Caveolins/caveolae protect adipocytes from fatty acid-mediated lipotoxicity

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 detergentresistant membrane domains

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

<div><p>Background</p><p>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.</p><p>Methods</p><p>Differentiated human preadipocytes were exposed to the redox couples, lactate (L) and pyruvate (P), β–hydroxybutyrate (βOHB) and acetoacetate (Acoc), and the thiol-disulfides cysteine/ cystine (Cys/CySS) and GSH/GSSG for 1.5–4 hours. ROS measurements were done with CM-H₂DCFDA. Lipid peroxidation (LPO) was assessed by a modification of the thiobarbituric acid method. Lipolysis was measured as glycerol release. Lipid synthesis was measured as ¹⁴C-glucose incorporated into lipid. Respiration was assessed using the SeaHorse XF24 analyzer and the proton leak was determined from the difference in respiration with oligomycin and antimycin A.</p><p>Results</p><p>Metabolites with increasing oxidation potentials (GSSG, CySS, Acoc) increased adipocyte ROS. In contrast, P caused a decrease in ROS compared with L. Acoc also induced a significant increase in both LPO and lipid synthesis. L and Acoc increased lipolysis. βOHB increased respiration, mainly due to an increased proton leak. GSSG, when present throughout 14 days of differentiation significantly increased fat accumulation, but not when added later.</p><p>Conclusions</p><p>We demonstrated that in human adipocytes changes in the external redox state impacted ROS production, LPO, energy efficiency, lipid handling, and differentiation. A more oxidized state generally led to increased ROS, LPO and lipid turnover and more reduction led to increased respiration and a proton leak. However, not all of the redox couples were the same suggesting compartmentalization. These data are consistent with the concept of the circulating redox metabolome as a master metabolic regulatory system.</p></div

Acetoacetate induced Lipid synthesis.

<p>Fourteen days after differentiation, adipocytes were exposed to either 20 mM ßOHB and Acoc, 10 μM Forskolin, or 5 nM insulin (positive control) in a Krebs solution containing 5 mM glucose (of which 15 μM was ¹⁴C-glucose radiolabelled). After a 4 hour incubation period, cells were extracted in a chloroform-methanol. The organic layer (25 μl) was then placed in a LabLogic 300SL Liquid Scintillation counter (Brandon, Florida) to analyze β-particle emission to determine how much of the radiolabelled glucose was incorporated into lipid. Due to variations in the counts between experiments, data are represented as a percentage of the control condition average (41577±1630 counts). Data are presented as the mean ± SEM (N = 4). ANOVA analysis with a post-hoc Dunnetts test was used for statistics Forskolin p = 0.0001, Acoc p = 0.004.</p

Model illustrating ROS and redox interactions in the mitochondria.

<p>Others have come to similar conclusions: the antioxidant activity of pyruvate was also shown by: O'Donnell-Tormey et al [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164011#pone.0164011.ref041" target="_blank">41</a>] who conclude that in mouse and human cells exogenous pyruvate, in concentrations that approximated physiological plasma and serum, protects cells from lysis by H₂0₂. Bassenge et al [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164011#pone.0164011.ref042" target="_blank">42</a>] also find in postischemic hearts, that pyruvate (0.1–5.0 mM) dose dependently inhibits ROS up to 80% while L-lactate (1.0–15.0 mM) stimulates both basal and postischemic ROS several fold. Thus cytotoxicities due to cardiac ischemia-reperfusion-generated ROS may also be alleviated by redox reactants such as pyruvate. Mallet et al [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164011#pone.0164011.ref043" target="_blank">43</a>] reported that pyruvate promoted robust contractile recovery of H₂O₂-challenged myocardium. Thus, studies with variation in the L/P ratio cannot help to differentiate cytosolic from mitochondrial targets. Further studies will require compartment specific alteration of ROS-scavenging enzymes to address this issue. In addition, we cannot differentiate between pyruvate directly scavenging ROS in a non-enzymatic manner [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164011#pone.0164011.ref041" target="_blank">41</a>] or through NADH or NADPH production. Regardless of the exact mechanism by which pyruvate inhibits lipolysis, it is clear that it involves reducing ROS generation (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164011#pone.0164011.g001" target="_blank">Fig 1D</a>).</p

Extracellular oxidation by Acoc and GSH/GSSG but not Cys/CySS increased lipid peroxidation.

<p>LPO was assessed in adipocytes using modified TBARs. Cells were grown and maintained as for ROS measurements. Test compounds were added as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164011#pone.0164011.g001" target="_blank">Fig 1</a> legend, for 90 min (to match the time cells were treated in the ROS production assay). LPO was measured as described in the materials and methods, A) Total concentration of active ßOHB plus Acoc was 20mM, B) GSH plus GSSG 110 μM glutathione (reduced + oxidized glutathione), C) 200 μM cysteine (cysteine + cystine). Data are pooled from 2 separate experiments done in triplicate. Results are expressed as % of reduced form. P = 0.02 comparing Acoc to ßOHB (mean ± SD: 122 ± 1.7%) and p = 0.046 comparing oxidized to reduced glutathione (mean ± SD: 115 ± 6.4%). Fifty μM tert-butyl hydroperoxide (tBH), an inducer of hydrogen peroxide production added as a positive control, increased lipid peroxidation (mean ± SD: 115.7 ± 8.3%) p = 0.03 n = 3 experiments. P-values calculated using ANOVA analysis with a post-hoc Dunnetts.</p

Redox couples altered lipolysis.

<p>Lipolysis was measured as glycerol release [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164011#pone.0164011.ref018" target="_blank">18</a>]. Adipocytes day 14 after differentiation were incubated in KRB with 0.5 mM oleate complexed to 150 μM BSA with or without the test solutions, as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164011#pone.0164011.g001" target="_blank">Fig 1</a> legend, for 4 hours. Forskolin (5 μM) was used as a positive control (not shown). Aliquots were removed and the glycerol content was measured using an NADH-linked assay. DPI was added as an inhibitor of flavin oxidases at 10 μM (B). Data were pooled from 3 independent experiments done in triplicate. Results are expressed as means ± SD. A) Difference between ßOHB and Acoc was significant p = 0.042. C) Pyruvate decreased lipolysis when the L/P ratio was 10:1 p = 0.048, 5:1 p = 0.027 and pyruvate alone p = 0.015. P-values were calculated using ANOVA analysis with a post-hoc Dunnetts test.</p