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1.  Cavin-3 Knockout Mice Show that Cavin-3 Is Not Essential for Caveolae Formation, for Maintenance of Body Composition, or for Glucose Tolerance 
PLoS ONE  2014;9(7):e102935.
The cavins are a family of proteins associated with caveolae, cavin-1, -2 and -3 being widely expressed while cavin-4 is restricted to striated muscle. Deletion of cavin-1 results in phenotypes including metabolic changes consistent with adipocyte dysfunction, and caveolae are completely absent. Deletion of cavin-2 causes tissue-specific loss of caveolae. The consequences of cavin-3 deletion are less clear, as there are divergent data on the abundance of caveolae in cavin-3 null mice. Here we examine the consequences of cavin-3 deficiency in vivo by making cavin-3 knockout mice. We find that loss of cavin-3 has minimal or no effects on the levels of other caveolar proteins, does not appear to play a major role in formation of protein complexes important for caveolar morphogenesis, and has no significant effect on caveolae abundance. Cavin-3 null mice have the same body weight and fat mass as wild type animals at ages 8 through 30 weeks on both normal chow and high fat diets. Likewise, the two mouse strains exhibit identical glucose tolerance tests on both diets. Microarray analysis from adipose tissue shows that the changes in mRNA expression between cavin-3 null and wild type mouse are minimal. We conclude that cavin-3 is not absolutely required for making caveolae, and suggest that the mechanistic link between cavin-3 and metabolic regulation remains uncertain.
doi:10.1371/journal.pone.0102935
PMCID: PMC4103889  PMID: 25036884
2.  IDOL Stimulates Clathrin-Independent Endocytosis and Multivesicular Body-Mediated Lysosomal Degradation of the Low-Density Lipoprotein Receptor 
Molecular and Cellular Biology  2013;33(8):1503-1514.
The low-density lipoprotein receptor (LDLR) is a critical determinant of plasma cholesterol levels that internalizes lipoprotein cargo via clathrin-mediated endocytosis. Here, we show that the E3 ubiquitin ligase IDOL stimulates a previously unrecognized, clathrin-independent pathway for LDLR internalization. Real-time single-particle tracking and electron microscopy reveal that IDOL is recruited to the plasma membrane by LDLR, promotes LDLR internalization in the absence of clathrin or caveolae, and facilitates LDLR degradation by shuttling it into the multivesicular body (MVB) protein-sorting pathway. The IDOL-dependent degradation pathway is distinct from that mediated by PCSK9 as only IDOL employs ESCRT (endosomal-sorting complex required for transport) complexes to recognize and traffic LDLR to lysosomes. Small interfering RNA (siRNA)-mediated knockdown of ESCRT-0 (HGS) or ESCRT-I (TSG101) components prevents IDOL-mediated LDLR degradation. We further show that USP8 acts downstream of IDOL to deubiquitinate LDLR and that USP8 is required for LDLR entry into the MVB pathway. These results provide key mechanistic insights into an evolutionarily conserved pathway for the control of lipoprotein receptor expression and cellular lipid uptake.
doi:10.1128/MCB.01716-12
PMCID: PMC3624246  PMID: 23382078
3.  Deletion of Cavin/PTRF causes global loss of caveolae, dyslipidemia and glucose intolerance 
Cell metabolism  2008;8(4):310-317.
SUMMARY
Caveolae are specialized invaginations of the plasma membrane found in numerous cell types. They have been implicated as playing a role in a variety of physiological processes and are typically characterized by their association with the caveolin family of proteins. We show here by means of targeted gene disruption in mice, that a distinct caveolae-associated protein, Cavin/PTRF, is an essential component of caveolae. Animals lacking Cavin have no morphologically detectable caveolae in any cell type examined and have markedly diminished protein expression of all three caveolin isoforms whilst retaining normal or above normal caveolin mRNA expression. Cavin knockout mice are viable and of normal weight but have higher circulating triglyceride levels, significantly reduced adipose tissue mass, glucose intolerance and hyperinsulinemia, which characteristics constitute a lipodystrophic phenotype. Our results underscore the multi-organ role of caveolae in metabolic regulation and the obligate presence of Cavin for caveolae formation.
doi:10.1016/j.cmet.2008.07.008
PMCID: PMC2581738  PMID: 18840361
4.  Role of Insulin-dependent Cortical Fodrin/Spectrin Remodeling in Glucose Transporter 4 Translocation in Rat Adipocytes 
Molecular Biology of the Cell  2006;17(10):4249-4256.
Fodrin or nonerythroid spectrin is an abundant component of the cortical cytoskeletal network in rat adipocytes. Fodrin has a highly punctate distribution in resting cells, and insulin causes a dramatic remodeling of fodrin to a more diffuse pattern. Insulin-mediated remodeling of actin occurs to a lesser extent than does that of fodrin. We show that fodrin interacts with the t-soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) syntaxin 4, and this interaction is increased by insulin stimulation and decreased by prior latrunculin A treatment. Latrunculin A disrupts all actin filaments, inhibits glucose transporter 4 (GLUT4) translocation, and causes fodrin to partially redistribute from the plasma membrane to the cytosol. In contrast, cytochalasin D disrupts only the short actin filament signal, and cytochalasin D neither inhibits GLUT4 translocation nor fodrin redistribution in adipocytes. Together, our data suggest that insulin induces remodeling of the fodrin–actin network, which is required for the fusion of GLUT4 storage vesicles with the plasma membrane by permitting their access to the t-SNARE syntaxin 4.
doi:10.1091/mbc.E06-04-0278
PMCID: PMC1635356  PMID: 16870704
5.  p115 Interacts with the GLUT4 Vesicle Protein, IRAP, and Plays a Critical Role in Insulin-stimulated GLUT4 Translocation 
Molecular Biology of the Cell  2005;16(6):2882-2890.
Insulin-regulated aminopeptidase (IRAP) is an abundant cargo protein of Glut4 storage vesicles (GSVs) that traffics to and from the plasma membrane in response to insulin. We used the amino terminus cytoplasmic domain of IRAP, residues 1–109, as an affinity reagent to identify cytosolic proteins that might be involved in GSV trafficking. In this way, we identified p115, a peripheral membrane protein known to be involved in membrane trafficking. In murine adipocytes, we determined that p115 was localized to the perinuclear region by immunofluorescence and throughout the cell by fractionation. By immunofluorescence, p115 partially colocalizes with GLUT4 and IRAP in the perinuclear region of cultured fat cells. The amino terminus of p115 binds to IRAP and overexpression of a N-terminal construct results in its colocalization with GLUT4 throughout the cell. Insulin-stimulated GLUT4 translocation is completely inhibited under these conditions. Overexpression of p115 C-terminus has no significant effect on GLUT4 distribution and translocation. Finally, expression of the p115 N-terminus construct has no effect on the distribution and trafficking of GLUT1. These data suggest that p115 has an important and specific role in insulin-stimulated Glut4 translocation, probably by way of tethering insulin-sensitive Glut4 vesicles at an as yet unknown intracellular site.
doi:10.1091/mbc.E05-01-0072
PMCID: PMC1142432  PMID: 15800058
6.  Co-Regulation of Cell Polarization and Migration by Caveolar Proteins PTRF/Cavin-1 and Caveolin-1 
PLoS ONE  2012;7(8):e43041.
Caveolin-1 and caveolae are differentially polarized in migrating cells in various models, and caveolin-1 expression has been shown to quantitatively modulate cell migration. PTRF/cavin-1 is a cytoplasmic protein now established to be also necessary for caveola formation. Here we tested the effect of PTRF expression on cell migration. Using fluorescence imaging, quantitative proteomics, and cell migration assays we show that PTRF/cavin-1 modulates cellular polarization, and the subcellular localization of Rac1 and caveolin-1 in migrating cells as well as PKCα caveola recruitment. PTRF/cavin-1 quantitatively reduced cell migration, and induced mesenchymal epithelial reversion. Similar to caveolin-1, the polarization of PTRF/cavin-1 was dependent on the migration mode. By selectively manipulating PTRF/cavin-1 and caveolin-1 expression (and therefore caveola formation) in multiple cell systems, we unveil caveola-independent functions for both proteins in cell migration.
doi:10.1371/journal.pone.0043041
PMCID: PMC3418245  PMID: 22912783
7.  Fat caves: caveolae, lipid trafficking and lipid metabolism in adipocytes 
Caveolae are subdomains of the eukaryotic cell surface that are so-called because they resemble little caves, small omega-shaped invaginations of the plasma membrane into the cytosol. They are present in many cell types and are especially abundant in adipocytes where they have been implicated as playing a role in lipid metabolism. Thus mice and humans lacking caveolae have small adipocytes and exhibit lipodystrophies along with other physiological abnormalities. Here we review the evidence supporting the role of caveolae in adipocyte lipid metabolism in the context of the protein and lipid composition of these structures.
doi:10.1016/j.tem.2011.04.001
PMCID: PMC3149783  PMID: 21592817
8.  Cholesterol Depletion in Adipocytes Causes Caveolae Collapse Concomitant with Proteosomal Degradation of Cavin-2 in a Switch-Like Fashion 
PLoS ONE  2012;7(4):e34516.
Caveolae, little caves of cell surfaces, are enriched in cholesterol, a certain level of which is required for their structural integrity. Here we show in adipocytes that cavin-2, a peripheral membrane protein and one of 3 cavin isoforms present in caveolae from non-muscle tissue, is degraded upon cholesterol depletion in a rapid fashion resulting in collapse of caveolae. We exposed 3T3-L1 adipocytes to the cholesterol depleting agent methyl-β-cyclodextrin, which results in a sudden and extensive degradation of cavin-2 by the proteasome and a concomitant movement of cavin-1 from the plasma membrane to the cytosol along with loss of caveolae. The recovery of cavin-2 at the plasma membrane is cholesterol-dependent and is required for the return of cavin-1 from the cytosol to the cell surface and caveolae restoration. Expression of shRNA directed against cavin-2 also results in a cytosolic distribution of cavin-1 and loss of caveolae. Taken together, these data demonstrate that cavin-2 functions as a cholesterol responsive component of caveolae that is required for cavin-1 localization to the plasma membrane, and caveolae structural integrity.
doi:10.1371/journal.pone.0034516
PMCID: PMC3321009  PMID: 22493697
9.  Caveolae, Fenestrae and Transendothelial Channels Retain PV1 on the Surface of Endothelial Cells 
PLoS ONE  2012;7(3):e32655.
PV1 protein is an essential component of stomatal and fenestral diaphragms, which are formed at the plasma membrane of endothelial cells (ECs), on structures such as caveolae, fenestrae and transendothelial channels. Knockout of PV1 in mice results in in utero and perinatal mortality. To be able to interpret the complex PV1 knockout phenotype, it is critical to determine whether the formation of diaphragms is the only cellular role of PV1. We addressed this question by measuring the effect of complete and partial removal of structures capable of forming diaphragms on PV1 protein level. Removal of caveolae in mice by knocking out caveolin-1 or cavin-1 resulted in a dramatic reduction of PV1 protein level in lungs but not kidneys. The magnitude of PV1 reduction correlated with the abundance of structures capable of forming diaphragms in the microvasculature of these organs. The absence of caveolae in the lung ECs did not affect the transcription or translation of PV1, but it caused a sharp increase in PV1 protein internalization rate via a clathrin- and dynamin-independent pathway followed by degradation in lysosomes. Thus, PV1 is retained on the cell surface of ECs by structures capable of forming diaphragms, but undergoes rapid internalization and degradation in the absence of these structures, suggesting that formation of diaphragms is the only role of PV1.
doi:10.1371/journal.pone.0032655
PMCID: PMC3293851  PMID: 22403691
10.  Caveolae and lipid trafficking in adipocytes 
Clinical lipidology  2011;6(1):49-58.
The abundance of caveolae in adipocytes suggests a possible cell-specific role for these structures, and because these cells take up and release fatty acids as their quantitatively most robust activity, modulation of fatty acid movement is one such role that is supported by substantial in vitro and in vivo data. In addition, caveolae are particularly rich in cholesterol and sphingolipids, and indeed, fat cells harbor more cholesterol than any other tissue. In this article, we review the role of adipocyte caveolae with regard to these important lipid classes.
PMCID: PMC3103140  PMID: 21625349
adipocyte; caveolin; cavin; cholesterol; fatty acid; lipid droplet
11.  Insulin Resistance and Altered Systemic Glucose Metabolism in Mice Lacking Nur77 
Diabetes  2009;58(12):2788-2796.
OBJECTIVE
Nur77 is an orphan nuclear receptor with pleotropic functions. Previous studies have identified Nur77 as a transcriptional regulator of glucose utilization genes in skeletal muscle and gluconeogenesis in liver. However, the net functional impact of these pathways is unknown. To examine the consequence of Nur77 signaling for glucose metabolism in vivo, we challenged Nur77 null mice with high-fat feeding.
RESEARCH DESIGN AND METHODS
Wild-type and Nur77 null mice were fed a high-fat diet (60% calories from fat) for 3 months. We determined glucose tolerance, tissue-specific insulin sensitivity, oxygen consumption, muscle and liver lipid content, muscle insulin signaling, and expression of glucose and lipid metabolism genes.
RESULTS
Mice with genetic deletion of Nur77 exhibited increased susceptibility to diet-induced obesity and insulin resistance. Hyperinsulinemic-euglycemic clamp studies revealed greater high-fat diet–induced insulin resistance in both skeletal muscle and liver of Nur77 null mice compared with controls. Loss of Nur77 expression in skeletal muscle impaired insulin signaling and markedly reduced GLUT4 protein expression. Muscles lacking Nur77 also exhibited increased triglyceride content and accumulation of multiple even-chained acylcarnitine species. In the liver, Nur77 deletion led to hepatic steatosis and enhanced expression of lipogenic genes, likely reflecting the lipogenic effect of hyperinsulinemia.
CONCLUSIONS
Collectively, these data demonstrate that loss of Nur77 influences systemic glucose metabolism and highlight the physiological contribution of muscle Nur77 to this regulatory pathway.
doi:10.2337/db09-0763
PMCID: PMC2780886  PMID: 19741162
12.  MURC/Cavin-4 and cavin family members form tissue-specific caveolar complexes 
The Journal of Cell Biology  2009;185(7):1259-1273.
Polymerase I and transcript release factor (PTRF)/Cavin is a cytoplasmic protein whose expression is obligatory for caveola formation. Using biochemistry and fluorescence resonance energy transfer–based approaches, we now show that a family of related proteins, PTRF/Cavin-1, serum deprivation response (SDR)/Cavin-2, SDR-related gene product that binds to C kinase (SRBC)/Cavin-3, and muscle-restricted coiled-coil protein (MURC)/Cavin-4, forms a multiprotein complex that associates with caveolae. This complex can constitutively assemble in the cytosol and associate with caveolin at plasma membrane caveolae. Cavin-1, but not other cavins, can induce caveola formation in a heterologous system and is required for the recruitment of the cavin complex to caveolae. The tissue-restricted expression of cavins suggests that caveolae may perform tissue-specific functions regulated by the composition of the cavin complex. Cavin-4 is expressed predominantly in muscle, and its distribution is perturbed in human muscle disease associated with Caveolin-3 dysfunction, identifying Cavin-4 as a novel muscle disease candidate caveolar protein.
doi:10.1083/jcb.200903053
PMCID: PMC2712963  PMID: 19546242
13.  Nur77 coordinately regulates expression of genes linked to glucose metabolism in skeletal muscle 
Innervation is important for normal metabolism in skeletal muscle, including insulin-sensitive glucose uptake. However, the transcription factors that transduce signals from the neuromuscular junction to the nucleus and affect changes in metabolic gene expression are not well defined. We demonstrate here that the orphan nuclear receptor Nur77 is a regulator of gene expression linked to glucose utilization in muscle. In vivo, Nur77 is preferentially expressed in glycolytic compared to oxidative muscle and is responsive to β-adrenergic stimulation. Denervation of rat muscle compromises expression of Nur77 in parallel with that of numerous genes linked to glucose metabolism, including GLUT4 and genes involved in glycolysis, glycogenolysis, and the glycerophosphate shuttle. Ectopic expression of Nur77, either in rat muscle or in C2C12 muscle cells, induces expression of a highly overlapping set of genes, including GLUT4, muscle phosphofructokinase, and glycogen phosphorylase. Furthermore, selective knockdown of Nur77 in rat muscle by shRNA or genetic deletion of Nur77 in mice reduces the expression of a battery of genes involved in skeletal muscle glucose utilization in vivo. Finally, we show that Nur77 binds the promoter regions of multiple innervation-dependent genes in muscle. These results identify Nur77 as a potential mediator of neuromuscular signaling in the control of metabolic gene expression.
doi:10.1210/me.2007-0169
PMCID: PMC2602962  PMID: 17550977
Nur77; glucose metabolism; skeletal muscle
14.  Glut4 Storage Vesicles without Glut4: Transcriptional Regulation of Insulin-Dependent Vesicular Traffic 
Molecular and Cellular Biology  2004;24(16):7151-7162.
Two families of transcription factors that play a major role in the development of adipocytes are the CCAAT/enhancer-binding proteins (C/EBPs) and the peroxisome proliferator-activated receptors (PPARs), in particular PPARγ. Ectopic expression of either C/EBPα or PPARγ in NIH 3T3 fibroblasts results in the conversion of these cells to adipocyte-like cells replete with fat droplets. NIH 3T3 cells ectopically expressing C/EBPα (NIH-C/EBPα) differentiate into adipocytes and exhibit insulin-stimulated glucose uptake, whereas NIH 3T3 cells ectopically expressing PPARγ (NIH-PPARγ) differentiate but do not exhibit any insulin-stimulated glucose uptake, nor do they express any C/EBPα. The reason for the lack of insulin-responsive glucose uptake in the NIH-PPARγ cells is their virtual lack of the insulin-responsive glucose transporter, Glut4. The NIH-PPARγ cells express functionally active components of the insulin receptor-signaling pathway (the insulin receptor, IRS-1, phosphatidylinositol 3-kinase, and Akt2) at levels comparable to those in responsive cell lines. They also express components of the insulin-sensitive vesicular transport machinery, namely, VAMP2, syntaxin-4, and IRAP, the last of these being the other marker of insulin-regulated vesicular traffic along with Glut4. Interestingly, the NIH-PPARγ cells show normal insulin-dependent translocation of IRAP and form an insulin-responsive vesicular compartment as assessed by cell surface biotinylation and sucrose velocity gradient analysis, respectively. Moreover, expression of a Glut4-myc construct in the NIH-PPARγ cells results in its insulin-dependent translocation to the plasma membrane as assessed by immunofluorescence and Western blot analysis. Based on these data, we conclude that major role of C/EBPα in the context of the NIH-PPARγ cells is to regulate Glut4 expression. The differentiated cells possess a large insulin-sensitive vesicular compartment with negligible Glut4, and Glut4 translocation can be reconstituted on expression of this transporter.
doi:10.1128/MCB.24.16.7151-7162.2004
PMCID: PMC479711  PMID: 15282314
15.  Effect of Thyroid Status on Insulin Action in Rat Adipocytes and Skeletal Muscle 
Journal of Clinical Investigation  1980;66(3):574-582.
Isolated adipocytes and soleus muscles prepared from mature rats, rendered hypothyroid by a low iodine diet and propylthiouracil, markedly resisted the ability of insulin to increase glucose utilization. In adipocytes, the sum of basal d-(1-14C)-glucose conversion to CO2, glyceride-glycerol, and fatty acid was unaltered by hypothyroidism, although conversion to fatty acid was decreased. The response of each of these metabolic pathways to insulin at all concentrations tested was greatly diminished in hypothyroid rat adipocytes. 3-O-Methylglucose transport rates in the presence of insulin were not significantly different in adipocytes from hypothyroid as compared with euthyroid rats, although basal transport rates were significantly higher in the hypothyroid state. Lipolysis and cyclic AMP accumulation in adipocytes from hypothyroid rats in response to theophylline were markedly diminished compared with euthyroid controls, but insulin was about as effective in inhibiting lipolysis in these cells as in those derived from euthyroid animals. The binding of 125I-insulin to adipocytes at several hormone concentrations was also shown to be unaffected by hypothyroidism.
In soleus muscle, basal glucose conversion to H2O and glycogen was unaltered in the hypothyroid state, whereas insulin action on these pathways was markedly inhibited. The decrease in muscle insulin responsiveness was less marked than that observed in adipocytes. Uptake of either 2-deoxyglucose or l-arabinose in the presence or absence of insulin was similar in soleus muscles derived from euthryoid vs. hypothyroid rats. Similarly, insulin action on the conversion of soleus muscle glycogen synthase D to the I form in the absence of glucose was unaltered by hypothyroidism. We conclude that (a) hypothyroidism in mature rats leads to a marked decrease in the responsiveness of glucose metabolism in adipocytes and skeletal muscle to insulin; (b) no detectable impairment of the membrane insulin effector systems that mediate the regulation of adipocyte hexose transport and glycogen synthase is caused by hypothyroidism in this animal model; and (c) the cellular defect that leads to apparent insulin resistance of adipocyte and soleus muscle glucose utilization resides at the level of one or more intracellular enzymes involved in glucose catabolism.
PMCID: PMC371686  PMID: 6249852
16.  The Formation of an Insulin-responsive Vesicular Cargo Compartment Is an Early Event in 3T3-L1 Adipocyte Differentiation 
Molecular Biology of the Cell  1999;10(5):1581-1594.
Differentiating 3T3-L1 cells exhibit a dramatic increase in the rate of insulin-stimulated glucose transport during their conversion from proliferating fibroblasts to nonproliferating adipocytes. On day 3 of 3T3-L1 cell differentiation, basal glucose transport and cell surface transferrin binding are markedly diminished. This occurs concomitant with the formation of a distinct insulin-responsive vesicular pool of intracellular glucose transporter 1 (GLUT1) and transferrin receptors as assessed by sucrose velocity gradients. The intracellular distribution of the insulin-responsive aminopeptidase is first readily detectable on day 3, and its gradient profile and response to insulin at this time are identical to that of GLUT1. With further time of differentiation, GLUT4 is expressed and targeted to the same insulin-responsive vesicles as the other three proteins. Our data are consistent with the notion that a distinct insulin-sensitive vesicular cargo compartment forms early during fat call differentiation and its formation precedes GLUT4 expression. The development of this compartment may result from the differentiation-dependent inhibition of constitutive GLUT1 and transferrin receptor trafficking such that there is a large increase in, or the new formation of, a population of postendosomal, insulin-responsive vesicles.
PMCID: PMC25345  PMID: 10233164

Results 1-16 (16)