three-dimensional-adipocyte-culture-as-model-to-study-cachexia

Three-Dimensional Adipocyte Culture as a Model to Study Cachexia-Induced White Adipose Tissue Remodeling

Cancer cachexia (CC) presents itself as a syndrome with multiple manifestations, causing a marked multi-organ metabolic imbalance. Recently, cachectic ...

Boston University School of Medicine

Adipocytes are robust protein secretors, most notably of adipokines, hormone-like polypeptides, which act in an endocrine and paracrine fashion to affect numerous physiological processes such as energy balance and insulin sensitivity. To understand how such proteins are assembled for secretion we describe the function of a novel endoplasmic reticulum oxidoreductase, adiporedoxin (Adrx).

Adiporedoxin, an upstream regulator of ER oxidative folding and protein secretion in adipocytes.

Neutropenia is considered a significant risk factor for invasive aspergillosis but is almost always associated with concurrent thrombocytopenia. Studies determined that platelets, like neutrophils, attached to cell walls of the invasive hyphal form of Aspergillus fumigatus. Organisms were damaged as shown by loss of cell wall integrity in scanning laser confocal microscopy and release of defined hyphal surface glycoproteins. Rapid expression appearance of surface antigen CD63 and release of markers of platelet degranulation confirmed activation during attachment to hyphae. Optimal platelet activation required opsonization of hyphae with fresh or heat-inactivated whole plasma. These effects of opsonization with whole plasma could not be duplicated by pooled human serum, immunoglobulin G, or fibrinogen, whether used separately or combined. Thus, platelets in the presence of whole plasma have the potential to play an important role in normal host defenses against invasive aspergillosis.

Human platelets damage Aspergillus fumigatus hyphae and may supplement killing by neutrophils.

The presence of cell surface caveolin/caveolae has been postulated to influence the localization, expression levels, and kinase activity of numerous receptors, including the insulin receptor. However, there are conflicting data concerning the effects of caveolin on insulin receptor expression and function. To help clarify this issue, we created a gain of function situation by expressing caveolin-1 at various levels in HEK-293 cells where the endogenous level of caveolin-1 is very low. We generated four permanent lines of this cell expressing amounts of caveolin-1 ranging from 10 to 40 times that of parental cells. The amount of caveolin-1 in the human embryonic kidney cells expressing the highest caveolin levels is comparable with that of adipocytes, cells that naturally express one of the highest levels of caveolin-1. We measured insulin receptor amount and insulin-dependent receptor autophosphorylation as well as insulin receptor substrate 1 (IRS1) tyrosine phosphorylation as an index of insulin signaling. We found that the insulin receptor level was essentially the same in the parental and all four derived cell lines. Likewise, we determined that insulin-dependent insulin receptor and IRS1 tyrosine phosphorylation was not significantly different in the four cell lines representing parental, low, medium, and high levels of caveolin-1 expression. We conclude that insulin receptor expression and ligand-dependent signaling is independent of caveolin-1 expression.

Dissociation of insulin receptor expression and signaling from caveolin-1 expression.

We have created by transfection a series of HEK 293 cell lines that express varying amounts of caveolin-1 to test the possible effect of this protein on the transport and metabolism of long chain fatty acids (FA) in cells with this gain of function. We used an extracellular fluorescent probe (ADIFAB) to monitor binding of exogenous FA to the plasma membrane and an intracellular pH probe to monitor FA equilibration across the plasma membrane. Real-time fluorescence measurements showed rapid binding of oleic acid to the extracellular side of the plasma membrane and a rapid translocation across the lipid bilayer by the flip-flop mechanism (<5 s). Two cell lines expressing levels of caveolin-1 roughly comparable to that of adipocytes, which have a very high level of endogenous expression of caveolin-1, showed a relatively slow change in intracellular pH (t(1/2) < 100 s) in addition to the fast changes in fluorescence. We interpret this additional second phase to represent translocation of additional FA from the outer to inner leaflet of the plasma membrane. The slower kinetics could represent either slower flip-flop of FA across highly organized, rigid regions of the plasma membrane or binding of FA to caveolin-1 in the intracellular leaflet of the plasma membrane. The kinetics of palmitate and elaidate (a trans FA) transmembrane movement were identical to that for oleate. These results were observed in the absence of the putative FA transport protein, CD36, and in the absence of any changes in expression of fatty acid transport proteins (FATP) 2 and 4, and are in direct correlation with increased cellular free cholesterol content. FA metabolism was slow in all cell lines and was not enhanced by caveolin-1 expression. We conclude that transport of FA across the plasma membrane is modulated by caveolin-1 and cholesterol and is not dependent on the putative FA transport proteins CD36 and FATP.

Role of caveolin-1 and cholesterol in transmembrane fatty acid movement.

Ectopic expression of caveolin-1 in HEK293 cells enhances FA sequestration in membranes as measured by a pH-sensitive fluorescent dye (1). We hypothesized that sequestration of FA is due to the enrichment of caveolin in the cytosolic leaflet and its ability to facilitate the formation of lipid rafts to buffer high FA levels. Here we show that ec-topic expression of caveolin-3 also results in enhanced FA sequestration. To further discriminate the effect that caveolins have on transmembrane FA movement and distribution, we labeled the outer membrane leaflet with fluorescein-phosphatidylethanolamine (FPE), whose emission is quenched by the presence of FA anions. Real-time measurements made with FPE and control experiments with positively charged fatty amines support our hypothesis that caveolins promote localization of FA anions through interactions with basic amino acid residues (lysines and arginines) present at the C termini of caveolins-1 and -3.

Caveolins sequester FA on the cytoplasmic leaflet of the plasma membrane, augment triglyceride formation, and protect cells from lipotoxicity.

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.

Caveolae and lipid trafficking in adipocytes.

Mice and humans lacking functional caveolae are dyslipidemic and have reduced fat stores and smaller fat cells. To test the role of caveolins/caveolae in maintaining lipid stores and adipocyte integrity, we compared lipolysis in caveolin-1 (Cav1)-null fat cells to that in cells reconstituted for caveolae by caveolin-1 re-expression. We find that the Cav1-null cells have a modestly enhanced rate of lipolysis and reduced cellular integrity compared with reconstituted cells as determined by the release of lipid metabolites and lactic dehydrogenase, respectively, into the media. There are no apparent differences in the levels of lipolytic enzymes or hormonally stimulated phosphorylation events in the two cell lines. In addition, acute fasting, which dramatically raises circulating fatty acid levels in vivo, causes a significant upregulation of caveolar protein constituents. These results are consistent with the hypothesis that caveolae protect fat cells from the lipotoxic effects of elevated levels fatty acids, which are weak detergents at physiological pH, by virtue of the property of caveolae to form detergent-resistant membrane domains.

Caveolins/caveolae protect adipocytes from fatty acid-mediated lipotoxicity.

Many tissues play an important role in metabolic homeostasis and the development of diabetes and obesity. We hypothesized that the circulating redox metabolome is a master metabolic regulatory system that impacts all organs and modulates reactive oxygen species (ROS) production, lipid peroxidation, energy production and changes in lipid turnover in many cells including adipocytes.

Extracellular Redox Regulation of Intracellular Reactive Oxygen Generation, Mitochondrial Function and Lipid Turnover in Cultured Human Adipocytes.

Medium chain triglycerides (MCT) have unique metabolic properties which may improve insulin sensitivity (Si) and beta cell function but data in humans are limited. We conducted a 6-week clinical trial of MCT oil supplementation.

Article	Year
Adiporedoxin, an upstream regulator of ER oxidative folding and protein secretion in adipocytes. Molecular metabolism\| PubMed ID: 26629401	2015
Human platelets damage Aspergillus fumigatus hyphae and may supplement killing by neutrophils. Infection and immunity\| PubMed ID: 9488412	1998
Dissociation of insulin receptor expression and signaling from caveolin-1 expression. The Journal of biological chemistry\| PubMed ID: 15699039	2005
Role of caveolin-1 and cholesterol in transmembrane fatty acid movement. Biochemistry\| PubMed ID: 16503643	2006
Caveolins sequester FA on the cytoplasmic leaflet of the plasma membrane, augment triglyceride formation, and protect cells from lipotoxicity. Journal of lipid research\| PubMed ID: 20388923	2010
Caveolae and lipid trafficking in adipocytes. Clinical lipidology\| PubMed ID: 21625349	2011
Caveolins/caveolae protect adipocytes from fatty acid-mediated lipotoxicity. Journal of lipid research\| PubMed ID: 21652731	2011
Extracellular Redox Regulation of Intracellular Reactive Oxygen Generation, Mitochondrial Function and Lipid Turnover in Cultured Human Adipocytes. PloS one\| PubMed ID: 27741233	2016
Effects of medium chain triglycerides supplementation on insulin sensitivity and beta cell function: A feasibility study. PloS one\| PubMed ID: 31869355	2019

Tova Meshulam

Biography

JoVE Journal Publications

Other Publications