acid oxidation

Applied Physiology Nutrition and Metabolism ◽

10.1139/h07-172 ◽

2008 ◽

Vol 33 (1) ◽

pp. 141-142

Author(s):

Graham Paul Holloway

Keyword(s):

Skeletal Muscle ◽

Plasma Membrane ◽

Fatty Acid ◽

Muscle Contraction ◽

Fatty Acid Oxidation ◽

Acid Oxidation ◽

Mitochondrial Content ◽

Palmitate Oxidation ◽

Long Chain

This thesis is an investigation of the role of fatty acid translocase (FAT/CD36), plasma membrane associated fatty acid binding protein (FABPpm), and carnitine palmitoyltransferase I (CPTI) in transporting long-chain fatty acids (LCFAs) across mitochondrial membranes. Maximal CPTI activity, as well as the sensitivity of CPTI for its substrate palmitoyl-CoA (P-CoA) and its inhibitor malonyl-CoA (M-CoA), were measured in mitochondria isolated from human vastus lateralis muscles at rest and following muscle contraction. Exercise did not alter maximal CPTI activity or the sensitivity of CPTI for P-CoA. In contrast, exercise progressively attenuated the ability of M-CoA to inhibit CPTI activity. Mitochondrial FAT/CD36 protein content was also measured at rest, during, and following 2 h of cycling at ~60% maximal oxygen uptake. Exercise progressively increased the content of mitochondrial FAT/CD36 (+59%), which was significantly (p < 0.05) correlated with palmitate oxidation during exercise (r = 0.52), while palmitate oxidation was inhibited ~80% by the administration of a specific FAT/CD36 inhibitor. These data suggest that alterations in CPTI M-CoA sensitivity and increases in mitochondrial FAT/CD36 coordinate exercise-induced increases in fatty acid oxidation. FABPpm, another plasma membrane transport protein, has identical amino acid sequence to mitochondrial aspartate aminotransferase (mAspAT). Since FABPpm contributes to plasma membrane fatty acid transport, the role of FABPpm with respect to mitochondrial LCFA transport was investigated. However, unlike FAT/CD36, muscle contraction did not induce an increase in mitochondrial FABPpm protein in rat or human skeletal muscle. In addition, electrotransfecting FABPpm cDNA into rat skeletal muscle upregulated this protein in mitochondria by 80% without altering mitochondrial palmitate oxidation. In contrast, electrotransfection increased mAspAT activity by 90%, and this was correlated (r = 0.75; p < 0.01) with FABPpm protein. These data suggest that FABPpm does not contribute to the regulation of mitochondrial LCFA transport. Previously, it has been suggested that mitochondria from obese individuals contain an inherent dysfunction to oxidize LCFAs. In age-matched lean (BMI = 23.3 ± 0.7 kg·m–2) and obese (BMI = 37.6 ± 2.2 kg·m–2) individuals, isolated mitochondrial palmitate oxidation was not altered. In addition, mitochondrial FAT/CD36 content was not different in lean and obese individuals. In contrast, citrate synthase and β-hydroxyacyl-CoA dehydrogenase, common markers of total mitochondrial content, were decreased with obesity. Therefore, the decrease in mitochondrial content appeared to account for the observed reductions in whole-muscle LCFA oxidation.

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Insulin and Hypertonicity Recruit GLUT4 to the Plasma Membrane of Muscle Cells by Using N-Ethylmaleimide-sensitive Factor-dependent SNARE Mechanisms but Different v-SNAREs: Role of TI-VAMP

Molecular Biology of the Cell ◽

10.1091/mbc.e04-03-0266 ◽

2004 ◽

Vol 15 (12) ◽

pp. 5565-5573 ◽

Cited By ~ 43

Author(s):

Varinder K. Randhawa ◽

Farah S.L. Thong ◽

Dawn Y. Lim ◽

Dailin Li ◽

Rami R. Garg ◽

...

Keyword(s):

Plasma Membrane ◽

Muscle Cells ◽

Dominant Negative ◽

Attachment Protein ◽

Insulin Effect ◽

Tetanus Neurotoxin ◽

Protein Receptor ◽

Sensitive Factor ◽

Interfering Rna

Insulin and hypertonicity each increase the content of GLUT4 glucose transporters at the surface of muscle cells. Insulin enhances GLUT4 exocytosis without diminishing its endocytosis. The insulin but not the hypertonicity response is reduced by tetanus neurotoxin, which cleaves vesicle-associated membrane protein (VAMP)2 and VAMP3, and is rescued upon introducing tetanus neurotoxin-resistant VAMP2. Here, we show that hypertonicity enhances GLUT4 recycling, compounding its previously shown ability to reduce GLUT4 endocytosis. To examine whether the canonical soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) mechanism is required for the plasma membrane fusion of the tetanus neurotoxin-insensitive GLUT4 vesicles, L6 myoblasts stably expressing myc-tagged GLUT4 (GLUT4myc) were transiently transfected with dominant negative N-ethylmaleimide-sensitive factor (NSF) (DN-NSF) or small-interfering RNA to tetanus neurotoxin-insensitive VAMP (TI-VAMP siRNA). Both strategies markedly reduced the basal level of surface GLUT4myc and the surface gain of GLUT4myc in response to hypertonicity. The insulin effect was abolished by DN-NSF, but only partly reduced by TI-VAMP siRNA. We propose that insulin and hypertonicity recruit GLUT4myc from partly overlapping, but distinct sources defined by VAMP2 and TI-VAMP, respectively.

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Phosphorylated HSP27 essential for acetylcholine-induced association of RhoA with PKCα

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00261.2003 ◽

2004 ◽

Vol 286 (4) ◽

pp. G635-G644 ◽

Cited By ~ 15

Author(s):

Suresh B. Patil ◽

Mercy D. Pawar ◽

Khalil N. Bitar

Keyword(s):

Smooth Muscle ◽

Smooth Muscle Cells ◽

Muscle Contraction ◽

Membrane Fraction ◽

Muscle Cells ◽

Smooth Muscle Contraction ◽

Cell Length ◽

Sustained Contraction ◽

Hsp 27

Reorganization of the cytoskeleton and association of contractile proteins are important steps in modulating smooth muscle contraction. Heat shock protein (HSP) 27 has significant effects on actin cytoskeletal reorganization during smooth muscle contraction. We investigated the role of phosphorylated HSP27 in modulating acetylcholine-induced sustained contraction of smooth muscle cells from the rabbit colon by transfecting smooth muscle cells with phosphomimic (3D) or nonphosphomimic (3G) HSP27. In 3G cells, the initial peak contractile response at 30 s was inhibited by 25% (24.0 ± 4.5% decrease in cell length, n = 4). The sustained contraction was greatly inhibited by 75% [9.3 ± .9% decreases in cell length ( n = 4)]. Furthermore, in 3D cells, translocation of both PKCα and of RhoA was greatly enhanced and resulted in a greater association of PKCα-RhoA in the membrane fraction. In 3G transfected cells, PKCα and RhoA failed to translocate in response to stimulation with acetylcholine, resulting in an inhibition of association of PKCα-RhoA in the membrane fraction. Studies using GST-RhoA fusion protein indicate that there is a direct association of RhoA with PKCα and with HSP27. The results suggest that phosphorylated HSP27 plays a crucial role in the maintenance of association of PKCα-RhoA in the membrane fraction and in the maintenance of acetylcholine-induced sustained contraction.

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The exocyst complex regulates insulin-stimulated glucose uptake of skeletal muscle cells

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00109.2019 ◽

2019 ◽

Vol 317 (6) ◽

pp. E957-E972

Author(s):

Brent A. Fujimoto ◽

Madison Young ◽

Lamar Carter ◽

Alina P. S. Pang ◽

Michael J. Corley ◽

...

Keyword(s):

Skeletal Muscle ◽

Plasma Membrane ◽

Cell Surface ◽

Glucose Uptake ◽

Glucose Transporter ◽

Muscle Cells ◽

Skeletal Muscle Cells ◽

Cell Surface Receptor ◽

Exocyst Complex

Skeletal muscle handles ~80–90% of the insulin-induced glucose uptake. In skeletal muscle, insulin binding to its cell surface receptor triggers redistribution of intracellular glucose transporter GLUT4 protein to the cell surface, enabling facilitated glucose uptake. In adipocytes, the eight-protein exocyst complex is an indispensable constituent in insulin-induced glucose uptake, as it is responsible for the targeted trafficking and plasma membrane-delivery of GLUT4. However, the role of the exocyst in skeletal muscle glucose uptake has never been investigated. Here we demonstrate that the exocyst is a necessary factor in insulin-induced glucose uptake in skeletal muscle cells as well. The exocyst complex colocalizes with GLUT4 storage vesicles in L6-GLUT4myc myoblasts at a basal state and associates with these vesicles during their translocation to the plasma membrane after insulin signaling. Moreover, we show that the exocyst inhibitor endosidin-2 and a heterozygous knockout of Exoc5 in skeletal myoblast cells both lead to impaired GLUT4 trafficking to the plasma membrane and hinder glucose uptake in response to an insulin stimulus. Our research is the first to establish that the exocyst complex regulates insulin-induced GLUT4 exocytosis and glucose metabolism in muscle cells. A deeper knowledge of the role of the exocyst complex in skeletal muscle tissue may help our understanding of insulin resistance in type 2 diabetes.

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Deciphering caveolar functions by syndapin III KO-mediated impairment of caveolar invagination

eLife ◽

10.7554/elife.29854 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 23

Author(s):

Eric Seemann ◽

Minxuan Sun ◽

Sarah Krueger ◽

Jessica Tröger ◽

Wenya Hou ◽

...

Keyword(s):

Plasma Membrane ◽

Physical Exercise ◽

Mouse Models ◽

Skeletal Muscles ◽

Molecular Mechanisms ◽

Physiological Role ◽

Muscle Cells ◽

Coat Proteins ◽

Bona Fide

Several human diseases are associated with a lack of caveolae. Yet, the functions of caveolae and the molecular mechanisms critical for shaping them still are debated. We show that muscle cells of syndapin III KO mice show severe reductions of caveolae reminiscent of human caveolinopathies. Yet, different from other mouse models, the levels of the plasma membrane-associated caveolar coat proteins caveolin3 and cavin1 were both not reduced upon syndapin III KO. This allowed for dissecting bona fide caveolar functions from those supported by mere caveolin presence and also demonstrated that neither caveolin3 nor caveolin3 and cavin1 are sufficient to form caveolae. The membrane-shaping protein syndapin III is crucial for caveolar invagination and KO rendered the cells sensitive to membrane tensions. Consistent with this physiological role of caveolae in counterpoising membrane tensions, syndapin III KO skeletal muscles showed pathological parameters upon physical exercise that are also found in CAVEOLIN3 mutation-associated muscle diseases.

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