•Role of prohibitins (PHBs) in macrophage signaling was studied using PHB knockdown.•PHB knockdown decreases expression and signaling of lipid raft-dependent receptors.•Plasma membrane packing and ...raft formation are altered in PHB knockdown macrophages.•PHB controls the composition of cellular fatty acids.•PHBs impact macrophage signaling by regulating lipid rafts.
Prohibitins (PHB1 and PHB2) are ubiquitously expressed proteins which play critical roles in multiple biological processes, and together form the ring-like PHB complex found in phospholipid-rich cellular compartments including lipid rafts. Recent studies have implicated PHB1 as a mediator of fatty acid transport as well as a membrane scaffold mediating B lymphocyte and mast cell signal transduction. However, the specific role of PHBs in the macrophage have not been characterized, including their role in fatty acid uptake and lipid raft-mediated inflammatory signaling. We hypothesized that the PHB complex regulates macrophage inflammatory signaling through the formation of lipid rafts. To evaluate our hypothesis, RAW 264.7 macrophages were transduced with shRNA against PHB1, PHB2, or scrambled control (Scr), and then stimulated with lipopolysaccharide (LPS) or tumor necrosis factor-alpha (TNF-α), which activate lipid raft-dependent receptor signaling (CD14/TLR4 and TNFR1, respectively). PHB1 knockdown was lethal, whereas PHB2 knockdown (PHB2kd), which also resulted in decreased PHB1 expression, led to attenuated nuclear factor-kappa-B (NF-κB) activation and subsequent cytokine and chemokine production. PHB2kd macrophages also had decreased cell surface TNFR1, CD14, TLR4, and lipid raft marker ganglioside GM1 at baseline and post-stimuli. Post-LPS, PHB2kd macrophages did not increase the concentration of cellular saturated, monounsaturated, and polyunsaturated fatty acids. This was accompanied by decreased lipid raft formation and modified plasma membrane molecular packing, further supporting the PHB complex's importance in lipid raft formation. Taken together, these data suggest a critical role for PHBs in regulating macrophage inflammatory signaling via maintenance of fatty acid composition and lipid raft structure.
Prohibitins are proteins found in phospholipid-rich cellular compartments, including lipid rafts, that play important roles in signaling, transcription, and multiple other cell functions. Macrophages are key cells in the innate immune response and the presence of membrane lipid rafts is integral to signal transduction, but the role of prohibitins in macrophage lipid rafts and associated signaling is unknown. To address this question, prohibitin knockdown macrophages were generated and responses to lipopolysaccharide and tumor necrosis factor-alpha, which act through lipid raft-dependent receptors, were analyzed. Prohibitin knockdown macrophages had significantly decreased cytokine and chemokine production, transcription factor activation, receptor expression, lipid raft assembly and membrane packing, and altered fatty acid remodeling. These data indicate a novel role for prohibitins in macrophage inflammatory signaling through regulation of fatty acid composition and lipid raft formation.
Alterations to branched-chain keto acid (BCKA) oxidation have been implicated in a wide variety of human diseases, ranging from diabetes to cancer. Although global shifts in BCKA metabolism-evident ...by gene transcription, metabolite profiling, and
flux analyses have been documented across various pathological conditions, the underlying biochemical mechanism(s) within the mitochondrion remain largely unknown.
experiments using isolated mitochondria represent a powerful biochemical tool for elucidating the role of the mitochondrion in driving disease. Such analyses have routinely been utilized across disciplines to shed valuable insight into mitochondrial-linked pathologies. That said, few studies have attempted to model
BCKA oxidation in isolated organelles. The impetus for the present study stemmed from the knowledge that complete oxidation of each of the three BCKAs involves a reaction dependent upon bicarbonate and ATP, both of which are not typically included in respiration experiments. Based on this, it was hypothesized that the inclusion of exogenous bicarbonate and stimulation of respiration using physiological shifts in ATP-free energy, rather than excess ADP, would allow for maximal BCKA-supported respiratory flux in isolated mitochondria. This hypothesis was confirmed in mitochondria from several mouse tissues, including heart, liver and skeletal muscle. What follows is a thorough characterization and validation of a novel biochemical tool for investigating BCKA metabolism in isolated mitochondria.
Estrogen receptor-α (ERα) is a nuclear receptor family member thought to substantially contribute to the metabolic regulation of skeletal muscle. However, previous mouse models utilized to assess the ...necessity of ERα signaling in skeletal muscle were confounded by altered developmental programming and/or influenced by secondary effects, making it difficult to assign a causal role for ERα. The objective of this study was to determine the role of skeletal muscle ERα in regulating metabolism in the absence of confounding factors of development.
A novel mouse model was developed allowing for induced deletion of ERα in adult female skeletal muscle (ERαKOism). ERαshRNA was also used to knockdown ERα (ERαKD) in human myotubes cultured from primary human skeletal muscle cells isolated from muscle biopsies from healthy and obese insulin-resistant women.
Twelve weeks of HFD exposure had no differential effects on body composition, VO2, VCO2, RER, energy expenditure, and activity counts across genotypes. Although ERαKOism mice exhibited greater glucose intolerance than wild-type (WT) mice after chronic HFD, ex vivo skeletal muscle glucose uptake was not impaired in the ERαKOism mice. Expression of pro-inflammatory genes was altered in the skeletal muscle of the ERαKOism, but the concentrations of these inflammatory markers in the systemic circulation were either lower or remained similar to the WT mice. Finally, skeletal muscle mitochondrial respiratory capacity, oxidative phosphorylation efficiency, and H2O2 emission potential was not affected in the ERαKOism mice. ERαKD in human skeletal muscle cells neither altered differentiation capacity nor caused severe deficits in mitochondrial respiratory capacity.
Collectively, these results suggest that ERα function is superfluous in protecting against HFD-induced skeletal muscle metabolic derangements after postnatal development is complete.
•Induced skeletal muscle specific ERαKO (ERαKOism) examines the role of ERα without confounding factors of development.•Skeletal muscle glucose uptake is not impaired in ERαKOism.•Skeletal muscle mitochondrial function is not impaired in ERαKOism.•ERαKD in human myotubes does not severely affect mitochondrial respiratory capacity.
Inflammation is a complex mechanism primarily driven by the immune system to eradicate pathogens/foreign substances and restore tissue homeostasis. Despite the beneficial effects that inflammation ...employs, signaling can often become dysregulated leading to uncontrolled systemic inflammation and irreversible host tissue damage. Therefore, regulating the cellular and physiological mechanisms of inflammation constitutes a viable avenue of research to mitigate inflammatory disease progression. Herein, we evaluated prohibitins (PHB1 and PHB2), pleiotropic homologous proteins with known anti-inflammatory and antioxidant capabilities, in the context of systemic inflammation as well as macrophage-specific inflammatory signaling. Using two in vivo models of systemic inflammation, we found that PHB1 levels were increased in serum, suggesting a potential signaling role for PHB. Moreover, recombinant PHB1 treatment mitigated systemic inflammation and tissue/organ injury and modulated the phenotype of circulating immune cells. When investigating the role of PHB specifically in monocytes/macrophages, we found that PHB not only increased populations of pro-inflammatory monocytes in vivo but also regulated vital macrophage inflammatory signaling (as shown in vitro). We determined that PHB is a scaffold protein important for macrophage lipid raft formation and subsequent receptor trafficking. PHB modulation of macrophages influenced cell surface display of lipid-raft-dependent receptors and downstream inflammatory signaling cascades. To our knowledge, these are the first data to reveal PHB’s pro-inflammatory effects in macrophages and its mechanistic operation of lipid-raft-dependent signal transduction in macrophages. In this report, we provide insight into the diverse yet complementary roles of PHB in regulating various aspects of immune-driven inflammatory processes.
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