Inhibition Of Fatty Acid Oxidation Research Articles

Gold Nanoparticle Transforms Activated Cancer-Associated Fibroblasts to Quiescence.

Activated cancer-associated fibroblasts (CAFs) play a major role in the poor outcome in many diseases including pancreatic cancer. Normally quiescent with high lipid content and low proliferative capacity, CAFs receiving cues from cancer cells in the tumor microenvironment become activated and transformed into a lipid-deprived and highly proliferative myofibroblast type phenotype. Therefore, reversal of activated fibroblasts to the quiescence state is an important area of investigation that may help the therapeutic management of a number of diseases including pancreatic cancer. Here, we describe a unique biological function of gold nanoparticles (GNPs) and demonstrate that GNPs may be used to transform activated CAFs to quiescence and provide insights into the underlying molecular mechanisms. Using immortalized and primary patient derived CAFs, we demonstrate that GNPs enhanced lipid content in the cells by inducing expression of lipogenesis genes such as FASN, SREBP2, and FABP3. Using pharmacological inhibitors of lipolysis, lipophagy, and fatty acid oxidation, we further demonstrate that CAFs utilized a GNP-induced endogenously synthesized lipid to maintain the quiescent phenotype. Consequently, treatment with GNP sensitizes CAF to FASN inhibitor or FASN siRNA. Hence, GNPs may be used as a tool to probe mechanisms of quiescence in CAFs and help device strategies to target the stromal compartment exploiting the mechanisms of lipid utilization.

Role of metabolic reprogramming toward fatty acid oxidation on tumor lymph node metastasis.

e14501 Background: Metastasis of tumors to lymph node (LN) is a strong predictor of cancer patient mortality and determinant of disease progression and treatment options. Despite the clinical significance of tumor LN metastasis, the mechanisms underlying this process remain largely unexplored. Methods: We used comparative transcriptomics and metabolomics analyses of the primary tumor and LN micro- and macro-metastatic tumors from B16F10 murine melanoma model. B16F10 melanoma footpad implantation model and MMTV-PyMT spontaneous breast cancer genetic mouse models were analyzed in vivo. We also investigated the LN-metastatic tumors from sentinel LNs in 21 patients with melanoma. Results: We found that tumor cells undergo dramatic metabolic reprogramming towards fatty acid oxidation (FAO) for successful LN metastasis. Mechanistically, transcription co-activator YAP is selectively activated in LN metastatic tumors, leading to stimulation of FAO gene programs. Pharmacological inhibition of FAO or genetic ablation of YAP in tumors markedly suppressed LN metastasis. Unexpectedly, metastatic tumors highly stimulated the bile acid synthesis pathway upon arrival at the LN, and these bile acids activated YAP of tumor cells via nuclear vitamin D receptor. Lastly, YAP activation in the metastatic LNs of melanoma patients was reversely correlated with distant metastasis–free survival. Conclusions: This study propose that inhibition of FAO or YAP signaling to prevent the metabolic reprogramming of metastatic tumors offers a potential therapeutic strategy for mitigating tumor LN metastasis.

Pilot study to enhance FDG-PET imaging of prostate cancers with the metabolic inhibitor ranolazine.

e16551 Background: 2-deoxy-2-[18F]fluoro-D-glucose positron emission tomography (FDG-PET) is a widely-used imaging modality for many cancers; however, its utility in prostate cancer is limited. Fatty acid oxidation (FAO) is a primary source of energy for early prostate cancer. We previously demonstrated that FAO inhibition in prostate cancer mouse models resulted in increased glucose metabolism and enhanced tumor FDG uptake, with peak uptake at 24 hours. To validate these preclinical findings, we conducted a pilot study to evaluate whether a partial FAO inhibitor, ranolazine, increases tumor FDG uptake on PET imaging for prostate cancer. Methods: Prostate cancer patients with untreated localized cancer (arm 1) and with metastatic disease on hormonal or other therapy (arm 2) were enrolled and underwent baseline and post-treatment FDG-PET/CT scans (standard dose of 10 mCi FDG). Ranolazine 1000mg PO BID x 2 doses was given within 24-48 hours of the second scan. The primary objective was to evaluate the rate of successful enhancement of FDG uptake on PET imaging, based on one or more of the following criteria: 30% increase in maximum SUV with an absolute change of 2 units; 30% increase in mean SUV with an absolute change of 0.75 units; or 20% increase in mean SUV with an absolute change of 1 unit. Results: Eleven patients (four in arm 1, seven in arm 2) were enrolled. Ranolazine was well tolerated by all participants, with no adverse effects observed. Both increases and decreases in SUV uptake were observed on the post-ranolazine scans. No patient met the predefined criteria for successful enhancement of FDG uptake. There was an incidental finding of thyroid cancer seen in one patient that was discovered on PET imaging. The study was closed early due to the emerging clinical availability of alternative and effective PET imaging modalities such as [11C] choline, [18F] fluciclovine, [68Ga] PSMA, and [18F] sodium fluoride. Conclusions: Given the small sample size, we were not able to make any firm conclusions. In this limited study, ranolazine treatment did not result in enhanced FDG-PET-tumor detection. ClinicalTrials.gov identifier: NCT01992016. Supported by the William Meyn Foundation; NIH/NCI P30CA46934, 5K12CA086913, CA168934; ACS RSG-16-256-01-TBE; Colorado Translational Research Imaging Center Pilot Award; Paul Sandoval Cancer Research Summer Fellowship. Clinical trial information: NCT01992016.

An HK2 Antisense Oligonucleotide Induces Synthetic Lethality in HK1-HK2+ Multiple Myeloma.

Although the majority of adult tissues express only hexokinase 1 (HK1) for glycolysis, most cancers express hexokinase 2 (HK2) and many coexpress HK1 and HK2. In contrast to HK1+HK2+ cancers, HK1-HK2+ cancer subsets are sensitive to cytostasis induced by HK2shRNA knockdown and are also sensitive to synthetic lethality in response to the combination of HK2shRNA knockdown, an oxidative phosphorylation (OXPHOS) inhibitor diphenyleneiodonium (DPI), and a fatty acid oxidation (FAO) inhibitor perhexiline (PER). The majority of human multiple myeloma cell lines are HK1-HK2+. Here we describe an antisense oligonucleotide (ASO) directed against human HK2 (HK2-ASO1), which suppressed HK2 expression in human multiple myeloma cell cultures and human multiple myeloma mouse xenograft models. The HK2-ASO1/DPI/PER triple-combination achieved synthetic lethality in multiple myeloma cells in culture and prevented HK1-HK2+ multiple myeloma tumor xenograft progression. DPI was replaceable by the FDA-approved OXPHOS inhibitor metformin (MET), both for synthetic lethality in culture and for inhibition of tumor xenograft progression. In addition, we used an ASO targeting murine HK2 (mHK2-ASO1) to validate the safety of mHK2-ASO1/MET/PER combination therapy in mice bearing murine multiple myeloma tumors. HK2-ASO1 is the first agent that shows selective HK2 inhibition and therapeutic efficacy in cell culture and in animal models, supporting clinical development of this synthetically lethal combination as a therapy for HK1-HK2+ multiple myeloma. SIGNIFICANCE: A first-in-class HK2 antisense oligonucleotide suppresses HK2 expression in cell culture and in in vivo, presenting an effective, tolerated combination therapy for preventing progression of HK1-HK2+ multiple myeloma tumors. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/79/10/2748/F1.large.jpg.

Inhibition of fatty acid metabolism by etomoxir or TOFA suppresses murine dendritic cell activation without affecting viability

Objective: Dendritic cells (DCs) are important players in immunity against pathogens, but overactive DCs have been implicated in autoimmune diseases, like lupus, in which a paucity of targeted therapies remains. Recent research shows that DCs upregulate their immunometabolism when activating. We explored whether modulating fatty acid (FA) metabolism needed for oxidative phosphorylation can affect the activation of two main DC subsets.Material and methods: Sorted murine plasmacytoid DCs (pDCs) and conventional DCs (cDCs), generated in FLT3-L medium, were treated with etomoxir, an inhibitor of FA oxidation, or TOFA, an inhibitor of FA synthesis, then stimulated with TLR9 agonist CpGA. Surface activation markers and viability were analyzed by flow cytometry, cytokine, and chemokine production and were measured by ELISA.Results: Modulation of FA metabolism suppressed the upregulation of costimulatory molecules and the production of proinflammatory cytokine IL-6 and type I Interferon-dependent chemokine CXCL10 by both subsets of DCs, without affecting DC viability, neither of resting DCs or upon activation. Etomoxir inhibited pDCs at lower doses than cDCs, suggesting that pDCs may be more susceptible to FA metabolic modulation.Conclusions: Both cDCs, the primary antigen presenting cell, and pDCs, the primary type I IFN producer, exhibit a suppressed ability to activate but normal viability when their FA metabolism is inhibited by etomoxir or TOFA. Our findings indicate that FA metabolism plays an important role in the activation of both pDCs and cDCs and suggest that its modulation is an exploitable therapeutic target to suppress DC activation in inflammation or autoimmunity.

OR34-4 ATF3 Is A Converging Point For AR Signaling And Fatty Acid Oxidation In Prostate Cancer

Prostate cancer (PCa) is the second most prevalent cancer and leading causes of death among men worldwide. Androgen deprivation is the first line of treatment for locally advanced and metastatic PCa. For men who develop metastatic, castration-resistant PCa (CRPC), survival is limited. Thus, there is a need to identify synergistic pathways to either prevent or re-sensitize the castration-resistant phenotype. PCa has a long natural history of development and there is a significant correlation between age, diet, metabolism and progress of PCa. We have previously reported that treating PCa cells with fatty acid oxidation inhibitors (FAOi) sensitizes them to androgen blockade. Here we set out to evaluate how the two disparate pathways: AR signaling and FAO, influence one another hoping to identify new therapeutic vulnerabilities. We used CRPC cell models (LNCaP-MDVres, and LNCaP-C42), as well as the FAOi (etomoxir and ranolazine) and the antiandrogen enzalutamide (MDV3100). Cells were treated for 48 hours, followed by gene expression, androgen signaling and growth analysis. Both FAOi strongly suppressed the androgen response hallmark (FDR q-val < 0.001) in MDVres cells, and this was associated with a significant upregulation of the ATF3 gene (etomoxir; 30-fold, p<0.001 vs vehicle; ranolazine; 2-fold, p< 0.03). Interestingly, knockdown (KD) of ATF3 with shRNAs resulted in differential responses to FAOi in these genetically-related cells. In the MDVres cells, ATF3 KD resulted in increased PSA expression with FAOi treatment (etomoxir: 2-fold, p= 0.002; ranolazine 4-fold, p< 0.001 vs. control shRNA cells). On the other hand, in the C42 cells, ATF3 KD resulted in decreased PSA expression with FAOi treatment (etomoxir: 3-fold, p< 0.001; ranolazine 1.5-fold, p< 0.004 vs. control cells). These results suggested that ATF3 is a convergent point for AR and FAO pathways that is cell-context dependent, and its expression status could be exploited to sensitize these cells to MDV3100 and/or the FAOi. Cell growth assays confirmed that MDVres cells with ATF3 KD were more sensitive to MDV3100 alone and showed a synergistic combination effect with FAOi (p< 0.01, combinatorial index (CI) = 0.6). The C42 cells with ATF3 KD were more sensitive to the FAOi alone and showed a synergistic effect with MDV3100 (p< 0.001, CI= 0.4). Examination of patient databases revealed an inverse correlation between AR and ATF3, a low expression of ATF3 in advanced cancers (primary tumors n=64, metastatic tumors n=24), and better disease-free survival in patients with high ATF3 expression (MSKCC; n=157, Log rank p= 0.01). These data support the role for FAOi to re-sensitize CRPC to endocrine therapies. In conclusion, we have identified ATF3 as a modulator of FAO and AR signaling that has predictive value for clinical outcomes using fat oxidation inhibitors in CRPC. Sources of research support: NIH, ACS-RSG

Role of Fatty Acid Oxidation and Glucose Oxidation in Endothelial Cell Inflammation and Permeability Associated with Acute Lung Injury

Acute lung injury (ALI) is a common cause of respiratory failure in critically ill patients with a mortality rate of 38.5%. ALI can be precipitated by either direct insults such as pneumonia, aspiration or via indirect insults such as sepsis and multiple trauma, to the lungs. The vascular endothelium forming the innermost lining of all pulmonary blood vessels is the major barrier that protects air spaces against vascular fluid entry. Because of their localization, endothelial cells (EC) are constantly exposed to oxygen and nutrients that fuel cell metabolism. However, very little is known about the role of EC metabolism in the regulation of EC inflammation and permeability, the two major pathogenic features of ALI. Recent studies have highlighted that fatty acid oxidation (FAO) is central to the tight regulation of EC permeability required to maintain vessel functionality. The purpose of the present study is to understand the mechanistic link between cell metabolism and thrombin‐induced EC inflammation and permeability and to understand the relevance of this linkage in the pathophysiology of ALI. Human Pulmonary Artery Endothelial cells (HPAECs) were stimulated with proinflammatory mediator thrombin in the presence and absence of Etomoxir, an inhibitor of FAO or dichloroacetate (DCA), an inhibitor of pyruvate hydrogenase kinase (PHK). Our results indicate that both Etomoxir and DCA inhibited thrombin‐induced inflammatory responses as observed by decrease in the secretion of cytokines IL‐6 and IL‐8. In addition real time changes in transendothelial electrical resistance (TER) were measured to monitor endothelial permeability after exposure of HPAECs to Etomoxir or DCA, followed by thrombin treatment. Our data indicate that Etomoxir had no effect on thrombin‐induced decrease in TER however it delayed the recovery after 1 hour of thrombin challenge. DCA in contrast showed no effect on thrombin‐induced EC permeability. Together our data suggest that metabolic pathways are critical to EC inflammation and permeability induced by thrombin.Support or Funding InformationSupported by: NIH (HL130870)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Limited Fatty Acid Oxidation (FAO) in CPT2 Knockout Myocytes Associates with Insulin Resistance and Cell Stress: possible role of acylcarnitine lipotoxicity

Lipotoxicity (the accumulation of fat‐derived metabolites that activate cell stress pathways) has been implicated in muscle insulin resistance. We have proposed that lipotoxicity, and in particular accumulation of long‐chain acylcarnitines (LCACs), also contributes to the episodic myopathy and rhabdomyolysis that is evident in persons with inherited disorders of long‐chain FAO. Many studies in the arena of LCAC bioactivity have employed exogenous application of LCACs to cells; few studies have modified endogenously‐produced LCACs to determine myocyte outcomes. Thus, conclusions about the (patho)physiological relevance of LCAC‐associated lipotoxicity have been difficult to determine. Herein, we used CRISPR‐Cas9 technology to knock out carnitine palmitoyltransferase‐2 (CPT2) in murine C2C12 myotubes: as expected, this maneuver led to almost complete inhibition of FAO, plus significant cellular and media accumulation of palmitoylcarnitine in palmitate‐treated cells. In CPT2 KO myotubes, treatment with 500 μM palmitate led to ~2‐fold higher production of IL‐6 (a myocyte cell stress marker) and a ~1.5 to 2‐fold increased release of adenylate kinase (cell death and permeability marker) at 3 and 6 hr post‐treatment (when compared to palmitate‐treated wildtype control cells; n=4/treatment). Furthermore, CPT2 KO myotubes displayed higher sensitivity to palmitate‐induced insulin resistance: the % inhibition in terms of insulin‐induced p‐Akt abundance was 60%, 80%, and 95% in CPT2 KO cells (at 200, 500, and 750 μM palmitate, respectively), vs. 38%, 53%, and 67% in controls (p&lt;0.01, n=8/treatment). The results indicate that limitation of long‐chain FAO by loss of CPT2 in muscle cells elicits cell stress and insulin resistance, consistent with a working model in which lipotoxicity and accumulation of LCAC trigger these outcomes. Further studies are needed to determine if endogenously‐produced LCACs contribute to the phenotypes observed in CPT2 KO myotubes, or if LCAC‐independent mechanisms are involved. It remains to be established whether these phenomena underlie the cardiac and skeletal muscle myopathies typical of inherited disorders of FAO, or contribute to the insulin resistance accompanying type 2 diabetes.Support or Funding InformationThis work was supported by USDA‐ARS Project 6026‐51000‐010‐05S.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

PPAR\u03b1-Mediated Positive-Feedback Loop Contributes to Cold Exposure Memory

Fluctuations in food availability and shifts in temperature are typical environmental changes experienced by animals. These environmental shifts sometimes portend more severe changes; e.g., chilly north winds precede the onset of winter. Such telltale signs may be indicators for animals to prepare for such a shift. Here we show that HEK293A cells, cultured under starvation conditions, can “memorize” a short exposure to cold temperature (15 °C), which was evidenced by their higher survival rate compared to cells continuously grown at 37 °C. We refer to this phenomenon as “cold adaptation”. The cold-exposed cells retained high ATP levels, and addition of etomoxir, a fatty acid oxidation inhibitor, abrogated the enhanced cell survival. In our standard protocol, cold adaptation required linoleic acid (LA) supplementation along with the activity of Δ-6-desaturase (D6D), a key enzyme in LA metabolism. Moreover, supplementation with the LA metabolite arachidonic acid (AA), which is a high-affinity agonist of peroxisome proliferator-activated receptor-alpha (PPARα), was able to underpin the cold adaptation, even in the presence of a D6D inhibitor. Cold exposure with added LA or AA prompted a surge in PPARα levels, followed by the induction of D6D expression; addition of a PPARα antagonist or a D6D inhibitor abrogated both their expression, and reduced cell survival to control levels. We also found that the brief cold exposure transiently prevents PPARα degradation by inhibiting the ubiquitin proteasome system, and starvation contributes to the enhancement of PPARα activity by inhibiting mTORC1. Our results reveal an innate adaptive positive-feedback mechanism with a PPARα-D6D-AA axis that is triggered by a brief cold exposure in cells. “Cold adaptation” could have evolved to increase strength and resilience against imminent extreme cold temperatures.

MSC-regulated lncRNA MACC1-AS1 promotes stemness and chemoresistance through fatty acid oxidation in gastric cancer

Chemotherapy is the preferred treatment for advanced stage gastric cancer (GC) patients and chemotherapy resistance is the major obstacle to effective cancer therapy. Increasing evidence suggests that mesenchymal stem cells (MSCs) make important contributions to development of drug resistance. However, the underlying mechanism remains elusive. In this study, we discovered that abundant MSCs in tumor tissues predicted a poor prognosis in GC patients. MSCs promoted stemness and chemoresistance in GC cells through fatty acid oxidation (FAO) in vitro and in vivo. Mechanically, transforming growth factor β1 (TGF-β1) secretion by MSCs activated SMAD2/3 through TGF-β receptors and induced long non-coding RNA (lncRNA) MACC1-AS1 expression in GC cells, which promoted FAO-dependent stemness and chemoresistance through antagonizing miR-145-5p. Moreover, pharmacologic inhibition of FAO with etomoxir (ETX) attenuated MSC-induced FOLFOX regiment resistance in vivo. These results suggest that FAO plays an important role in MSC-mediated stemness and chemotherapy resistance in GC and FAO inhibitors in combination with chemotherapeutic drugs present as a promising strategy to overcome chemoresistance.

Tumor metastasis to lymph nodes requires YAP-dependent metabolic adaptation.

In cancer patients, metastasis of tumors to sentinel lymph nodes (LNs) predicts disease progression and often guides treatment decisions. The mechanisms underlying tumor LN metastasis are poorly understood. By using comparative transcriptomics and metabolomics analyses of primary and LN-metastatic tumors in mice, we found that LN metastasis requires that tumor cells undergo a metabolic shift toward fatty acid oxidation (FAO). Transcriptional coactivator yes-associated protein (YAP) is selectively activated in LN-metastatic tumors, leading to the up-regulation of genes in the FAO signaling pathway. Pharmacological inhibition of FAO or genetic ablation of YAP suppressed LN metastasis in mice. Several bioactive bile acids accumulated to high levels in the metastatic LNs, and these bile acids activated YAP in tumor cells, likely through the nuclear vitamin D receptor. Inhibition of FAO or YAP may merit exploration as a potential therapeutic strategy for mitigating tumor metastasis to LNs.

Fueling lymph node metastases

Cancer Metastatic cells can migrate from a primary tumor to distant organs through two routes: They can enter the bloodstream directly, or they can enter a lymph node adjacent to the primary tumor. Little is known about the biological mechanisms that allow tumor cells to survive and grow within lymph nodes. Studying mouse models, Lee et al. found that tumor cells adapt to the lymph node microenvironment by shifting their metabolism toward fatty acid oxidation. This occurs through activation of a signaling pathway driven by the yes-associated protein (YAP) transcription factor. Importantly, inhibition of fatty acid oxidation or YAP signaling suppressed lymph node metastasis in the mice. Science , this issue p. [644][1] [1]: /lookup/doi/10.1126/science.aav0173

Liposome-Mediated Drug Delivery in Larval Zebrafish to Manipulate Macrophage Function

Chemical interventions are regularly used to examine and manipulate macrophage function in larval zebrafish. Given chemicals are typically administered by simple immersion or injection, it is not possible to resolve whether their impact on macrophage function is direct or indirect. Liposomes provide an attractive strategy to target drugs to specific cellular compartments, including macrophages. As an example, injecting liposomal clodronate into animal models, including zebrafish, is routinely used to deliver toxic levels of clodronate specifically to macrophages for targeted cell ablation. Here we show that liposomes can also target the delivery of drugs to zebrafish macrophages to selectively manipulate their function. We utilized the drugs etomoxir (a fatty acid oxidation inhibitor) and MitoTEMPO (a scavenger of mitochondrial reactive oxygen species [mROS]), that we have previously shown, through free drug delivery, suppress monosodium urate (MSU) crystal-driven macrophage activation. We generated poloxamer 188 modified liposomes that were readily phagocytosed by macrophages, but not by neutrophils. Loading these liposomes with etomoxir or MitoTEMPO and injecting into larvae suppressed macrophage activation in response to MSU crystals, as evidenced by proinflammatory cytokine expression and macrophage-driven neutrophil recruitment. This work reveals the utility of packaging drugs into liposomes as a strategy to selectively manipulate macrophage function.

LXRα Promotes Hepatosteatosis in Part Through Activation of MicroRNA-378 Transcription and Inhibition of Ppargc1β Expression.

Nonalcoholic fatty liver disease (NAFLD) is a major risk factor of many end‐stage liver diseases. Alterations in microRNA expression have been reported in patients with NAFLD. However, the transcriptional mechanism(s) of dysregulated microRNAs under the state of NAFLD is poorly described, and microRNAs that regulate the pathogenesis of NAFLD synergistically with their regulators remain unknown. Here we report that microRNA‐378 expression is significantly increased in fatty livers of mice and patients with NAFLD. Although microRNA‐378 locates within the intron of Ppargc1β (peroxisome proliferator‐activated receptor γ coactivator 1‐beta), there was a significant uncoupling of Ppargc1β mRNA and microRNA‐378 levels in both sources of fatty livers. Further studies identified a full‐length primary transcript of microRNA‐378. LXRα (liver X receptor alpha) functioned as a transcription activator of microRNA‐378 and a repressor of Ppargc1β transcription. It is known that miR‐378 is an inhibitor of fatty acid oxidation (FAO) and the function of Ppargc1β is opposite to that of miR‐378. GW3965 treatment (LXRα agonist) of murine hepatocytes and mice increased microRNA‐378 and reduced Ppargc1β, which subsequently impaired FAO and aggravated hepatosteatosis. In contrast, additional treatment of miR‐378 inhibitor or Ppargc1β, which knocked down increased miR‐378 or recovered expression of Ppargc1β, offset the effects of GW3965. Liver‐specific ablation of Lxrα led to decreased miR‐378 and increased Ppargc1β, which subsequently improved FAO and reduced hepatosteatosis. Conclusion: Our findings indicated that miR‐378 possesses its own transcription machinery, which challenges the well‐established dogma that miR‐378 transcription is controlled by the promoter of Ppargc1β. LXRα selectively activates transcription of miR‐378 and inhibits expression of Ppargc1β, which synergistically impairs FAO. In addition to lipogenesis, impaired FAO by miR‐378 in part contributes to LXRα‐induced hepatosteatosis.

Cardiac-specific deficiency of the mitochondrial calcium uniporter augments fatty acid oxidation and functional reserve

The mitochondrial calcium uniporter (MCU) relays cytosolic Ca2+ transients to the mitochondria. We examined whether energy metabolism was compromised in hearts from mice with a cardiac-specific deficiency of MCU subjected to an isoproterenol (ISO) challenge. Surprisingly, isolated working hearts from cardiac MCU-deficient mice showed higher cardiac work, both in the presence or absence of ISO. These hearts were not energy-starved, with ISO inducing a similar increase in glucose oxidation rates compared to control hearts, but a greater increase in fatty acid oxidation rates. This correlated with lower levels of the fatty acid oxidation inhibitor malonyl CoA, and to an increased stimulatory acetylation of its degrading enzyme malonyl CoA decarboxylase and of the fatty acid β-oxidation enzyme β-hydroxyacyl CoA dehydrogenase. We conclude that impaired mitochondrial Ca2+ uptake does not compromise cardiac energetics due to a compensatory stimulation of fatty acid oxidation that provides a higher energy reserve during acute adrenergic stress.

Noninvasive evaluation of fat-carbohydrate metabolic switching in heart and contracting skeletal muscle.

The ability of heart and skeletal muscle (SM) to switch between fat and carbohydrate oxidation is of high interest in the study of metabolic diseases and exercise physiology. Positron emission tomography (PET) imaging with the glucose analog 2-[18F]fluoro-2-deoxy-glucose (18F-FDG) provides a noninvasive means to quantitate glucose metabolic rates. However, evaluation of fatty acid oxidation (FAO) rates by PET has been limited by the lack of a suitable FAO probe. We have developed a metabolically trapped oleate analog, ( Z)-18-[18F]fluoro-4-thia-octadec-9-enoate (18F-FTO), and investigated the feasibility of using 18F-FTO and 18F-FDG to measure FAO and glucose uptake, respectively, in heart and SM of rats in vivo. To enhance the metabolic rates in SM, the vastus lateralis (VL) muscle was electrically stimulated in fasted rats for 30 min before and 30 min following radiotracer injection. The responses of radiotracer uptake patterns to pharmacological inhibition of FAO were assessed by pretreatment of the rats with the carnitine palmitoyl-transferase-1 (CPT-1) inhibitor sodium 2-[5-(4-chlorophenyl)-pentyl]oxirane-2-carboxylate (POCA). Small-animal PET images and biodistribution data with 18F-FTO and 18F-FDG demonstrated profound metabolic switching for energy provision in the myocardium from exogenous fatty acids to glucose in control and CPT-1-inhibited rats, respectively. Uptake of both radiotracers was low in unstimulated SM. In stimulated VL muscle, 18F-FTO and 18F-FDG uptakes were increased 4.4- and 28-fold, respectively, and CPT-1 inhibition only affected 18F-FTO uptake (66% decrease). 18F-FTO is a FAO-dependent PET probe that may allow assessment of energy substrate metabolic switching in conjunction with 18F-FDG and other metabolic probes.

Control of Region-Specific Intestinal Metabolism and Maintenance by PRDM16

Distinct metabolic programs regulate intestinal stem cell renewal and differentiation. However, the mechanisms that rewire metabolism during differentiation are poorly defined. It is also unclear whether progenitors in different intestinal regions utilize specialized metabolic programs. Here, we identify the transcription factor PRDM16 as a critical region-specific regulator of small intestinal metabolism and progenitor differentiation. Acute deletion of Prdm16 in mice causes severe intestinal atrophy, apoptosis, and an accumulation of poorly differentiated cells. Genomic and metabolic studies show that PRDM16 controls the levels of fatty acid oxidation (FAO) within the stem cell niche. Notably, PRDM16 levels and FAO are highest in the upper small intestine and decline distally. Accordingly, deletion of Prdm16 or inhibition of FAO selectively impairs the differentiation of upper-intestinal enteroid cultures. Collectively, these data reveal that PRDM16 specifies a region-specific FAO metabolic switch in intestinal progenitor cells to maintain tissue homeostasis.

The Role of AMPK in Chemotherapy Response in AML

Introduction: Acute myeloid leukemia is an aggressive malignancy characterized by relapse and resistance to therapy. When treated with standard chemotherapy 60-80% of patients will achieve a remission however most will suffer a relapsed and once relapsed the disease is much more resistant to repeated treatment. Despite the central role of resistance little is known about the mechanism of chemotherapy responsiveness in AML. Our laboratory has previous shown a metabolic shift occurs in AML cells following chemotherapy exposure with increased mitochondrial oxygen consumption leading to resistance. Adenosine monophosphate activated protein kinase (AMPK) is a central metabolic regulator and leading candidate to coordinate this metabolic shift. However, the role of AMPK in chemotherapy response in AML is unknown.Methods: A Cas9 expressing AML model was generated and partially infected with a lentiviral vector coding an sgRNA targeting the alpha 1 subunit of AMPK, PRKAA1. This subunit was chosen as cell line and clinical expression datasets demonstrated that AML cells expressed little or none of the second alpha subunit PRKAA2. PRKAA1 deleted clones (AMPK KO) were isolated and loss of PRKAA1 expression and AMPK activity were confirmed. AMPK KO cells were characterized for responsiveness to chemotherapy, metabolic inhibitors and the DNA damage response.Results: Consistent with a role in response to chemotherapy AML cells treated with cytarabine demonstrated a significant increase in AMPK activation. When partially AMPK deleted populations were treated with cytarabine or doxorubicin a significant enrichment was seen in the deleted cells suggesting that loss of AMPK promotes therapy resistance. To confirm this finding isogeneic AML cell lines were treated with chemotherapy. AMPK KO cells were significantly more resistant to doxorubicin and cytarabine compared to isogeneic controls expressing an sgRNA targeting the ROSA26 (ROSA KO) safe harbor locus. When the mechanism of this resistance was explored AMPK KO cells were found to have diminished H2AX, p53 and p21 induction following exposure to doxorubicin culminating in a decrease in apoptosis. To assess if this resistant phenotype was present in vivo, AMPK KO or ROSA KO cells were tail vein injected into sub-lethally irradiated mice. Upon confirmation of engraftment mice were treated with a combination of cytarabine and doxorubicin. AMPK KO mice had a significantly decreased survival following treatment then treated ROSA controls. Additionally, AMPK KO and ROSA KO cells were treated with the glycolysis inhibitor 2-Deoxyglucose, the autophagy inhibitor chloroquine and the fatty acid oxidation inhibitor extomoxir. In all cases AMPK KO cells were significantly more resistant to these agents than the ROSA KO cells suggesting a proapoptotic role of AMPK following exposure to metabolic inhibitors. Finally, the clinical relevance of these findings was assessed by examining outcomes of AML patients treated with chemotherapy in relationship to their levels of PRKAA1 expression. Patients with the lowest expression of PRKAA1 had a significantly worse overall survival consistent with our findings.Conclusions: In AML cells activated AMPK provides pro-apoptotic signaling in response to DNA damaging agents allowing incorporation of metabolic status into cell fate decisions following treatment. Utilization of metabolic inhibitors that activate AMPK in combination with chemotherapy should be explored in the treatment of AML. DisclosuresPardee:Karyopharm: Research Funding; Novartis: Speakers Bureau; Rafael Pharmaceuticals: Employment; Celgene: Speakers Bureau; Amgen: Speakers Bureau.

70 - Role of fatty acid oxidation in hyperoxia-induced endothelial-to-mesenchymal transition in lung endothelial cells

Bronchopulmonary dysplasia (BPD) is a chronic lung disease, which is characterized by alveolar dysplasia and impaired vascularization in premature infants. BPD can develop pulmonary hypertension (PH) in adolescence and adulthood. This is corroborated by previous reports showing that hyperoxic exposure in newborn mice caused PH in adult mice. Endothelial-to-mesenchymal cells (EndoMT), a biological process where endothelial cells (ECs) progressively lose their specific markers and gain mesenchymal phenotype, is one of the contributing factors for development of PH. It has been shown that inhibition of fatty acid oxidation (FAO) augments the magnitude of EndoMT in ECs. This was attenuated by enhancing FAO via overexpression of carnitine palmitoyltransferase 1a (Cpt1a), a rate-limiting step of the carnitine shuttle. However, no information is available on whether hyperoxic exposure reduces FAO, resulting in EndoMT in lung ECs. We hypothesized that hyperoxic exposure causes EndoMT by modulating FAO in lung ECs. To test this hypothesis, mouse fetal lung EC line (MFLM-91U) and primary lung microvascular endothelial cells (LMVECs) were exposed to hyperoxia (95% O2/5% CO2) for 24 h. Some cultures were placed in air for 1, 3, or 7 days post exposure (air recovery). We found that hyperoxic exposure followed by air recovery reduced FAO as determined by the Seahorse XF24 Analyzer. The levels of markers for mesenchymal cells (i.e., vimentin, CD44, Snail1, Snug, Twist1, and α-SMA) were increased, while the EC markers (i.e., vWF, PECAM-1/CD31 and CD34) were reduced in lung ECs exposed to hyperoxia followed by air recovery. Surprisingly, gene deletion of Cpt1a attenuated hyperoxia-induced reduction of CD34 and CD31 gene expression. In conclusion, hyperoxic exposure followed by air recovery causes EndoMT in lung ECs, and inhibition of FAO rescues this phenotype. These findings may provide therapeutic approach targeting Cpt1a to preventing PH in BPD patients.

Excess Hydrocortisone Hampers Placental Nutrient Uptake Disrupting Cellular Metabolism.

Low birth weight increases neonatal morbidity and mortality, and surviving infants have increased risk of metabolic and cardiovascular disturbances later in life, as well as other neurological, psychiatric, and immune complications. A gestational excess of glucocorticoids (GCs) is a well-known cause for fetal growth retardation, but the biological basis for this association remains elusive. Placental growth is closely related to fetal growth. The placenta is the main regulator of nutrient transport to the fetus, resulting from the difference between placental nutrient uptake and the placenta's own metabolism. The aim of this study was to analyze how excess hydrocortisone affects placental glucose and lipid metabolism. Human placenta explants from term physiological pregnancies were cultured for 18 hours under different hydrocortisone concentrations (2.75, 5.5, and 55 mM; 1, 2, and 20 mg/ml). Placental glucose and lipid uptake and the metabolic partitioning of fatty acids were quantified by isotopic techniques, and expression of specific glucose transporter GLUT1 was quantified by western blot. Cell viability was assessed by MTT, immunohistochemistry and caspase activity. We found that excess hydrocortisone impairs glucose uptake and lipoprotein lipase (LPL) activity, coincident with a GC-dose dependent inhibition of fatty acid oxidation and esterification. None of the experimental conditions showed an increased cell death. In conclusion, our results show that GC overexposure exerts a dysfunctional effect on lipid transport and metabolism and glucose uptake in human placental explants. These findings could well be directly related to a reduced placental growth and possibly to a reduced supply of nutrients to the fetus and the consequent fetal growth retardation and metabolic programming.