Mitochondria-Lipid-droplet interaction: Where Mitochondrial Dynamics Meet Bioenergetics

Mitochondria surrounding lipid droplets (Peri-Droplet Mitochondria;PDM) maintain a unique proteome which is not equilibrated with the rest of the mitochondrial population. We find that PDM remain stationary and elongated on lipid droplets, fuel lipid droplet expansion, and do not fuse with cytosolic mitochondria. Compared to cytosolic mitochondria, PDM have higher capacity to metabolize pyruvate but reduced capacity to oxidize fatty acids. We reveal for the first time the mechanism by which the elongated mitochondrial shape of PDM differentiate their fuel preference from that of cytosolic mitochondria. We find that mitochondrial elongation reduces fatty acid utilization by inhibiting CPT1 activity. On the other hand, we find that mitochondrial fragmentation, such as observed in NASH, increases mitochondrial lipid utilization and may act as a compensatory mechanism to reduce lipotoxicity. Indeed, inhibition of fission in a model of NASH exacerbated NASH phenotype. We developed a cell-based imaging for the quantification of mitochondria-lipid droplet association and identified a set of small molecules that detach mitochondria from lipid droplets (PDM-Detachers). New unpublished data demonstrate PDM detachers induce lipolysis and lipid droplet shrinkage. Using these tools we assessed the role of lipid droplets in SARS-CoV2 expansion. We find that proliferation of various coronaviruses can be strongly inhibited by PDM-detachers and enhanced by attaching mitochondria to lipid droplets. We demonstrate that PDM can be found in humans. We studied adipose tissue from pheochromocytoma patients, a model of white adipose browning characterized by enhanced capacity for energy expenditure. Remarkably, bioenergetic changes associated with browning were primarily localized to PDM. Similar changes were found in a mouse model of re-browning, where PDM increased in whitened brown adipose tissue upon reducing housing temperature. Copyright © 2022

PATHOPHYSIOLOGY OF LACTIC ACIDOSIS IN PATIENTS WITH ASTHMA

SESSION TITLE: Challenges in Asthma SESSION TYPE: Rapid Fire Case Reports PRESENTED ON: 10/18/2022 10:15 am - 11:10 am INTRODUCTION: Asthma is a chronic illness affecting 334 million people worldwide[1]. Asthma affects the respiratory gas exchange, which plays a significant role in acid-base balance. Acid-base disorders in asthma involve respiratory alkalosis, respiratory acidosis, and AG acidosis[2]. CASE PRESENTATION: A 37 years old Hispanic male with a PMH of intermittent asthma presents with progressive dyspnea for three days, worse with activity and decreases with rest. He reported no [cough, fever, rhinorrhea, chest pain]. No orthopnea. He is vaccinated for COVID ( 2 Pfizer doses), has no sickness exposure, and works as a driver. The patient is not a smoker. Physical Exam: Blood pressure 124/72 mmHg. Heart Rate 100 PPM. Temperature 97.1 F.Respiratory Rate 21BPM.SPO2 90% General appearance: acute distress with nasal flaring. Heart: Normal S1, S2. RRR. Lung: Poor air entry with diffuse wheeze bilaterally. He was placed on a 6 LPM NC. CBC and differential were unremarkable. He was started on methylprednisone, Ceftriaxone, and azithromycin. The patient was started on inhaled Salbutamol and Budesonide. Chest X-ray was unremarkable, Chemistry was unremarkable except for elevated Lactic acid 4.7, There was no concern for reduced tissue perfusion or hypoxia, with no evidence of an infectious process because both viral and bacterial causes for pneumonia were excluded, and antibiotics were stopped. A serial lactic acid level trend was 4.5/4.3/ 4.1/ 4 on the first day, while on the next day, it was 3.1/ 2.9/ 2.7/ 2.5/ 3.5, we stopped trending his lactic acid level. He improved and was discharged on an oral taper steroid and inhaled steroids with a B2 agonist. DISCUSSION: There are two types of Lactic acidosis in patients with asthma: 1- Type-A results from impaired oxygen delivery to tissues and reduced tissue perfusion in severe acute asthma may be accompanied by reduced cardiac output. 2- Type B where oxygen delivery is normal, but the cellular function is impaired due to increased norepinephrine in plasma, increasing metabolic rate and lactate production, drugs like beta-agonists increase glycogenolysis leading to an increased pyruvate concentration;pyruvate is converted to lactic acid. B2 agonist increases lipolysis and increases Acetyl CoA, this increase in Acetyl CoA inhibits the conversion of pyruvate to Acetyl CoA, increasing pyruvate which will be converted to lactic acid[2], Theophylline is a non-selective 5'-phosphodiesterase inhibitor and potentiates the activity of ß-adrenergic agents by increasing the intracellular concentration of cAMP, Glucocorticoids are also known to increase the ß-receptor's sensitivity to ß-adrenergic agonists. CONCLUSIONS: Providers are increasingly challenged by hyperlactatemia,it is not harmful but elevated Lactic acid levels and clearance rate is used for prognostication,hyperlactatemia might be misleading,and all possible causes of elevated lactic acid levels must be explored. Reference #1: 10.5334/aogh.2412 Reference #2: https://doi.org/10.3390/jcm8040563 Reference #3: Edwin B. Liem, Stephen C. Mnookin, Michael E. Mahla;Albuterol-induced Lactic Acidosis. Anesthesiology 2003;99:505–506 doi: https://doi.org/10.1097/00000542-200308000-00036 DISCLOSURES: No relevant relationships by Vasudev Malik Daliparty No relevant relationships by Abdallah Khashan No relevant relationships by Samer Talib No relevant relationships by MATTHEW YOTSUYA

Triglyceride levels in COVID-19 patients during admission in intensive care correlate with disease severity and mortality

Introduction: It has been established that plasma triglyceride levels are raised during infection and inflammation, due to increased adipose tissue lipolysis, fatty acid synthesis and suppressed fatty acid oxidation.1 Increased triglycerides have also been linked to the 'cytokine storm' underlying the pathophysiology of coronavirus disease 2019 (COVID-19).2 Severe outcomes in COVID-19 patients have been found to be associated with higher triglyceride levels before the infection.3 Another study found triglycerides to be significantly higher after recovery than during the acute phase of infection.4 There is limited data on the trajectory of triglyceride levels in COVID-19 patients during admission in intensive care. Investigating whether triglyceride levels are a useful predictor of disease severity and mortality would enable earlier detection of high-risk patients. While triglyceride levels in COVID-19 patients with mild or severe infections were found to be elevated, this was not the case in those with critical illness which included respiratory or multiple organ failure and septic shock.5 The differences in lipid metabolism between COVID-19 patients and patients with critical illness are yet to be elucidated. Objectives: In this study, triglyceride levels of COVID-19 patients during admission in the general intensive care (GICU) were examined in relation to disease severity and mortality. Triglyceride levels at the beginning of hospitalisation were compared between GICU patients with COVID-19, acute respiratory distress syndrome (ARDS) and critical illness, and healthy controls. Methods: Data was obtained from medical records of 93 patients with COVID-19 admitted to GICU at University Hospital Southampton, between March 2020 and May 2020. Triglyceride levels were recorded for each day of hospitalisation. Data was also obtained from medical records of 8 critically ill patients, 10 patients with ARDS and 10 healthy volunteers. Results: The trajectory of triglyceride levels in this cohort of COVID-19 patients started low, then increased over the course of admission (Figure 1). Increased average triglyceride levels correlated with increased risk for severe disease outcome, as indicated by the Sequential Organ Failure Assessment (SOFA) score calculated at the beginning of GICU admission (p<0.05) (Figure 2). Increased triglyceride levels in the first week of GICU admission also correlated with mortality (p<0.05). When compared with healthy controls and patients admitted to GICU with ARDS, patients with COVID-19 and critical illness had raised triglycerides (Figure 3). Conclusions: Increased triglyceride levels in COVID-19 patients over the duration of GICU admission are associated with worse disease outcomes and increased mortality. This provides further evidence for the role of dyslipidaemia in the progression of COVID-19.

SPECIALIZED PRO-RESOLVING MEDIATORS (SPMS) and INFLAMMATORY NETWORKS in PATIENTS AFFECTED by ADULT ONSET STILL'S DISEASE (AOSD) and COVID-19

Background: Uncontrolled systemic infammation characterizes COVID-19 and autoinfammatory diseases such as adult-onset Still's disease (AOSD). Biosynthesis of pro-resolving mediators (SPMs), i.e. lipoxins (LX), resolvins (Rv), pro-tectins (PD), and maresins (MaR), ensures infammation shutdown and tissue repair, limiting neutrophils recruitment and stimulating macrophages to remove apoptotic cells. Among protectins, reduction of PD1 was found in the lungs of mice infected with the H5N1 infuenza virus and experimental treatment with PD1 resulted in increased animals' survival (Morita M et al 2013). Objectives: We investigated the effects of SPMs in pathogenesis and clinical evolution of AOSD and compared these data with mild and severe COVID-19. Finally, we analyzed the potential role of PD1 in modulating the infammatory response of macrophages obtained from AOSD patients, COVID-19 patients and healthy donors (HDs). Methods: 21 patients hospitalized for COVID-19 (10 ICU and 11 hospitalized in medical clinical unit) and 13 patients with AOSD were enrolled. Plasma PD1 levels from patients and controls were analyzed by ELISA, and mono-cytes-derived macrophages were polarized into M1 and M2 phenotype. We analyzed the effect of PD-1 on macrophages differentiation. At 10 days, macrophages were analyzed for surface expression of subtypes markers by flow cytometry. Cytokines production was measured in supernatants by Bio-Plex Assays. Peripheral blood mononuclear cells (PBMCS) from 3 AOSD patients, 2 COVID-19 patients and 3 HDs were obtained. Next-generation deep sequencing was then performed to identify the differences in PBMCs transcripts profiles. Results: AOSD patients with systemic scored (SS) ≥1 showed an increase of PD1 levels compared to AOSD patients with lower systemic score (p=0.04) (Figure 1A). Similarly, plasma levels of PD1 were increased in COVID-19 patients independently from their clinical subsets, compared to HDs (p=0.02). In vitro treatment with PD1 of monocytes-derived macrophages from AOSD and COVID-19 patients induced a signifcant increase of M2 polarization vs control (p<0.05) (Figure 1B). Furthermore, a signifcant release of IL-10 and CCL4 from M2 macrophages was observed when compared to control (p<0.05) (Figure 1C). In the transcriptomes from 3 AOSD patients (2 mild and 1 severe), 2 COVID-19 patients (1 mild and 1 ICU) and 2 HDs, we observed that genes involved in infammation, lipid catabolism and monocytes activation were spe-cifcally dysregulated in AOSD and COVID-19 patients when compared to HDs. Among them pla2g15, pla2g12a, pla2g2d, involved in mobilization of SPMs precursors, were signifcant upregulated in patients compared to HDs (p<.01, ;log2FoldChange;>1.2) (Figure 1D). The largest part of the genes involved in infammation, lipid catabolism, and monocytes activation are less expressed in AOSD patients when compared to COVID19 patients, as reported in Table 1. Conclusion: The counterbalance by SPMs during infammation is still a largely unexplored pathway. Our study suggests that an imbalance of SPMs in autoin-fammatory diseases as well as COVID-19. The modulation of SPMs as observed in our experiments, might represent a new possible therapeutic strategy during AOSD and COVID-19.

Anti-adipogenic and Pro-lipolytic Effects on 3T3-L1 Preadipocytes by CX-4945, an Inhibitor of Casein Kinase 2.

Casein kinase 2 (CK2) is a ubiquitously expressed serine/threonine kinase and is upregulated in human obesity. CX-4945 (Silmitasertib) is a CK2 inhibitor with anti-cancerous and anti-adipogenic activities. However, the anti-adipogenic and pro-lipolytic effects and the mode of action of CX-4945 in (pre)adipocytes remain elusive. Here, we explored the effects of CX-4945 on adipogenesis and lipolysis in differentiating and differentiated 3T3-L1 cells, a murine preadipocyte cell line. CX-4945 at 15 µM strongly reduced lipid droplet (LD) accumulation and triglyceride (TG) content in differentiating 3T3-L1 cells, indicating the drug's anti-adipogenic effect. Mechanistically, CX-4945 reduced the expression levels of CCAAT/enhancer-binding protein-α (C/EBP-α), peroxisome proliferator-activated receptor-γ (PPAR-γ), fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC), and perilipin A in differentiating 3T3-L1 cells. Strikingly, CX-4945 further increased the phosphorylation levels of cAMP-activated protein kinase (AMPK) and liver kinase B-1 (LKB-1) while decreasing the intracellular ATP content in differentiating 3T3-L1 cells. In differentiated 3T3-L1 cells, CX-4945 had abilities to stimulate glycerol release and elevate the phosphorylation levels of hormone-sensitive lipase (HSL), pointing to the drug's pro-lipolytic effect. In addition, CX-4945 induced the activation of extracellular signal-regulated kinase-1/2 (ERK-1/2), and PD98059, an inhibitor of ERK-1/2, attenuated the CX4945-induced glycerol release and HSL phosphorylation in differentiated 3T3-L1 cells, indicating the drug's ERK-1/2-dependent lipolysis. In summary, this investigation shows that CX-4945 has strong anti-adipogenic and pro-lipolytic effects on differentiating and differentiated 3T3-L1 cells, mediated by control of the expression and phosphorylation levels of CK2, C/EBP-α, PPAR-γ, FAS, ACC, perilipin A, AMPK, LKB-1, ERK-1/2, and HSL.

POS-092 SGLT2 INHIBITOR – INDUCED EUGLYCEMIC DIABETIC KETOACIDOSIS IN GUILLAIN-BARRE SYNDROME: A CASE REPORT

Introduction: Introduction: Euglycemic diabetic ketoacidosis (EuDKA) is a rare but increasingly reported serious adverse effect of SGLT2 inhibitors. There is not much published literature on the incidence of EuDKA and the factors associated with it. Though SGLT2 inhibitors were introduced as glucose lowering agents, recent trials have demonstrated their favourable cardiovascular outcome in heart failure and ability to retard progression of proteinuric kidney disease, including in non-diabetics. Hence use of this class of drugs is anticipated to increase exponentially, given the combined high global burden of diabetes, coronary artery disease and chronic kidney disease. As we embark to use the SGLT2 inhibitors in different clinical scenarios, it becomes imperative to report their adverse effects encountered in uncommon clinical conditions as well. Methods: Case history: A 42-year-old gentleman with history of type 2 diabetes mellitus for 15 years and coronary artery disease, presented with difficulty in climbing stairs and walking for 5 days with progressive difficulty in getting up from bed. He did not have past history of covid infection and had been immunised with 1 dose of covid vaccine. On examination, patient had normal hemodynamics. There was flaccid quadriparesis with areflexia and truncal muscle weakness. Nerve conduction study confirmed acute demyelinating polyradiculoneuropathy. His baseline laboratory investigations revealed normal renal parameters but metabolic acidosis was noted at the time of admission. Patient was started on iv immunoglobulin 2mg/kg and the motor weakness improved from grade 2/5 to 4/5. However, the high anion gap metabolic acidosis worsened over the next 4 days and patient developed acidotic breathing. His sugars were within normal limits and the patient was on metformin, glimeperide, vildagliptin, voglibose and dapagliflozin. As blood lactate levels were normal with urine acetone positivity, euglycemic diabetic ketoacidosis secondary to SGLT2 inhibitor was suspected and all the oral hypoglycemic agents were stopped. He was started on hydration and insulin infusion. After 48 hours of stopping dapagliflozin, acidosis resolved completely and the patient was reintroduced back on the other 4 class drugs. At follow-up, there was no recurrence of acidosis and patient was able to walk with support and physiotherapy. [Formula presented] Results: Discussion: SGLT2 inhibitors cause glycosuria and directly induce glucagon release from pancreas. Combined with insulin deficiency, this results in lipolysis, fatty acid oxidation and ketogenesis. They also cause increased renal reabsorption of ketone bodies. The precipitating factors for EuDKA identified so far include abrupt reduction in insulin dosage, reduced oral intake, infections, surgery, excess alcohol use, volume depletion, type 1 diabetes and heavy physical exercise. This is the first reported case of SGLT2 inhibitor-induced EuDKA in a patient with Guillain-Barre syndrome. As symptoms of dehydration may not be significant due to lack of hyperglycemia in EuDKA, there may be a delay in the diagnosis of this complication. Conclusions: Conclusion: The possibility of EuDKA to be kept in mind while evaluating metabolic acidosis in a diabetic patient on SGLT2 inhibitors. Temporarily withholding the SGLT2 inhibitors during an intercurrent illness will prevent the occurrence of the above serious adverse effect. No conflict of interest