Prior studies have implicated accumulation of ceramide in blood vessels as a basis for vascular dysfunction in diet-induced obesity via a mechanism involving type 2 protein phosphatase (PP2A) dephosphorylation of endothelial nitric oxide synthase (eNOS). The current study sought to elucidate the mechanisms linking ceramide accumulation with PP2A activation and determine whether pharmacological inhibition of PP2A in vivo normalizes obesity-associated vascular dysfunction and limits the severity of hypertension. We show in endothelial cells that ceramide associates with the inhibitor 2 of PP2A (I2PP2A) in the cytosol, which disrupts the association of I2PP2A with PP2A leading to its translocation to the plasma membrane. The increased association between PP2A and eNOS at the plasma membrane promotes dissociation of an Akt-Hsp90-eNOS complex that is required for eNOS phosphorylation and activation. A novel small-molecule inhibitor of PP2A attenuated PP2A activation, prevented disruption of the Akt-Hsp90-eNOS complex in the vasculature, preserved arterial function, and maintained normal blood pressure in obese mice. These findings reveal a novel mechanism whereby ceramide initiates PP2A colocalization with eNOS and demonstrate that PP2A activation precipitates vascular dysfunction in diet-induced obesity. Therapeutic strategies targeted to reducing PP2A activation might be beneficial in attenuating vascular complications that exist in the context of type 2 diabetes, obesity, and conditions associated with insulin resistance.
The reprogramming of lipid metabolism is a hallmark of many cancers that has been shown to promote breast cancer progression. While several lipid signatures associated with breast cancer aggressiveness have been identified, a comprehensive lipidomic analysis specifically targeting the triple-negative subtype of breast cancer (TNBC) may be required to identify novel biomarkers and therapeutic targets for this most aggressive subtype of breast cancer that still lacks effective therapies. In this current study, our global LC-MS-based lipidomics platform was able to measure 684 named lipids across 15 lipid classes in 70 TNBC tumors. Multivariate survival analysis found that higher levels of sphingomyelins were significantly associated with better disease-free survival in TNBC patients. Furthermore, analysis of publicly available gene expression datasets identified that decreased production of ceramides and increased accumulation of sphingoid base intermediates by metabolic enzymes were associated with better survival outcomes in TNBC patients. Our LC-MS lipidomics profiling of TNBC tumors has, for the first time, identified sphingomyelins as a potential prognostic marker and implicated enzymes involved in sphingolipid metabolism as candidate therapeutic targets that warrant further investigation.
African-American (AA) men are more than twice as likely to die of prostate cancer (PCa) than European American (EA) men. Previous in silico analysis revealed enrichment of altered lipid metabolic pathways in pan-cancer AA tumors. Here, we performed global unbiased lipidomics profiling on 48 matched localized PCa and benign adjacent tissues (30 AA, 24 ancestry-verified, and 18 EA, 8 ancestry verified) and quantified 429 lipids belonging to 14 lipid classes. Significant alterations in long chain polyunsaturated lipids were observed between PCa and benign adjacent tissues, low and high Gleason tumors, as well as associated with early biochemical recurrence, both in the entire cohort, and within AA patients. Alterations in cholesteryl esters, and phosphatidyl inositol classes of lipids delineated AA and EA PCa, while the levels of lipids belonging to triglycerides, phosphatidyl glycerol, phosphatidyl choline, phosphatidic acid, and cholesteryl esters distinguished AA and EA PCa patients with biochemical recurrence. These first-in-field results implicate lipid alterations as biological factors for prostate cancer disparities.
In whole cell lysates we showed that palmitate disrupts the interaction between inhibitor 2 (I2) of protein phosphatase 2A (PP2A) and PP2A and that this event initiates PP2A colocalization with eNOS. Both responses were negated when ceramide synthesis was inhibited. We sought to confirm this palmitate evoked, ceramide‐mediated effect using immunofluorescent approaches. Bovine aortic endothelial cells (BAECs) were treated for 3 h with BSA (V), V + 500 μM palmitate (P), V + ceramide inhibition (10 uM myriocin; M), or P+M (PM). P‐induced increases (p<0.05) in ceramide were prevented (p<0.05) in cells exposed to P+M (n=8). P increased PP2A association with eNOS (70±9% vs. V) and decreased p‐eNOS(S)1177 to eNOS (35±5% vs. V) in isolated membrane fractions (both p<0.05, n=8), and these responses were prevented in cells exposed to P+M. We then permeabilized cells that were treated as above, and incubated them with 1° antibodies for eNOS, PP2A, I2PP2A, or ceramide, followed by incubation with fluorescent 2° antibodies to assess subcellular localization in intact cells using confocal microscopy. Relative to V‐treatment, P: (i) increased ceramide association with I2PP2A in the cytosol by 70±6%; (ii) decreased PP2A association with I2PP2A in the cytosol by 45±5%; and (iii) increased PP2A association with eNOS in the membrane by 80±9% (all p<0.05; n=6 per combination). All responses were negated in P+M –treated cells. Endogenous ceramide disrupts the cytosolic interaction between I2PP2A and PP2A and causes translocation of PP2A to the membrane where it associates with eNOS. ADA1–12‐BS‐208, 2R15HL091493
We have shown in bovine aortic endothelial cells (BAECs) treated with palmitate (P) and in arteries from obese mice that protein phosphatase 2A (PP2A) associates directly with eNOS. When PP2A co‐localizes with eNOS, interactions among Akt‐Hsp90‐eNOS are disrupted, p‐eNOS/eNOS is decreased, and NO bioavailability is impaired. We hypothesized that PP2A activation is sufficient to disrupt interactions among Akt‐Hsp90‐eNOS in arteries from fat‐fed mice. To test efficacy of the in vivo PP2A inhibitor Lixte Biotechnology 1 (LB1, Setauket, NY), BAECs were treated (3h) ± 500 uM P ± 4 uM LB1. P‐induced increases (p<0.05) in PP2A activity, and reductions (p<0.05) in p‐eNOS/eNOS and NO production (ELISA and EPR), were prevented by LB1. To determine the safety / efficacy of LB1, mice were treated (1 mg/kg/day IP) ± LB1 for 21 days. PP2A activity was reduced (p<0.05) and p‐PP2A Y307 to PP2A was elevated (p<0.05) in arteries from LB1 vs. vehicle‐treated mice after 3 and 21 days. To confirm the importance of PP2A vs. other phosphatases, BAECs incubated ± PP1 (3 uM tautomycin), PP2B (100 nM cyclosporine), or PP2A (4 uM LB1) inhibition. P‐induced reductions (p<0.05) in p‐eNOS/eNOS were prevented only by LB1. Finally, 7‐week old, male C57B16 mice consumed standard (CON, n=20) or high‐fat (HF, n=20) chow for 12‐weeks. Subgroups (n=10) of CON and HF mice received IP injections of saline (vehicle; V) or LB1 for the last 14‐days. HF mice gained weight and developed peripheral glucose intolerance vs. CON mice regardless of LB1 treatment. p‐eNOS/eNOS was impaired (53±6%; p<0.05) in vascular homogenates from HF‐V vs. all groups. After eNOS immunoprecipitation (IP), the association between: (i) PP2A:eNOS increased 95±11%, and the co‐localization between (ii) Akt:eNOS decreased 53±8%, and (iii) Hsp90:eNOS decreased 56±12%, in HF‐V vs. CON mice (all p<0.05). These findings suggest that PP2A activation is sufficient to disrupt interactions among Akt‐Hsp90‐eNOS in arteries from fat‐fed mice. Grant Funding Source: ADA 7‐08‐RA‐164, ADA1‐12‐BS‐208, 2R15HL091493
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