PPARα, δ and FOXO1 Gene Silencing Overturns Palmitate-Induced Inhibition of Pyruvate Oxidation Differentially in C2C12 Myotubes

The molecular mechanisms by which free fatty acids (FFA) inhibit muscle glucose oxidation is still elusive. We recently showed that C2C12 myotubes treated with palmitate (PAL) presented with greater protein expression levels of PDK4 and transcription factors PPARα and PPARδ and lower p-FOXO/t-FOXO protein ratios when compared to control. This was complemented with the hallmarks of metabolic inflexibility (MI), i.e., reduced rates of glucose uptake, PDC activity and maximal pyruvate-derived ATP production rates (MAPR). However, the relative contribution of these transcription factors to the increase in PDK4 and reduced glucose oxidation could not be established. Therefore, by using a similar myotube model, a series of individual siRNA gene silencing experiments, validated at transcriptional and translation levels, were performed in conjunction with measurements of glucose uptake, PDC activity, MAPR and concentrations of metabolites reflecting PDC flux (lactate and acetylcarnitine). Gene silencing of PPARα, δ and FOXO1 individually reduced PAL-mediated inhibition of PDC activity and increased glucose uptake, albeit by different mechanisms as only PPARδ and FOXO1 silencing markedly reduced PDK4 protein content. Additionally, PPARα and FOXO1 silencing, but not PPARδ, increased MAPR with PAL. PPARδ silencing also decreased FOXO1 protein. Since FOXO1 silencing did not alter PPARδ protein, this suggests that FOXO1 might be a PPARδ downstream target. In summary, this study suggests that the molecular mechanisms by which PAL reduces PDC-mediated glucose-derived pyruvate oxidation in muscle occur primarily through increased PPARδ and FOXO1 mediated increases in PDK4 protein expression and secondarily through PPARα mediated allosteric inhibition of PDC flux. Furthermore, since PPARδ seems to control FOXO1 expression, this may reflect an important role for PPARδ in preventing glucose oxidation under conditions of increased lipid availability.

Lactate Dehydrogenase B and Pyruvate Oxidation Pathway Associated With Carfilzomib-Related Cardiotoxicity in Multiple Myeloma Patients: Result of a Multi-Omics Integrative Analysis

Multiple myeloma (MM) is the second most frequent hematologic cancer in the United States. Carfilzomib (CFZ), an irreversible proteasome inhibitor being used to treat relapsed and refractory MM, has been associated with cardiotoxicity, including heart failure. We hypothesized that a multi-omics approach integrating data from different omics would provide insights into the mechanisms of CFZ-related cardiovascular adverse events (CVAEs). Plasma samples were collected from 13 MM patients treated with CFZ (including 7 with CVAEs and 6 with no CVAEs) at the University of Florida Health Cancer Center. These samples were evaluated in global metabolomic profiling, global proteomic profiling, and microRNA (miRNA) profiling. Integrative pathway analysis was performed to identify genes and pathways differentially expressed between patients with and without CVAEs. The proteomics analysis identified the up-regulation of lactate dehydrogenase B (LDHB) [fold change (FC) = 8.2, p = 0.01] in patients who experienced CVAEs. The metabolomics analysis identified lower plasma abundance of pyruvate (FC = 0.16, p = 0.0004) and higher abundance of lactate (FC = 2.4, p = 0.0001) in patients with CVAEs. Differential expression analysis of miRNAs profiling identified mir-146b to be up-regulatein (FC = 14, p = 0.046) in patients with CVAE. Pathway analysis suggested that the pyruvate fermentation to lactate pathway is associated with CFZ-CVAEs. In this pilot multi-omics integrative analysis, we observed the down-regulation of pyruvate and up-regulation of LDHB among patients who experienced CVAEs, suggesting the importance of the pyruvate oxidation pathway associated with mitochondrial dysfunction. Validation and further investigation in a larger independent cohort are warranted to better understand the mechanisms of CFZ-CVAEs.

Lactic Acid Fermentation Is Required for NLRP3 Inflammasome Activation

Activation of the Nod-like receptor 3 (NLRP3) inflammasome is important for activation of innate immune responses, but improper and excessive activation can cause inflammatory disease. We previously showed that glycolysis, a metabolic pathway that converts glucose into pyruvate, is essential for NLRP3 inflammasome activation in macrophages. Here, we investigated the role of metabolic pathways downstream glycolysis – lactic acid fermentation and pyruvate oxidation—in activation of the NLRP3 inflammasome. Using pharmacological or genetic approaches, we show that decreasing lactic acid fermentation by inhibiting lactate dehydrogenase reduced caspase-1 activation and IL-1β maturation in response to various NLRP3 inflammasome agonists such as nigericin, ATP, monosodium urate (MSU) crystals, or alum, indicating that lactic acid fermentation is required for NLRP3 inflammasome activation. Inhibition of lactate dehydrogenase with GSK2837808A reduced lactate production and activity of the NLRP3 inflammasome regulator, phosphorylated protein kinase R (PKR), but did not reduce the common trigger of NLRP3 inflammasome, potassium efflux, or reactive oxygen species (ROS) production. By contrast, decreasing the activity of pyruvate oxidation by depletion of either mitochondrial pyruvate carrier 2 (MPC2) or pyruvate dehydrogenase E1 subunit alpha 1 (PDHA1) enhanced NLRP3 inflammasome activation, suggesting that inhibition of mitochondrial pyruvate transport enhanced lactic acid fermentation. Moreover, treatment with GSK2837808A reduced MSU-mediated peritonitis in mice, a disease model used for studying the consequences of NLRP3 inflammasome activation. Our results suggest that lactic acid fermentation is important for NLRP3 inflammasome activation, while pyruvate oxidation is not. Thus, reprograming pyruvate metabolism in mitochondria and in the cytoplasm should be considered as a novel strategy for the treatment of NLRP3 inflammasome-associated diseases.

Effects of insulin on adaptive capacity of rat pancreatic acinar cells mitochondria

Insulin increases the basal and agonist-stimulated secretion of pancreatic acinar cells, which leads to increase of energy demand and requires sufficient oxidative substrates supply. Cholecystokinin substantially increases the respiration rate of pancreatic acinar cells upon pyruvate oxidation. However, it is not clear how insulin affects mitochondrial oxidative processes at rest and upon secretory stimulation. Experiments were carried out on male Wistar rats (250–300 g) kept on standard diet. Animals were fasted 12 h before the experiment. Pancreatic acini were isolated with collagenase. Basal and FCCP-stimulated respiration of rat pancreatic acini was measured with Clark electrode. Adaptive capacity of mitochondria was assessed by the maximal rate of uncoupled respiration. Statistical significance (P) of differenced between the means was assessed either with a paired t-test or with repeated measures two-way ANOVA and post-hoc Turkey test. Adaptive capacity of pan­creatic acinar mitochondria was significantly higher when pyruvate (2 mM) was used as oxidative substrate comparing with glucose (10 mM). Incubation with insulin (100 nM) for 20 minutes elevated the basal respiration and adaptive capacity of pancreatic acinar mitochondria upon glucose, but not pyruvate, oxidation. Cholecystokinin (0.1 nM, 30 min) stimulated the rate of basal and maximal uncoupled respiration of acinar cells upon pyruvate oxidation, but insulin completely negated this increase of mitochondrial adaptive capacity. Thus, insulin increases the glucose oxidation in pancreatic acinar cells at resting state, but suppresses pyruvate oxidation upon secretory stimulation with cholecystokinin. The mechanisms of insulin action of pyruvate metabolism in pancreatic acinar cells require further elucidation.

Skeletal Muscle Atrophy in Chronic Kidney Disease (CKD)

Background: Skeletal muscle atrophy and dysfunction occur with chronic kidney disease (CKD) progression leading to morbidity, mortality, and falls. Skeletal muscle dysfunction may be due to impaired fatty acid (FA) oxidation and enhanced pyruvate oxidation as demonstrated in preliminary metabolomic data. We performed multiple techniques to assess the extent of muscle dysfunction and associated pathways, including: systematic review and meta-analysis, assays of disease progression and FA metabolism, expression of markers associated with skeletal muscle FA metabolism and pyruvate oxidation. Methods: Meta-analysis: Multiple databases were used to identify relevant studies of muscle atrophy in preclinical and clinical models. Experimental Study: 1)CKD rats and 2)Normal littermates (N=12/gr) at 35 weeks. Extensor digitorum longus (EDL) and soleus were harvested at sacrifice. Serum Biochemistry: Plasma BUN, calcium and phosphorus were analyzed using colorimetric assays. Carnitine Assay: Plasma carnitine levels was measured using ELISA kit. Protein Expression: Western blots with the lysate of EDL and soleus to determine the activity levels of PDH and PDK4. Results: A total of 4685 studies were screened in the meta-analysis, of which 646 were relevant. Subsequent steps are to perform full text review and data extraction. Animal studies: BUN and phosphorus were significantly increased in CKD compared to normal. Carnitine levels were significantly decreased in CKD rats compared to normal. PDH was not significantly different in the EDL or soleus. PDK4 is yet to be performed. Conclusions: The extent to which muscle atrophy occurs will be identified in the meta-analysis. Elevated BUN confirmed disease and carnitine assay confirmed low carnitine levels in CKD. Identifying low carnitine has led to an interventional study of carnitine supplementation to determine if there was improved FA oxidation. Testing of PDK4 is needed to determine significance of pyruvate regulation. Reviewing the literature and understanding the mechanism of skeletal muscle atrophy in CKD will allow future targeted therapeutics.