Expression Of CPT1A Research Articles

Dapagliflozin alleviates renal fibrosis in a mouse model of adenine-induced renal injury by inhibiting TGF-β1/MAPK mediated mitochondrial damage.

Renal fibrosis is a common pathological outcome of various chronic kidney diseases, and as yet, there is no specific treatment. Dapagliflozin has shown renal protection in some clinical trials as a glucose-lowering drug, but its role and mechanism on renal fibrosis remain unclear. In this study, we used a 0.2% adenine diet-induced renal fibrosis mouse model to investigate whether dapagliflozin could protect renal function and alleviate renal fibrosis in this animal model. In vivo, we found that dapagliflozin's protective effect on renal fibrosis was associated with 1) sustaining mitochondrial integrity and respiratory chain complex expression, maintained the amount of mitochondria; 2) improving fatty acid oxidation level with increased expression of CPT1-α, PPAR-α, ACOX1, and ACOX2; 3) reducing inflammation and oxidative stress, likely via regulation of IL-1β, IL-6, TNF-α, MCP-1, cxcl-1 expression, and glutathione (GSH) activity, superoxide dismutase (SOD) and malondialdehyde (MDA) levels; and 4) inhibiting the activation of the TGF-β1/MAPK pathway. In HK2 cells treated with TGF-β1, dapagliflozin reduced the expression of FN and α-SMA, improved mitochondrial respiratory chain complex expression, and inhibited activation of the TGF-β1/MAPK pathway.

Self-assembling polymer-based short chain fatty acid prodrugs ameliorate non-alcoholic steatohepatitis and liver fibrosis

With the preponderance of a high-calorie diet and sedentary lifestyle, the prevalence of non-alcoholic steatohepatitis (NASH), a state of abnormally elevated lipid accumulation in the liver with chronic inflammation, is increasing at an alarming rate worldwide. Hence, cost-effective therapeutic interventions are required to manage this disease at an early stage. Numerous reports have suggested a link between gut microbial dysbiosis, particularly a decrease in the abundance of short-chain fatty acids (SCFA)-producing microbiota and NASH pathogenesis. Considering these low molecular weight (LMW) SCFAs such as acetic, propionic, and butyric acids have been used to inhibit hepatic steatosis in mouse models. However, the poor pharmacokinetic (PK) profile of SCFAs, caused due to their LMW, renders them therapeutically ineffective. Thus, to improve the PK characteristic-based therapeutic efficacy of LMW SCFAs, we designed SCFA-based prodrugs that possess self-assembling characteristics in aqueous media. The designed SCFA prodrugs consist of enzyme-metabolizable amphiphilic block copolymers, [poly(ethylene glycol)-b-poly(vinyl ester)s] conjugated to propionic acid (PA) or butyric acid (BA) by an ester linkage, which self-assemble into stable nanosized micelles several tens of nanometers in diameter (NanoPA and NanoBA). Via pharmacological analysis, we confirmed that, after oral administration, LMW BA decreased to a physiological level within 24 h in the liver, whereas BA liberated from NanoBA was observed until 72 h post-administration, implying a sustained release profile. Here, we evaluated the therapeutic efficacy of NanoSCFA in a choline-deficient, L-amino acid-defined high-fat diet (CDAHFD)-induced NASH and liver fibrosis mouse model by ad libitum drinking. NanoSCFA, particularly NanoBA, exhibited the remarkable potential to ameliorate the phenotypic features of fatty liver disease by reducing hepatic lipogenesis and fibrosis, with negligible adverse effects. In contrast, conventional LMW SCFAs failed to prevent the pathogenesis of fatty liver disease, which plausibly can be explained by their rapid clearance and discernible adverse effects. Mechanistic studies revealed that NanoBA restored the nuclear expression of PPARα, a transcriptional factor regulating mitochondrial fatty acid oxidation, in the periportal hepatocytes and decreased the CPT1A expression level in the hepatic tissues, reflecting the therapeutic effects of NanoBA. Taken together, we confirmed that our NanoSCFA potentially improved the PK properties of SCFAs, and it consequently alleviated NASH symptoms and fibrotic liver compared to LMW SCFAs. Our study establishes NanoSCFA as a suitable nano-assembled prodrug for NASH treatment.

Elevated fatty acid β-oxidation by leptin contributes to the proinflammatory characteristics of fibroblast-like synoviocytes from RA patients via LKB1-AMPK pathway

Fibroblast-like synoviocytes (FLS) maintain chronic inflammation leading to joint destruction in rheumatoid arthritis (RA). Fatty acid β-oxidation (FAO) regulates cell function. Here, we aimed to investigate the effect of FAO enhanced by leptin on the characteristics of RA-FLS and elucidate the potential metabolic mechanism. Key enzymes involved in lipid metabolism were detected with qPCR in HSF, MH7A cell line and isolated RA-FLS treated with RA or healthy control (HC) serum. In some experiments, FAO inhibitor, etomoxir (ETO) or anti-leptin antibody were added into serum-treated RA-FLS. In other experiments, RA-FLS were stimulated with leptin together with ETO or AMP-activated protein kinase (AMPK) inhibitor compound C (CC) or silencing liver kinase B1 (LKB1). Cell proliferation, proinflammatory factor production, pro-angiogenesis, chemoattractive potential, FAO-related key enzymes, AMPK and LKB1 in FLS were analyzed. FAO-related key enzymes were evaluated in serum-treated RA-FLS with or without anti-leptin antibody. Related functions of leptin-stimulated RA-FLS were examined in the presence or absence of ETO. AMP-activated protein kinase (AMPK) and liver kinase B1 (LKB1) in leptin-stimulated RA-FLS were tested with western blot. Activation of AMPK in leptin-stimulated RA-FLS was detected after silencing LKB1. We found that MH7A cell line and RA serum-treated FLS exhibited upregulated FAO, and ETO could inhibit the proinflammatory phenotypes of RA-FLS. The addition of anti-leptin antibody suppressed the elevation of FAO mediated by RA serum. More importantly, leptin promoted the proinflammatory characteristics of RA-FLS, which was reversed by ETO. Leptin activated AMPK by upregulating LKB1. CC impaired leptin-induced CPT-1A expression in RA-FLS. Our study uncovers that elevated FAO mediated by leptin drives abnormal function of RA-FLS and suggests leptin or FAO inhibition may serve as a promising therapeutic strategy for RA.

The impact of variance in carnitine palmitoyltransferase-1 expression on breast cancer prognosis is stratified by clinical and anthropometric factors.

CPT1A is a rate-limiting enzyme in fatty acid oxidation and is upregulated in high-risk breast cancer. Obesity and menopausal status' relationship with breast cancer prognosis is well established, but its connection with fatty acid metabolism is not. We utilized RNA sequencing data in the Xena Functional Genomics Explorer, to explore CPT1A's effect on breast cancer patients' survival probability. Using [18F]-fluorothymidine positron emission tomography-computed tomography images from The Cancer Imaging Archive, we segmented these analyses by obesity and menopausal status. In 1214 patients, higher CPT1A expression is associated with lower breast cancer survivability. We confirmed a previously observed protective relationship between obesity and breast cancer in pre-menopausal patients and supported this data using two-sided Pearson correlations. Taken together, these analyses using open-access databases bolster the potential role of CPT1A-dependent fatty acid metabolism as a pathogenic factor in breast cancer.

Alleviation effect of conjugated linoleic acid on estradiol benzoate induced fatty liver hemorrhage syndrome in Hy-line male chickens.

The purpose of this study was to explore whether conjugated linoleic acid (CLA) could alleviate fatty liver hemorrhagic syndrome (FLHS) induced by estradiol benzoate intramuscular injection in laying hens. One hundred male Hy-Line white chickens were randomly divided into two groups, namely, the control (CON) and estradiol benzoate (E) groups, and both groups were fed the same basal diet. After injections of estradiol benzoate at 2 mg/kg every two days for a total of 7 times, chickens in the E group showed FLHS symptoms, including liver enlargement, hemorrhage, and steatosis. Then half of the chickens in the E group received an additional diet containing 5000 mg/kg CLA for 8 weeks. The results of morphological observations, hematoxylin and eosin staining, and Oil Red O staining showed that CLA alleviated liver enlargement, hemorrhage, and lipid accumulation in FLHS chickens. In addition, we measured liver function and lipid metabolism indicators, including ALT, AST, TG, TCH, HDL-C, and LDL-C, which further suggested that CLA mitigated the disturbance of serum and liver metabolism in FLHS chickens. Mechanistically, CLA inhibited hepatic de novo lipogenesis, cholesterol synthesis, and TG accumulation and increased TG hydrolysis in FLHS chickens by regulating the gene expression of CD36, ACC, FAS, SCD 1, DGAT2, LIPE, ATGL, CPT1A, SREBP-1c, SREBP-2, PPARγ, and PPARα. Furthermore, CLA ameliorated hepatic oxidative stress and inhibited NF-κB signaling pathway-mediated inflammation in FLHS chickens. In conclusion, CLA regulated lipid metabolism, thus further alleviating oxidative stress and inflammation to alleviate FLHS induced by estrogen in chickens.

Alfalfa Xeno-miR168b Target CPT1A to Regulate Milk Fat Synthesis in Bovine Mammary Epithelial Cells.

It was shown that microRNAs (miRNAs) play an important role in the synthesis of milk fat; thus, this manuscript evaluated whether exogenous miRNA (xeno-miRNAs) from alfalfa could influence the milk fat content in dairy cows. At first, mtr-miR168b was screened from dairy cow milk and blood. Then, EdU staining, flow cytometry, Oil Red O staining, qRT-PCR, and WB were applied to explore the effect of xeno-miR168b on the proliferation, apoptosis, and lipid metabolism of bovine mammary epithelial cells (BMECs). Finally, in order to clarify the pathway that regulated the lipid metabolism of BMECs using xeno-miR168b, a double-luciferase reporter assay was used to verify the target gene related to milk fat. These results showed that overexpression of xeno-miR168b inhibited cell proliferation but promoted apoptosis, which also decreased the expression of several lipid metabolism genes, including PPARγ, SCD1, C/EBPβ, and SREBP1, significantly inhibited lipid droplet formation, and reduced triglyceride content in BMECs. Furthermore, the targeting relationship between CPT1A and xeno-miR168b was determined and it was confirmed that CPT1A silencing reduced the expression of lipid metabolism genes and inhibited fat accumulation in BMECs. These findings identified xeno-miR168b from alfalfa as a cross-kingdom regulatory element that could influence milk fat content in dairy cows by modulating CPT1A expression.

Oolong tea of different years protects high-fat diet-fed mice against obesity by regulating lipid metabolism and modulating the gut microbiota.

Long-term stored oolong tea has recently attracted considerable attention concerning its salutary effect. In this study, the anti-obesity effect of different years' oolong tea on high-fat diet-fed mice was compared. Wuyi rock tea of 2001, 2011, and 2020 were chosen to be the representative samples of oolong tea. The results showed that eight-week administration of 2001 Wuyi rock tea (WRT01), 2011 Wuyi rock tea (WRT11), and 2020 Wuyi rock tea (WRT20) extracts (400 mg per kg per d) significantly decreased the body weight and attenuated the obesity in high-fat diet-fed mice. 2001 and 2011 Wuyi rock teas reduced obesity mainly through regulating lipid metabolism and activating the AMPK/SREBP-1 pathway, downregulating the expression of SREBP-1, FAS, and ACC and upregulating CPT-1a expression; while the 2011 and 2020 Wuyi rock teas by moderating the gut microbiota dysbiosis, reshaping the gut microbiota, and promoting the growth of beneficial bacteria, especially Akkermansia. 2011 Wuyi rock tea was proven to be more effective in reducing body weight gain and liver oxidative stress than the others. Collectively, all three Wuyi rock teas of different years alleviated high-fat diet-induced obesity by regulating lipid metabolism and modulating gut microbiota, whereas the emphasis of their internal mechanism is different with different storage ages.

ATL I, Acts as a SIRT6 Activator to Alleviate Hepatic Steatosis in Mice via Suppression of NLRP3 Inflammasome Formation.

Accumulating evidence has highlighted that sirtuin-6 (SIRT6) plays an important role in hepatic gluconeogenesis and lipogenesis. We aim to investigate the underlying mechanisms and pharmacological interventions of SIRT6 on hepatic steatosis treatment. Herein, our results showed that atractylenolide I (ATL I) activated the deacetylase activity of SIRT6 to promote peroxisome proliferator-activated receptor alpha (PPARα) transcription and translation, while suppressing nuclear factor NF-kappa-B (NFκB)-induced NACHT, LRR, and PYD domains containing protein 3 (NLRP3) inflammasome formation. Together, these decreased the infiltration of F4/80 and CD11B positive macrophages, accompanied by decreased mRNA expression and serum levels of tumor necrosis factor alpha (TNF-α), interleukin-6 (IL6), and interleukin-1 beta (IL1β). Additionally, these changes decreased sterol regulatory element-binding protein-1c (SREBP-1c) expression, while restoring carnitine O-palmitoyltransferase 1a (Cpt1a) expression, to decrease the size of adipocytes and adipose deposition, which, in turn, reversed high-fat diet (HFD)-induced liver weight and body weight accumulation in C57 mice. SIRT6 knockout or hepatic SIRT6 knockout in C57 mice largely abolished the effect of ATL I on ameliorating hepatic steatosis. Taken together, our results suggest that ATL I acts as a promising compound that activates SIRT6/PPARα signaling and attenuates the NLRP3 inflammasome to ameliorate hepatic inflammation and steatosis.

Cpt1a alleviates cigarette smoke‑induced chronic obstructive pulmonary disease.

The current study aimed to determine the expression of carnitine palmitoyltransferase 1A (Cpt1a) in the lung tissue of chronic obstructive pulmonary disease (COPD) patients and its correlation with lung function. An increase in Cpt1a expression improved lung function in patients with COPD by inhibiting apoptosis and the inflammatory response of lung endothelial cells. Lung tissues of 20 patients with COPD and 10 control patients were collected, their Cpt1a expression was determined by western blotting and apoptosis and inflammation were assessed by haematoxylin-eosin staining, TUNEL assay and ELISA. Mice with knockout or overexpression of Cpt1a were constructed by lentivirus in vivo. A COPD model was induced by cigarette smoke and the role of Cpt1a in COPD was determined in vivo and in vitro. Cpt1a expression was positively correlated with lung function and negatively correlated with apoptosis and inflammation. Patients with COPD with higher expression of Cpt1a in lung tissues had improved lung function indices and lung tissue morphology with less apoptosis and decreased inflammatory response. Compared with the control group, COPD mice with Cpt1a knockdown had aggravated lung dysfunction and increased lung inflammation and apoptosis. Overexpression of Cpt1a alleviated lung dysfunction and reduced inflammatory response and apoptosis of lung tissues in COPD mice. Pulmonary microvascular endothelial cells of mice were isolated in vitro and the results were consistent with the findings obtained in vivo. In conclusion, the clinical, in vivo and in vitro data confirmed for the first time that Cpt1a alleviated lung dysfunction of patients with COPD by inhibiting apoptosis of endothelial cells and inflammatory responses.

ACC1 Inhibition Enhances Treg Gvhd Treatment Efficacy through Reciprocal Fatty-Acid Synthesis and Oxidative Phosphorylation Regulation

Regulatory T-cells (Treg) play a critical role in preventing autoimmune and alloimmune reactions. Clinical trials demonstrated that Treg adoptive transfer significantly reduced but did not eliminate grades II-IV graft-vs-host disease (GVHD). We hypothesized that increasing fatty acid oxidation (FAO) would augment Treg potency, and ultimately enhance Treg-mediated GVHD reduction. Acetyl-CoA carboxylase-1 (ACC1) negatively regulates FAO by augmenting fatty acid synthesis (FAS), which results in downstream inhibition of CPT1a, the rate-limiting enzyme in FAO. ACC1 genetic deletion or inhibition increases T-cell FAO and skews naive CD4 T-cell differentiation from Th17 to Treg. However, the role of ACC1 in thymic Treg function and metabolic fitness is unknown. Here we show that a 2-hour treatment of murine Treg with ND630, a pharmacological inhibitor of ACC1 currently in clinical trials for hepatic steatosis, enhances Treg suppressive function. As compared to control Tregs, ND630 treatment also increased expression of suppressive molecules Lag3, TIGIT, and IL-10. Treatment of Tregs with either of two other ACC1 inhibitors (TOFA, sorafin A) resulted in similar augmentations in Treg function, disfavoring the likelihood of off-target effects. Additionally, ND630 treatment increased Treg oxygen consumption rate (OCR), a surrogate for mitochondrial oxidative phosphorylation (OXPHOS), a critical source of ATP for Treg. Similar to murine Treg, ND630 treatment of in vitro expanded human Treg enhanced their suppressive function and OCR/OXPHOS. IACS, an OXPHOS inhibitor, abrogated the positive ND630 effect on in vitro Treg suppressive function and OXPHOS. Given the augmentation in OXPHOS with ND630 treatment, we further evaluated ND630 effects on Treg mitochondria. Compared to controls, ND630 treatment increased mitochondrial mass and membrane potential, essential for energy storage during OXPHOS, as well as mitochondrial polarization, which is required for full Treg functionality. Microscopy analysis revealed enhanced mitochondrial fusion and elongation after ND630 or TOFA treatment (FigA). This mitochondrial structural change boosts OXPHOS, consistent with a direct effect of ACC1 inhibition on mitochondrial fitness. Since ACC1-driven FAS ultimately inhibits FAO, we next evaluated contributors to FAO. Expression of CPT1a, a critical enzyme for mitochondrial FAO, fatty acid binding protein 5, a key shuttle of fatty acids (FA), and exogenous FA uptake were all enhanced after ND630 treatment. Using an unbiased metabolomic screen, a metabolic signature was observed that was consistent with enhanced FAO. Together, these data suggest that ND630 increases mitochondrial fitness, and augments both FA uptake and FAO. To identify additional ND603-induced intracellular effects in Treg, activated murine Treg were subjected to RNAseq. Differential gene expression analysis indicated that 82 genes differed between DMSO and ND630 treated Treg. These included increased expression of genes associated with OXPHOS, as well as mTOR, consistent with high OXPHOS and suppressor cell function. Flow cytometry analysis of phosphorylated proteins showed reduced pFOXO3a and pAKT, both mTORC2 targets, but increased mTORC1 downstream phospho-proteins. An imbalance of mTORC1/2 is consistent to our previous studies reflective of higher Treg function and metabolic fitness (McDonald-Hyman C, JCI 2018). To exclude potential bystander effects of ND630 on Treg function, we utilized murine Treg with genetic deletion of ACC1 (ACC1KO). Supporting our studies with chemical ACC1 inhibitors, ACC1KO Treg were functionally and metabolically superior to wild-type Treg. Furthermore, as our goal is to use ACC1 inhibited or deleted Treg in treatment of GVHD, we tested ACC1KO Treg in treating established bronchiolitis obliterans (BOS) in a murine model of chronic GVHD. ACC1KO Treg were more potent than wild-type Treg in reversing BOS pathology (FigB), a finding consistent in each of 3 replicate transplants. In summary, ACC1 inhibition or deletion amplifies murine (and human) Treg potency resulting in superior metabolic fitness, and greater efficacy in treating established BOS in murine chronic GVHD. Since ND630 is already under investigation in patients, ACC1 inhibition with ND630 could be readily translatable for clinical trials with a goal of utilizing ACC1 inhibited Treg infusion to improve GVHD therapy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

CPT1A promotes anoikis resistance in esophageal squamous cell carcinoma via redox homeostasis

Anoikis resistance was a prominent hallmark of cancer metastasis, and lipo-genic characteristics have been identified as another metabolic alteration during tumorigenesis. However, their crosstalk has not been fully elucidated, especially in advanced esophageal squamous cell carcinoma (ESCC). In this study, we showed, for the first time, that the key enzyme carnitine O-palmitoyl transferase 1 (CPT1A), which is involved in fatty acid oxidation (FAO), was markedly upregulated in ESCC cells upon detached culture via a metabolism PCR array. Overexpression of CPT1A was associated with poor survival of ESCC patients and could protect ESCC cells from apoptosis via maintaining redox homeostasis through supply of GSH and NADPH. Mechanistically, detached culture conditions enhanced the expression of the transcription factor ETV4 and suppressed the expression of the ubiquitin enzyme RNF2, which were responsible for the elevated expression of CPT1A at the mRNA and protein levels, respectively. Moreover, genetic or pharmacologic disruption of CPT1A switched off the NADPH supply and therefore prevented the anchorage-independent growth of ESCC cells in vitro and lung metastases of xenografted tumor models in vivo. Collectively, our results provide novel insights into how ESCC cancer cells exploit metabolic switching to form distant metastases and some evidence for the link between anoikis and FAO.

Shatianyu (Citrus grandis L. Osbeck) Flavonoids and Dietary Fiber in Combination Are More Effective Than Individually in Alleviating High-Fat-Diet-Induced Hyperlipidemia in Mice by Altering Gut Microbiota

This study was aimed at exploring the separate and combined anti-hyperlipidemic effect of Shatianyu (Citrus grandis L. Osbeck) flavonoids (SPFEs) and DF (SPDF) on HFD-fed mice after 14-week administration in diet, together with the possible microbiota-mediated mechanisms. SPFEs and SPDF were more effective together than separately in improving serum lipid profiles, decreasing hepatic lipid accumulation, and upregulating the expression of hepatic CPT1a, CYP7A1, ABCG5, and ABCG8. Butyrate has been previously proved to have an anti-hyperlipidemic effect. The fecal butyrate contents were negatively correlative with serum/liver lipid but positively correlated with fecal total bile acids levels, and SPDF + SPFEs had the most fecal butyrate in this study. SPDF or SPFEs enriched microbiota related to acetic and propionic acids production, while SPDF + SPFEs also bloomed norank_f_Muribaculaceae, Dubosiella, Lachnoclostridium, and norank_f_Eubacterium_coprostanoligenes_group, which were positively correlated to fecal butyrate contents. Thus, SPFEs and SPDF might alleviate hyperlipidemia synergistically by regulating microbiota to produce butyrate, thereby regulating lipid metabolism.

ODP037 Effects of DHCR24 Inhibitor on Excessive Adrenal Steroidogenesis in db/db mouse

Abstract Background and purpose Patients with type 2 diabetes have an elevated basal level of cortisol, which has been suggested to be involved in the exacerbation of hyperglycemia and the progression of organ complications. Furthermore, it has been reported that adrenal steroidogenesis increases in db/db mice, an animal model of type 2 diabetes. However, the mechanism of the increased adrenal steroidogenesis in type 2 diabetes is unclear. In this study, we aimed to elucidate the mechanism of increased adrenal steroidogenesis in db/db mice and examine whether it is a therapeutic target. Experiment 1: Methods Gene expression in adrenal glands of 10-week-old male db/db mice and db/+ mice was examined by DNA microarray and real-time PCR. Results In the adrenal glands of db/db mice, expression of Acc and Scd1, which are involved in fatty acid synthesis, was decreased by 3.7-fold and 2.6-fold, respectively, compared with db/+ mice. However, expression of Cpt1α, which is involved in acetyl-CoA production by lipolysis, was increased by 1.8-fold and expression of Dhcr24, the final enzyme of the de novo cholesterol synthesis system, was increased by 2.5-fold. Thus, fatty acid degradation and endogenous cholesterol synthesis had increased in the adrenal glands of db/db mice. Experiment 2: Methods 8-week-old male db/db mice were divided into two groups, PBS administration (P) and 10mg/kg DHCR24 inhibitor (U18666A) administration (U) groups. PBS or U18666A was injected intraperitoneally into mice once a week. 4 weeks later, the mice were euthanized, blood corticosterone was measured by LC-MS/MS, and gene expression in adrenal glands was measured by real-time PCR. Results The blood corticosterone level was 196.6±12.7ng/mL in the U group and 245.8±15.4ng/mL in the P group, which was significantly lower in the U group (unpaired t-test, p<0. 05). mRNA expression of Acc and Scd1 was increased by 3.1-fold and 1.3-fold, respectively, in the U group compared with the P group. Conclusion These results suggest that increased endogenous cholesterol synthesis may be a cause of steroid overproduction in the adrenal glands of db/db mice and a DHCR24 inhibitor suppresses the increase in steroid hormone synthesis. This study indicates that changes in adrenal fatty acid metabolism and endogenous cholesterol synthesis might play a major role in understanding the mechanism of increased adrenal steroidogenesis and finding a new therapeutic target to prevent organ complications of type 2 diabetes. Presentation: No date and time listed

Effects of SGLT2 inhibitor dapagliflozin in patients with type 2 diabetes on skeletal muscle cellular metabolism

ObjectiveSGLT2 inhibitors increase urinary glucose excretion and have beneficial effects on cardiovascular and renal outcomes; the underlying mechanism may be metabolic adaptations due to urinary glucose loss. Here, we investigated the cellular and molecular effects of 5 weeks of dapagliflozin treatment on skeletal muscle metabolism in type 2 diabetes patients. MethodsTwenty-six type 2 diabetes mellitus patients were randomized to a 5-week double-blind, cross-over study with 6-8-week wash-out. Skeletal muscle acetylcarnitine levels, intramyocellular lipid (IMCL) content and phosphocreatine (PCr) recovery rate were measured by magnetic resonance spectroscopy (MRS). Ex vivo mitochondrial respiration was measured in skeletal muscle fibers using high resolution respirometry. Intramyocellular lipid droplet and mitochondrial network dynamics were investigated using confocal microscopy. Skeletal muscle levels of acylcarnitines, amino acids and TCA cycle intermediates were measured. Expression of genes involved in fatty acid metabolism were investigated. ResultsMitochondrial function, mitochondrial network integrity and citrate synthase and carnitine acetyltransferase activities in skeletal muscle were unaltered after dapagliflozin treatment. Dapagliflozin treatment increased intramyocellular lipid content (0.060 (0.011, 0.110) %, p = 0.019). Myocellular lipid droplets increased in size (0.03 μm2 (0.01–0.06), p < 0.05) and number (0.003 μm−2 (−0.001–0.007), p = 0.09) upon dapagliflozin treatment. CPT1A, CPT1B and malonyl CoA-decarboxylase mRNA expression was increased by dapagliflozin. Fasting acylcarnitine species and C4–OH carnitine levels (0.4704 (0.1246, 0.8162) pmoles∗mg tissue−1, p < 0.001) in skeletal muscle were higher after dapagliflozin treatment, while acetylcarnitine levels were lower (−40.0774 (−64.4766, −15.6782) pmoles∗mg tissue−1, p < 0.001). Fasting levels of several amino acids, succinate, alpha-ketoglutarate and lactate in skeletal muscle were significantly lower after dapagliflozin treatment. ConclusionDapagliflozin treatment for 5 weeks leads to adaptive changes in skeletal muscle substrate metabolism favoring metabolism of fatty acid and ketone bodies and reduced glycolytic flux.The trial is registered with ClinicalTrials.gov, number NCT03338855.

Triterpenoids from the genus Ilex attenuate free fatty acid-induced lipid accumulation in HepG2 cells by regulating lipid metabolism disorder and the AMPK signalling pathway

Ethnopharmacological relevanceVarious traditional Chinese medicines from the genus Ilex (Aquifoliaceae) have been reported to have excellent hypolipidaemic effects. Although triterpenoids have been found to be the main active components, the underlying mechanisms have not been clarified. Aim of the studyThis study aimed to investigate the lipid-lowering effect, structure-activity relationship and action mechanism of triterpenoids from the genus Ilex. Materials and methodsFFA was used to induce HepG2 cells to establish a classical lipid-lowering activity screening model for the activities of 31 triterpenoids, and the contents of intracellular lipids, TC, and TG were measured. Furthermore, the structure-activity relationship was discussed. Mechanistically, UPLC-Q/TOF-MS-based metabolomics and lipidomics studies were performed, and metabolic pathways were analysed to investigate the lipid-lowering mechanism. Moreover, western blotting was performed to analyse the expression of key proteins of lipid metabolism and predict the targets of action. ResultsThirteen triterpenoids significantly reduced intracellular lipid accumulation and decreased the levels of TG and TC. Among them, rotundic acid (RA) showed stronger lipid-lowering activity than the simvastatin-positive group, and structure-activity relationship analysis indicated that the hydroxyl groups at C-3 and C-19, hydroxymethyl groups at C-23, and carboxyl groups at C-28 may be the key groups for biological activity. Twenty-two metabolites in the metabolomics study and 19 metabolites in the lipidomics study were identified. The identified biomarkers were primarily glycerophosphocholine, LysoPCs, PCs, TAGs, LysoPEs, LysoPIs and sphingolipids, which are involved in glycerophospholipid and sphingolipid metabolism. Moreover, western blotting analysis showed that the expression of SREBP-1 and HMGCR decreased, while AMPK and ACC phosphorylation and the expression of CPT1A and CYP7A1 increased in the RA-treated group. ConclusionThe results suggested that triterpenoids from the genus Ilex showed significant lipid-lowering effects and that RA may be a novel hypolipidaemic drug candidate. Moreover, the underlying mechanism indicated that RA showed a lipid-lowering effect by regulating glycerophospholipid and sphingolipid metabolism and activating the AMPK pathway.

Long non-coding RNA FABP5P3/miR-22 axis improves TGFβ1-induced fatty acid oxidation deregulation and fibrotic changes in proximal tubular epithelial cells of renal fibrosis

ABSTRACT Severe hydronephrosis increases the risk of urinary tract infection and irretrievable renal fibrosis. While TGFβ1-mediated fibrotic changes in proximal tubular epithelial cells and fatty acid oxidation (FAO) deregulation contribute to renal fibrosis and hydronephrosis. Firstly, a few elements were analyzed in this paper, including differentially-expressed long non-coding RNAs (lncRNAs), and miRNAs correlated to CPT1A, RXRA, and NCOA1. This paper investigated TGFβ1 effects on lncRNA FABP5P3, CPT1A, RXRA, and NCOA1 expression and fibrotic changes in HK-2 cells and FABP5P3 overexpression effects on TGFβ1-induced changes. Moreover, this paper predicted and proved that miR-22 binding to lncRNA FABP5P3, 3’UTR of CPT1A, RXRA, and NCOA1 was validated. The dynamic effects of the FABP5P3/miR-22 axis on TGFβ1-induced changes were investigated. A Renal fibrosis model was established in unilateral ureteral obstruction (UUO) mice, and FABP5P3 effects were investigated. Eventually, this paper concluded that TGFβ1 inhibited lncRNA FABP5P3, CPT1A, RXRA, and NCOA1 expression, induced fibrotic changes in HK-2 cells, and induced metabolic reprogramming within HK-2 cells, especially lower FAO. FABP5P3 overexpression partially reversed TGFβ1-induced changes. miR-22 targeted lncRNA FABP5P3, CPT1A, RXRA, and NCOA1. LncRNA FABP5P3 counteracted miR-22 inhibition of CPT1A, NCOA1, and RXRA through competitive binding. TGFβ1 stimulation induced the activation of TGFβ/SMAD and JAG/Notch signaling pathways; Nocth2 knockdown reversed TGFβ1 suppression on lncRNA FABP5P3. FABP5P3 overexpression attenuated renal fibrosis in unilateral ureteral obstruction mice. The LncRNA FABP5P3/miR-22 axis might be a potent target for improving the FAO deregulation and fibrotic changes in proximal TECs under TGFβ1 stimulation.

Sinomenine Ameliorates Colitis-Associated Cancer by Modulating Lipid Metabolism via Enhancing CPT1A Expression.

Colitis-associated cancer (CAC), arising from long-lasting intestinal inflammation, is a common type of colorectal cancer. Sinomenine (SIN), the major active compound of Sinomenium acutum, displays excellent antitumor activity. In modern pharmacological research, SIN has been proved to arrest proliferation of human colon cancer cells in vitro, but its functional role and specific mechanism in CAC were still elusive. This study explored the molecular mechanism of SIN on CAC. The results showed that orally administered SIN could decrease the occurrence and development of CAC. Metabolomics results revealed SIN could reprogram metabolism in CAC mice by reversing 34 endogenous metabolites. Importantly, the most prominent metabolic alteration was lipid metabolism. Mechanistically, SIN improved lipid metabolism by enhancing the expression of CPT1A in CAC mice. Moreover, the inhibitory effect of SIN on the proliferation of human colon cancer cells was blunted via CPT1A inhibitor. The results of this study added further evidence of the molecular mechanisms that allow SIN to exert anti-CAC effect by facilitating lipid metabolism and reaffirmed its potential and distinctive role as a chemopreventive agent in CAC.

The role of CPT1A as a biomarker of breast cancer progression: a bioinformatic approach

Breast cancer is the commonest malignancy of women and with its incidence on the rise, the need to identify new targets for treatment is imperative. There is a growing interest in the role of lipid metabolism in cancer. Carnitine palmitoyl-transferase-1 (CPT-1); the rate limiting step in fatty acid oxidation, has been shown to be overexpressed in a range of tumours. There are three isoforms of CPT-1; A, B and C. It is CPT-1A that has been shown to be the predominant isoform which is overexpressed in breast cancer. We performed a bioinformatic analysis using readily available online platforms to establish the prognostic and predictive effects related to CPT-1A expression. These include the KM plotter, the Human Protein Atlas, the cBioPortal, the G2O, the MethSurvand the ROC plotter. A Network analysis was performed using the Oncomine platform and signalling pathways constituting the cancer hallmarks, including immune regulation as utilised by NanoString. The epigenetic pathways were obtained from the EpiFactor website. Spearman correlations (r) to determine the relationship between CPT-1A and the immune response were obtained using the TISIDB portal. Overexpression of CPT-1A largely confers a worse prognosis and CPT-1A progressively recruits a range of pathways as breast cancer progresses. CPT-1A’s interactions with cancer pathways is far wider than previously realised and includes associations with epigenetic regulation and immune evasion pathways, as well as wild-type moderate to high penetrant genes involved in hereditary breast cancer. Although CPT-1A genomic alterations are detected in 9% of breast carcinomas, both the alteration and the metagene associated with it, confers a poor prognosis. CPT-1A expression can be utilised as a biomarker of disease progression and as a potential therapeutic target.

MiR204 potentially promotes non-alcoholic fatty liver disease by inhibition of cpt1a in mouse hepatocytes

Non-alcoholic fatty liver disease (NAFLD) is associated with hepatic metabolism dysfunction. However, the mechanistic role of miR204 in the development of NAFLD is unknown. We investigate the functional significance of miR204 in the evolution of NAFLD. IDH2 KO mice feed a normal diet (ND) or HFD increased body weight, epididymal fat-pad weight, lipid droplet in liver, blood parameter and inflammation compared to WT mice fed a ND or HFD. Moreover, the expression of miR204 is increased in mice with IDH2 deficiency. Increased miR204 by IDH2 deficiency regulates carnitine palmitoyltransferase 1a (cpt1a) synthesis, which inhibits fatty acid β-oxidation. Inhibition of miR204 prevents the disassembly of two fatty acid-related genes by activating CPT1a expression, which decreases lipid droplet in liver, inflammatory cytokines, epididymal fat pad weight, blood parameters. Increased miR204 by IDH2 deficiency promotes the pathogenesis of HFD-induced NAFLD by regulating hepatic fatty acid metabolism and inflammation.

Abstract P1084: Mature Ipsc-derived Atrial Cardiomyocytes Show That Saturated Fatty Acids Differentially Modulate Atrial Remodeling In Obesity-induced Atrial Fibrillation Compared To Mono And Poly-unsaturated Fatty Acids.

Background: Increased free fatty acids (FFA) and FA infiltration, both major hallmarks of obesity can individually modulate changes leading to AF. For example, mass spectrometry on obese mouse heart tissues have shown that saturated FAs (SFA), monounsaturated FAs (MUFA), and polyunsaturated FAs (PUFA) are all significantly increased compared to controls. The highest increase was shown to be in linoleic acid (LA), a PUFA followed by palmitic acid (PA, SFA) and oleic acid (OA, MUFA). Although reports suggest that some FAs induce ventricular arrhythmias in mice, the differential electrophysiological effects of SFAs, MUFAs, and PUFAs on the atria remain unclear. Objective: To determine the individual roles of SFAs, MUFAs, and PUFAs in mediating atrial remodeling in obesity-induced AF using mature human induced pluripotent stem-derived atrial cardiomyocytes (iPSC-aCMs). Methods: We compared iPSC-aCMs and HL-1 cells treated with bovine serum albumin (BSA) with palmitic acid (PA), oleic acid (OA) and LA cells (600 uM each) for 24 hours and 1 week. Ion channel function were assessed using optical voltage mapping and whole-cell patch clamping, and we assessed atrial remodeling using qPCR, and western blotting. Results: iPSC-aCMs treated with PA, OA, and LA displayed higher expression of FABP3 and CPT1A suggesting increased FA metabolism (Figure A-C). In HL1 cells, protein expression of Cpt1a was increased in all groups after FA treatment (Figure D). PA differentially modulated expression of Kv7.1 and NCX1 in HL1 cells (Figure E-F) and ion channel mRNA expression in iPSC-aCMs (Figure G-L). After 1 week PA and LA treatment b-adrenergic signaling was significantly increased in iPSC-aCMs while OA decreased it compared to controls (Figure M-O). Lastly, 24-hour PA, OA, LA treatment differentially modulated the atrial action potential (AP). While PA and LA significantly shorten the action potential, there was no significant changes to the AP after OA treatment. Conclusions: Using mature iPSC-aCMs, we showed for the first time that SFA, MUFA, and PUFA not only increased FA metabolism but differentially affected b-adrenergic signaling and ion channel remodeling leading to differential effects on the atrial AP. Our findings may have important implications for the management of obesity-induced AF in patients.