Perilipin 5 protects the mitochondrial oxidative functions and improves the alcoholic liver injury in mice.

BACKGROUND AND AIMS: Alcohol consumption is a well-established risk factor for the onset and progression of hepatic steatosis. Perilipin 5 (Plin5), a lipid droplet protein, is an important protective factor against hepatic lipotoxicity induced by excessive lipolysis, but its role and molecular mechanism in alcoholic liver disease (ALD) are not fully elucidated. METHODS: The optimized National Institute on Alcohol Abuse and Alcoholism model was used to construct ALD model mice. Automatic biochemical analyser was used for Biochemical Parameters. The primary hepatocytes and Plin5-overexpressed HepG2 cells (including full-length Plin5 and Plin5 deleting 444-464 aa) were used for in vitro experiment. Haematoxylin and Eosin staining, Oil Red O staining, Bodipy 493/503 staining, Periodic Acid-Schiff staining, immunohistochemistry and JC-1 staining were used to evaluate cell morphology, lipids, glycogen, inflammation and membrane potential. Commercially kits are used to detect glycolipid metabolites, such as triglycerides, glycogen, glucose, reactive oxygen species, lactic acids, ketone bodies. Fluorescently labelled deoxyglucose, NBDG, was used for glucose intake. An XF96 extracellular flux analyser was used to determinate oxygen consumption rate in hepatocytes. The morphological and structural damage of mitochondria was evaluated by electron microscopy. Classical ultracentrifugation is used to separate the subcellular organelles of tissues and cells. Immunoblotting and qPCR were used to detect changes in mRNA and protein levels of related genes. RESULTS: Our results showed that the expression of Plin5 in mouse livers was enhanced by alcohol intake, and Plin5 deficiency aggravated the alcohol-induced liver injury. To clarify the mechanism, we found that Plin5 deficiency significantly elevated the hepatic NADH levels and ketone body production in the alcohol-treated mice. As NADH elevation could promote the reduction of pyruvate into lactate and then inhibit the gluconeogenesis, alcohol-treated Plin5-deficient mice exhibited more lactate production and severer hypoglycemia. These results implied that Plin5 deficiency impaired the mitochondrial oxidative functions in the presence of alcohol. In addition, we demonstrated that Plin5 could be recruited onto mitochondria by alcohol, while Plin5 without mitochondrial targeting sequences lost its mitochondrial protection functions. CONCLUSION: Collectively, this study demonstrated that the mitochondrial Plin5 could protect the alcohol-induced mitochondrial injury, which provides an important new insight on the roles of Plin5 in highly oxidative tissues.

Microglial SLC25A28 Deficiency Ameliorates the Brain Injury After Intracerebral Hemorrhage in Mice by Restricting Aerobic Glycolysis.

The microglia overactivation-induced neuroinflammation is a significant cause of the brain injury after intracerebral hemorrhage (ICH). Iron homeostasis is crucial for microglia activation, but the mechanism and causality still need further study. This study aimed to explore the roles and mechanism of the mitochondrial iron transporter SLC25A28 in microglia activation after ICH. Intrastriatal injection of autologous blood was used to establish ICH model, and the neuroinflammation, iron metabolism and brain injuries were assessed in wildtype or microglia-specific SLC25A28 knockout mice after ICH. Mitochondria iron levels and microglial function were determined in SLC25A28 overexpressed or deleted microglia. The extracellular acidification rate (ECAR), lactate production, and glycolytic enzyme levels were used to determine aerobic glycolysis. The results showed that ICH stimulated mitochondrial iron overload, and synchronously upregulated the SLC25A28 expression. In vitro, SLC25A28 overexpression increased mitochondrial iron levels in microglia. Interestingly, microglial SLC25A28 deficiency ameliorated neuroinflammation, brain edema, blood-brain barrier injury and ethological alterations in mice after ICH. Mechanically, SLC25A28 deficiency inhibited microglial activation by restricting the aerobic glycolysis. Moreover, zinc protoporphyrin could reduce SLC25A28 expression and mitigated brain injury. SLC25A28 plays crucial roles in mitochondrial iron homeostasis and microglia activation after ICH, and it might be a potential therapeutic target for ICH.

Perilipin 5 deficiency aggravates cardiac hypertrophy by stimulating lactate production in leptin-deficient mice.

BACKGROUND: Perilipin 5 (Plin5) is well known to maintain the stability of intracellular lipid droplets (LDs) and regulate fatty acid metabolism in oxidative tissues. It is highly expressed in the heart, but its roles have yet to be fully elucidated. METHODS: Plin5-deficient mice and Plin5/leptin-double-knockout mice were produced, and their histological structures and myocardial functions were observed. Critical proteins related to fatty acid and glucose metabolism were measured in heart tissues, neonatal mouse cardiomyocytes and Plin5-overexpressing H9C2 cells. 2-NBDG was employed to detect glucose uptake. The mitochondria and lipid contents were observed by MitoTracker and BODIPY 493/503 staining in neonatal mouse cardiomyocytes. RESULTS: Plin5 deficiency impaired glucose utilization and caused insulin resistance in mouse cardiomyocytes, particularly in the presence of fatty acids (FAs). Additionally, Plin5 deficiency increased the NADH content and elevated the expression of lactate dehydrogenase (LDHA) in cardiomyocytes, which resulted in increased lactate production. Moreover, when fatty acid oxidation was blocked by etomoxir or LDHA was inhibited by GSK2837808A in Plin5-deficient cardiomyocytes, glucose utilization was improved. Leptin-deficient mice exhibited myocardial hypertrophy, insulin resistance and altered substrate utilization, and Plin5 deficiency exacerbated myocardial hypertrophy in leptin-deficient mice. CONCLUSION: Our results demonstrated that Plin5 plays a critical role in coordinating fatty acid and glucose oxidation in cardiomyocytes, providing a potential target for the treatment of metabolic disorders in the heart.

The use of high-sensitivity cardiac troponin T and creatinine kinase-MB as a prognostic markers in patients with acute myocardial infarction and chronic kidney disease.

Background: High-sensitivity cardiac troponin T (hs-cTnT) and creatine kinase (CK)-MB are the most commonly used biomarkers for the diagnosis and prognosis of acute myocardial infarction (AMI). Chronic kidney disease (CKD) often leads to elevated hs-cTnT levels in non-AMI patients. However, studies comparing the prognostic value of both hs-cTnT and CK-MB in patients with AMI and CKD are lacking.Methods: We conducted a retrospective study on AMI patients diagnosed between January 2015 and October 2020. Patients were categorized based on renal function as normal or CKD. Peak hs-cTnT and CK-MB levels during hospitalization were collected, and their diagnostic value was evaluated using receiver operating characteristic (ROC) curves. The impact on in-hospital mortality was analyzed using multivariate logistic regression. The relationship between the hs-cTnT/CK-MB ratio and in-hospital death was examined using a restricted cubic spline (RCS) curve.Results: The study included 5022 AMI patients, of whom 797 (15.9%) had CKD. The AUCs of Hs-cTnT and CK-MB were higher in the CKD group [0.842 (95% CI: 0.789-0.894) and 0.821 (95% CI: 0.760-0.882)] than in the normal renal function group [0.695 (95% CI: 0.604-0.790) and 0.708 (95% CI: 0.624-0.793)]. After full adjustment for all risk factors, hs-cTnT (OR, 2.82; 95% CI, 1.03-9.86; p = 0.038) and CK-MB (OR, 4.91; 95% CI, 1.54-14.68; p = 0.007) above the cutoff values were independent predictors of in-hospital mortality in patients with CKD. However, in patients with normal renal function, only CK-MB above the cutoff (OR, 2.45; 95% CI, 1.02-8.24; p = 0.046) was a predictor of in-hospital mortality, whereas hs-cTnT was not. There was an inverted V-shaped relationship between the hs-cTnT/CK-MB ratio and in-hospital mortality, with an inflection point of 19.61. The ratio within the second quartile (9.63-19.6) was an independent predictor of in-hospital mortality in patients with CKD (OR 5.3, 95% CI 1.66-16.86, p = 0.005).Conclusions: Hs-cTnT independently predicted in-hospital mortality in AMI patients with CKD, whereas its predictive value was not observed in patients with normal renal function. CK-MB was an independent predictor of in-hospital mortality regardless of renal function. Moreover, the hs-cTnT/CK-MB ratio may aid in risk stratification of AMI patients with CKD.

IDH1 mutation contributes to myeloid dysplasia in mice by disturbing heme biosynthesis and erythropoiesis.

Isocitrate dehydrogenase (IDH) mutations are common genetic alterations in myeloid disorders, including acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Epigenetic changes, including abnormal histone and DNA methylation, have been implicated in the pathogenic build-up of hematopoietic progenitors, but it is still unclear whether and how IDH mutations themselves affect hematopoiesis. Here, we show that IDH1-mutant mice develop myeloid dysplasia in that these animals exhibit anemia, ineffective erythropoiesis, and increased immature progenitors and erythroblasts. In erythroid cells of these mice, D-2-hydroxyglutarate, an aberrant metabolite produced by the mutant IDH1 enzyme, inhibits oxoglutarate dehydrogenase activity and diminishes succinyl-coenzyme A (CoA) production. This succinyl-CoA deficiency attenuates heme biosynthesis in IDH1-mutant hematopoietic cells, thus blocking erythroid differentiation at the late erythroblast stage and the erythroid commitment of hematopoietic stem cells, while the exogenous succinyl-CoA or 5-ALA rescues erythropoiesis in IDH1-mutant erythroid cells. Heme deficiency also impairs heme oxygenase-1 expression, which reduces levels of important heme catabolites such as biliverdin and bilirubin. These deficits result in accumulation of excessive reactive oxygen species that induce the cell death of IDH1-mutant erythroid cells. Our results clearly show the essential role of IDH1 in normal erythropoiesis and describe how its mutation leads to myeloid disorders. These data thus have important implications for the devising of new treatments for IDH-mutant tumors.

Isocitrate dehydrogenase 1 mutation enhances 24(S)-hydroxycholesterol production and alters cholesterol homeostasis in glioma.

Isocitrate dehydrogenase (IDH) mutation is the most important initiating event in gliomagenesis, and the increasing evidence shows that IDH mutation is associated with the metabolic reprogramming in the tumor. Dysregulated cholesterol metabolism is a hallmark of tumor cells, but the cholesterol homeostasis in IDH-mutated glioma is still unknown. In this study, we found that astrocyte-specific mutant IDH1(R132H) knockin reduced the cholesterol contents and damaged the structure of myelin in mouse brains. In U87 and U251 cells, the expression of mutant IDH1 consistently reduced the cholesterol levels. Furthermore, we found that IDH1 mutation enhanced the production of 24(S)-hydroxycholesterol (24-OHC), which is not only the metabolite of cholesterol elimination, but also functions as an endogenous ligand for the liver X receptors (LXRs). In IDH1-mutant glioma cells, the elevated 24-OHC activated LXRs, which consequently accelerated the low-density lipoprotein receptor (LDLR) degradation by upregulating the inducible degrader of the LDLR (IDOL). The reduced LDLR expressions in IDH1-mutant glioma cells abated the uptakes of low-density lipoprotein (LDL) to decrease the cholesterol influx. In addition, the activated LXRs also promoted the cholesterol efflux by elevating the ATP-binding cassette transporter A1 (ABCA1), ABCG1, and apolipoprotein E (ApoE) in both IDH1-mutant astrocytes and glioma cells. As a feedback, the reduced cholesterol levels stimulated the cholesterol biosynthesis, which made IDH1-mutated glioma cells more sensitive to atorvastatin, an inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase. The altered cholesterol homeostasis regulated by mutant IDH provides a pivotal therapeutical strategy for the IDH-mutated gliomas.

Wild-Type IDH1 and Mutant IDH1 Opposingly Regulate Podoplanin Expression in Glioma.

Isocitrate dehydrogenase (IDH) mutations occur frequently in lower-grade gliomas, which result in genome-wide epigenetic alterations. The wild-type IDH1 is reported to participate in lipid biosynthesis and amino acid metabolism, but its role in tumorigenesis is still unclear. In this study, the expressions of IDH1 and podoplanin (Pdpn) were determined in IDH-mutated and IDH-wild-type gliomas, and their relationships in glioma were further analyzed. In addition, the regulation of wild-type IDH1 and mutant IDH1 on Pdpn expression was investigated by luciferase assays and promoter methylation analysis. Our study showed that Pdpn was almost undetectable in IDH-mutated glioma but strongly expressed in higher-grade IDH-wild-type glioma. Pdpn overexpression promoted the migration of glioma cells but had little effect on cell growth. Moreover, Pdpn expression was positively correlated with the increased wild-type IDH1 levels in IDH-wild-type glioma. Consistently, the wild-type IDH1 greatly promoted the transcription and expression of Pdpn, but the mutant IDH1 and D-2-hydroxyglutarate significantly suppressed Pdpn expression in glioma cells. Besides, our results revealed that the methylation of CpG islands in the Pdpn promoter was opposingly regulated by wild-type and mutant IDH1 in glioma. Collectively, our results indicated that wild-type and mutant IDH1 opposingly controlled the Pdpn expression in glioma by regulating its promoter methylation, which provides a basis for understanding the relationship between wild-type and mutant IDH1 in epigenetic regulation and tumorigenesis.

Current needs for the improved management of depressive disorder in community healthcare centres, Shenzhen, China: a view from primary care medical leaders.

BACKGROUND: The prevalence of depressive disorder in Shenzhen is higher than for any other city in China. Despite national health system reform to integrate mental health into primary care, the majority of depression cases continue to go unrecognized and untreated. Qualitative research was conducted with primary care medical leaders to describe the current clinical practice of depressive disorder in community healthcare centres (CHC) in Shenzhen and to explore the participants' perceptions of psychological, organizational and societal barriers and enablers to current practice with a view to identifying current needs for the improved care of depressive disorder in the community. METHODS: Seventeen semi-structured, audio-recorded interviews (approx. 1 h long) were conducted in Melbourne (n = 7) and Shenzhen (n = 10) with a convenience sample of primary care medical leaders who currently work in community healthcare centres (CHC) in Shenzhen and completed any one of the 3-month long, Melbourne-based, "Monash-Shenzhen Primary Healthcare Leaders Programs" conducted between 2015 and 2017. The interview guide was developed using the Theoretical Domain's Framework (TDF) and a directed content analysis (using Nvivo 11 software) was performed using English translations. RESULTS: Despite primary care medical leaders being aware of a mental health treatment gap and the benefits of early depression care for community wellbeing, depressive disorder was not perceived as a treatment priority in CHCs. Instead, hospital specialists were identified as holding primary responsibility for formal diagnosis and treatment initiation with primary care doctors providing early assessment and basic health education. Current needs for improved depression care included: (i) Improved professional development for primary care doctors with better access to diagnostic guidelines and tools, case-sharing and improved connection with mentors to overcome current low levels of treatment confidence. (ii) An improved consulting environment (e.g. allocated mental health resource; longer and private consultations; developed medical referral system; better access to antidepressants) which embraces mental health initiatives (e.g. development of mental health departments in local hospitals; future use of e-mental health; reimbursement for patients; doctors' incentives). (iii) Improved health literacy to overcome substantive mental health stigma in society and specific stigma directed towards the only public psychiatric hospital. CONCLUSIONS: Whilst a multi-faceted approach is needed to improve depression care in community health centres in Shenzhen, this study highlights how appropriate mental health training is central to developing a robust work-force which can act as key agents in national healthcare reform. The cultural adaption of the depression component of the World Health Organisation's mental health gap intervention guide (mhGAP-IG.v2) could provide primary care doctors with a future training tool to develop their assessment skills and treatment confidence.

Plin5 deficiency exacerbates pressure overload-induced cardiac hypertrophy and heart failure by enhancing myocardial fatty acid oxidation and oxidative stress.

While cardiac hypertrophy and heart failure are accompanied by significant alterations in energy metabolism, more than 50-70% of energy is obtained from fatty acid ß-oxidation (FAO) in adult hearts under physiological conditions. Plin5 is involved in the metabolism of lipid droplets (LDs) and is highly abundant in oxidative tissues including heart, liver and skeletal muscle. Plin5 protects the storage of triglyceride (TG) in LDs by inhibiting lipolysis, thereby suppressing excess FAO and preventing excessive oxidative stress in the heart. In this study, we investigated the roles of Plin5 in cardiac hypertrophy and heart failure in mice treated with transverse aortic constriction (TAC). The results indicated that Plin5 deficiency aggravated myocardial hypertrophy in the TAC-treated mice and exacerbated the TAC-induced heart failure. We also found that Plin5 deficiency reduced the cardiac lipid accumulation and upregulated the levels of PPARα and PGC-1α, which stimulate mitochondrial proliferation. Moreover, Plin5 deficiency aggravated the TAC-induced oxidative stress. We consistently found that Plin5 knockdown disrupted TG storage and elevated FAO and lipolysis in H9C2 rat cardiomyocytes. In addition, Plin5 knockdown also provoked mitochondrial proliferation and lipotoxic injury in H9C2 cells. In conclusion, Plin5 deficiency increases myocardial lipolysis, elevates FAO and oxidative burden, and thereby exacerbates cardiac hypertrophy and heart failure in TAC-treated mice.

Plin3 protects against alcoholic liver injury by facilitating lipid export from the endoplasmic reticulum.

Hepatic lipid accumulation is the most common pathological characteristic of alcoholic liver disease (ALD). In mammalian cells, excess neutral lipids are stored in lipid droplets (LDs). As a member of perilipin family proteins, Plin3 was recently found to regulate the LD biogenesis. However, the roles and mechanism of Plin3 in ALD progression remain unclear. Herein, we found that alcohol stimulated Plin3 expression in both mouse livers and cultured AML12 mouse hepatic cells, which was accompanied by excess LD accumulation in hepatocytes. The elevations of Plin3 in alcohol-treated hepatocytes paralleled with the levels of both PPARα and γ, and the protein degradation of Plin3 was also reduced after alcohol exposure. Moreover, Plin3 knockdown increased cellular sensitivity to alcohol-induced apoptosis, endoplasmic reticulum (ER) stress, and inflammatory cytokines release, including TNF-α, IL-1, and IL-6ß. Notably, alcohol exacerbated triglycerides (TG) accumulation in the ER and caused ER dilation in Plin3-knockdown AML12 cells. Finally, we observed that Plin3 interacted with dynein subunit Dync1i1 and mediated the colocalization of LDs and microtubules, while high concentration of alcohol disrupted microtubules and caused dispersion of excess small LDs in cytoplasm. Summarily, Plin3 promotes lipid export from the ER and reduces ER lipotoxic stress, thereby, protecting against alcoholic liver injury. Moreover, Plin3 could be an adapter protein mediating LD transport by microtubules. This study explored the roles of Plin3 in alcohol-induced hepatic injury, suggesting Plin3 as a potential target for the prevention of ALD progression.

Perilipin 5 alleviates HCV NS5A-induced lipotoxic injuries in liver.

BACKGROUND: The homeostasis of lipid droplets (LDs) plays a crucial role in maintaining the physical metabolic processes in cells, and is regulated by many LD-associated proteins, including perilipin 5 (Plin5) in liver. As the putative sites of hepatitis C virus (HCV) virion assembly, LDs are vital to viral infection. In addition, the hepatic LD metabolism can be disturbed by non-structural HCV proteins, such as NS5A, but the details are still inexplicit. METHODS: HCV NS5A was overexpressed in the livers and hepatocytes of wild-type and Plin5-null mice. BODIPY 493/503 and oil red O staining were used to detect the lipid content in mouse livers and hepatocytes. The levels of lipids, lipid peroxidation and inflammation biomarkers were further determined. Immunofluorescence assay and co-immunoprecipitation assay were performed to investigate the relationship of Plin5 and NS5A. RESULTS: One week after adenovirus injection, livers expressing NS5A showed more inflammatory cell aggregation and more severe hepatic injuries in Plin5-null mice than in control mice, which was consistent with the increased serum levels of IL-2 and TNF-α (P < 0.05) observed in Plin5-null mice. Moreover, Plin5 deficiency in the liver and hepatocytes aggravated the elevation of MDA and 4-HNE levels induced by NS5A expression (P < 0.01). The triglyceride (TG) content was increased approximately 25% by NS5A expression in the wild-type liver and hepatocytes but was unchanged in the Plin5-null liver and hepatocytes. More importantly, Plin5 deficiency in the liver and hepatocytes exacerbated the elevation of non-esterified fatty acids (NEFAs) stimulated by NS5A expression (P < 0.05 and 0.01 respectively). Using triacsin C to block acyl-CoA biosynthesis, we found that Plin5 deficiency aggravated the NS5A-induced lipolysis of TG. In contrast, Plin5 overexpression in HepG2 cells ameliorated the NS5A-induced lipolysis and lipotoxic injuries. Immunofluorescent staining demonstrated that NS5A expression stimulated the targeting of Plin5 to the surface of the LDs in hepatocytes without altering the protein levels of Plin5. By co-IP, we found that the N-terminal domain (aa 32-128) of Plin5 was pivotal for its binding with NS5A. CONCLUSIONS: Our data highlight a protective role of Plin5 against hepatic lipotoxic injuries induced by HCV NS5A, which is helpful for understanding the steatosis and injuries in liver during HCV infection.

Isocitrate dehydrogenase1 mutation reduces the pericyte coverage of microvessels in astrocytic tumours.

INTRODUCTION: Tumour-associated angiogenesis is associated with the malignancy and poor prognosis of glioma. Isocitrate dehydrogenase (IDH) mutations are present in the majority of lower-grade (WHO grade II and III) and secondary glioblastomas, but their roles in tumour angiogenesis remain unclear. METHODS: Using magnetic resonance imaging (MRI), the cerebral blood flow (CBF) of IDH-mutated glioma was measured and compared with the IDH-wildtype glioma. The densities of microvessels in IDH-mutated and wildtype astrocytoma and glioblastoma were assessed by immunohistochemical (IHC) staining with CD34, and the pericytes were labelled with α-smooth muscle antigen (α-SMA), neural-glial antigen 2 (NG2) and PDGF receptor-ß (PDGFR-ß), respectively. Furthermore, glia-specific mutant IDH1 knock-in mice were generated to evaluate the roles of mutant IDH1 on brain vascular architectures. The transcriptions of the angiogenesis-related genes were assessed in TCGA datasets, including ANGPT1, PDGFB and VEGFA. The expressions of these genes were further determined by western blot in U87-MG cells expressing a mutant IDH1 or treated with 2-HG. RESULTS: The MRI results indicated that CBF was reduced in the IDH-mutated gliomas. The IHC staining showed that the pericyte coverages of microvessels were significantly decreased, but the microvessel densities (MVDs) were only slightly decreased in IDH-mutated glioma. The mutant IDH1 knock-in also impeded the pericyte coverage of brain microvessels in mice. Moreover, the TCGA database showed the mRNA levels of angiogenesis factors, including ANGPT1, PDGFB and VEGFA, were downregulated, and their promoters were also highly hyper-methylated in IDH-mutated gliomas. In addition, both mutant IDH1 and D-2-HG could downregulate the expression of these genes in U87-MG cells. CONCLUSIONS: Our results suggested that IDH mutations could reduce the pericyte coverage of microvessels in astrocytic tumours by inhibiting the expression of angiogenesis factors. As vascular pericytes play an essential role in maintaining functional blood vessels to support tumour growth, our findings imply a potential avenue of therapeutic strategy for IDH-mutated gliomas.

Mutation of IDH1 aggravates the fatty acid­induced oxidative stress in HCT116 cells by affecting the mitochondrial respiratory chain.

Increasing evidence has indicated that mutations of isocitrate dehydrogenase 1/2 (IDH1/2) contribute to the metabolic reprogramming of cancer cells; however their functions in lipid metabolism remain unknown. In the present study, the parental and IDH1 (R132H/+) mutant HCT116 cells were treated with various concentrations of oleic acid (OA) or palmitic acid (PA) in the presence or absence of glucose. The results demonstrated that mutation of IDH1 exacerbated the effects of OA and PA on cell viability and apoptosis, and consistently elevated the production of reactive oxygen species in HCT116 cells, particularly in the absence of glucose. Furthermore, mutation of IDH1 inhibited the rate of fatty acid oxidation (FAO), but elevated the glucose consumption in HCT116 cells. The results of immunoblotting and reverse transcription­quantitative polymerase chain reaction (RT­qPCR) indicated that the expression of glucose transporter 1 was upregulated, whereas that of carnitine palmitoyl transferase 1 was downregulated in IDH1 mutant HCT116 cells. Although mitochondrial DNA quantification demonstrated that mutation of IDH1 had no effect on the quantity of mitochondria, immunoblotting and RT­qPCR revealed that mutation of IDH1 in HCT116 cells significantly downregulated the expression of cytochrome c (CYCS) and CYCS oxidase IV, two important components in mitochondrial respiratory chain. These results indicated that mutation of IDH1 aggravated the fatty acid­induced oxidative stress in HCT116 cells, by suppressing FAO and disrupting the mitochondrial respiratory chain. The results of the present study may provide novel insight into therapeutic strategies for the treatment of cancer types with IDH mutation.

Atorvastatin reduces lipid accumulation in the liver by activating protein kinase A-mediated phosphorylation of perilipin 5.

Statins have been proven to be effective in treating non-alcoholic fatty liver disease (NAFLD). Recently, it was reported that statins decreased the hepatic expression of perilipin 5 (Plin5), a lipid droplet (LD)-associated protein, which plays critical roles in regulating lipid accumulation and lipolysis in liver. However, the function and regulation mechanism of Plin5 have not yet been well-established in NAFLD treatment with statins. In this study, we observed that atorvastatin moderately reduced the expression of Plin5 in livers without changing the protein level of Plin5 in the hepatic LD fraction of mice fed with high-fat diet (HFD). Intriguingly, atorvastatin stimulated the PKA-mediated phosphorylation of Plin5 and reduced the triglyceride (TG) accumulation in hepatocytes with overexpression of wide type (Plin5-WT) compared to serine-155 mutant Plin5 (Plin5-S155A). Moreover, PKA-stimulated FA release of purified LDs carrying Plin5-WT but not Plin5-S155A. Glucagon, a PKA activator, stimulated the phosphorylation of Plin5-WT and inhibited its interaction with CGI-58. The results indicated that atorvastatin promoted lipolysis and reduced TG accumulation in the liver by increasing PKA-mediated phosphorylation of Plin5. This new mechanism of lipid-lowering effects of atorvastatin might provide a new strategy for NAFLD treatment.

Cideb Deficiency Aggravates Dextran Sulfate Sodium-induced Ulcerative Colitis in Mice by Exacerbating the Oxidative Burden in Colonic Mucosa.

BACKGROUND: Abnormal lipid metabolism is one of many factors that contribute to the development of ulcerative colitis (UC). As a lipid droplet-associated protein, Cideb facilitated the export of lipids from enterocytes and promoted intestinal lipid absorption. We found that Cideb was upregulated in the colonic mucosa of both UC patients and dextran sodium sulfate (DSS)-induced mouse colitis, but its roles in the pathogenesis of UC are still ill-defined. METHODS: Acute colitis was induced with DSS in Cideb-null and wild-type mice, and the inflammation and oxidative stress were evaluated in the colonic mucosa. Moreover, triglyceride accumulation and oxidative stress were further analyzed in polarized Caco-2 cells with overexpression of Cideb. RESULTS: Our present data indicated that Cideb-null mice were more susceptible to DSS-induced colitis, and consumption of a high-fat diet exacerbated the deterioration of DSS-induced colitis in Cideb-null mice. Moreover, Cideb deficiency increased the colonic oxidative stress in DSS-treated mice and more significant under a high-fat diet condition. In exploring the mechanism, we found that Cideb deficiency elevated the lipid content in both feces and the colonic mucosa of DSS-treated mice, especially those fed with a high-fat diet. The in vitro evidence proved that Cideb expression reduced triglyceride accumulation and oxidative stress in polarized Caco-2 cells in the presence of oleic acid. CONCLUSIONS: Our data suggest that Cideb plays a protective role against the development of UC by reducing the lipid accumulation and oxidative damage in the colonic mucosa. Therefore, Cideb could be a potential therapeutic target for UC.

Plin5 alleviates myocardial ischaemia/reperfusion injury by reducing oxidative stress through inhibiting the lipolysis of lipid droplets.

Myocardial ischaemia-reperfusion (I/R) injury is a complex pathophysiological process. Current research has suggested that energy metabolism disorders, of which the abnormal consumption of fatty acids is closely related, compose the main pathological basis for myocardial I/R injury. Lipid droplets (LD) are critical regulators of lipid metabolism by LD-associated proteins. Among the lipid droplet proteins, the perilipin family members regulate lipolysis and lipogenesis through different mechanisms. Plin5, an important perilipin protein, promotes LD generation and lowers fatty acid oxidation, thus protecting the myocardium from lipotoxicity. This study investigated the protective effects of Plin5 in I/R myocardium. Our results indicated that Plin5 deficiency exacerbated the myocardial infarct area, aggravated left ventricular systolic dysfunction, reduced lipid storage, and elevated free fatty acids. Plin5-deficient myocardium exhibited severely damaged mitochondria, elevated reactive oxygen species (ROS) and malondialdehyde (MDA) levels, and decreased superoxide dismutase (SOD) activity. Furthermore, the decreased phosphorylation of PI3K/Akt in Plin5-null cardiomyocytes might contribute to I/R injury aggravation. In conclusion, Plin5, a new regulator of myocardial lipid metabolism, decreases free fatty acid peroxidation by inhibiting the lipolysis of intracellular lipid droplets, thus providing cardioprotection against I/R injury and shedding new light on therapeutic solutions for I/R diseases.

IDH1 deficiency attenuates gluconeogenesis in mouse liver by impairing amino acid utilization.

Although the enzymatic activity of isocitrate dehydrogenase 1 (IDH1) was defined decades ago, its functions in vivo are not yet fully understood. Cytosolic IDH1 converts isocitrate to α-ketoglutarate (α-KG), a key metabolite regulating nitrogen homeostasis in catabolic pathways. It was thought that IDH1 might enhance lipid biosynthesis in liver or adipose tissue by generating NADPH, but we show here that lipid contents are relatively unchanged in both IDH1-null mouse liver and IDH1-deficient HepG2 cells generated using the CRISPR-Cas9 system. Instead, we found that IDH1 is critical for liver amino acid (AA) utilization. Body weights of IDH1-null mice fed a high-protein diet (HPD) were abnormally low. After prolonged fasting, IDH1-null mice exhibited decreased blood glucose but elevated blood alanine and glycine compared with wild-type (WT) controls. Similarly, in IDH1-deficient HepG2 cells, glucose consumption was increased, but alanine utilization and levels of intracellular α-KG and glutamate were reduced. In IDH1-deficient primary hepatocytes, gluconeogenesis as well as production of ammonia and urea were decreased. In IDH1-deficient whole livers, expression levels of genes involved in AA metabolism were reduced, whereas those involved in gluconeogenesis were up-regulated. Thus, IDH1 is critical for AA utilization in vivo and its deficiency attenuates gluconeogenesis primarily by impairing α-KG-dependent transamination of glucogenic AAs such as alanine.

[Metformin inhibits THP-1 macrophage-derived foam cell formation induced by lipopolysaccharide].

OBJECTIVE: To investigate the impact of metformin (Met) on THP-1 macrophage-derived foam cell formation induced by lipopolysaccharide (LPS) and observe the changes of lipid droplets (LDs) and LDs-associated proteins. METHODS: THP-1 cells were induced to differentiate into macrophages by 100 ng/mL phorbol 12-myristate 13-acetate (PMA) for 48 hours, and then the macrophages were further induced to generate foam cells by 50 µg/mL oxidized low-density lipoprotein (ox-LDL) and 1 µg/mL LPS. During this process, these foam cells were treated with 0, 100, 200 µmol/L Met. Under the fluorescence microscope, the effect of Met on foam cell formation was evaluated by Oil red O staining and the number and morphology of LDs were observed by BODIPY493/503 staining. Intracellular triglyceride (TG) were extracted and measured by TG quantitative kits. The expressions of adipose differentiation-related protein (ADRP) and tail-interacting protein of 47 kDa (TIP47) were detected by Western blotting. RESULTS: Compared with the untreated group, the LDs in foam cells were reduced significantly and the size became smaller after treated with 100 or 200 µmol/L Met. What's more, the quantitative data showed that the intracellular TG content decreased markedly in a dose-dependent manner, and the TG content decreased about 25% in foam cells treated with 200 µmol/L Met. Western blotting showed that Met reduced the expression of ADRP, but not TIP47 in the THP-1 macrophage-derived foam cells. CONCLUSION: Met could inhibit THP-1-derived foam cell formation induced by LPS, reduce intracellular lipid accumulation, and down-regulate the expression of ADRP.

[The expression of IDH1 (R132H) is positively correlated with cell proliferation and angiogenesis in glioma samples].

OBJECTIVE: To explore the correlations of the expression of mutant isocitrate dehydrogenase (IDH1) (R132H) protein with angiogenesis and cell proliferation in glioma. METHODS: We performed polymerase chain reaction-based IDH gene mutation screening in 385 glioma samples, and the subcellular localization and expression levels of IDH1 (R132H) was examined by immunohistochemistry (IHC). Ki-67 labeling index was introduced to determine the proliferation of glioma cells, and the microvessel density was measured through CD34 staining. Statistical analyses were performed to show the correlations of IDH1 mutation with cell proliferation and microvessel density. RESULTS: The mutant rates of IDH1 were about 50%-60% in grade II-III gliomas and secondary glioblastomas, which were significantly higher than those in pilocytic astrocytoma (grade I) and primary glioblastoma (grade IV). Moreover, the level of IDH1 (R132H) protein was positively correlated with Ki-67 labeling index and microvessel density. CONCLUSION: IDH mutation was common in grade II-III glioma and secondary glioblastoma, and the mutant IDH1 (R132H) might play a critical role in the cell proliferation and angiogenesis of glioma.

Perilipin 5 improves hepatic lipotoxicity by inhibiting lipolysis.

UNLABELLED: Abnormal metabolism of nonesterified fatty acids (NEFAs) and their derivatives has been reported to be the main cause of intracellular lipotoxic injury. Normally, NEFAs are stored in lipid droplets (LDs) in the form of triglyceride (TG), which could reduce the lipotoxicity of cytosolic NEFAs. Previous studies have implicated that Perilipin 5 (Plin5), an LD-binding protein, regulates the storage and hydrolysis of TG in LD. However, its roles and underlying mechanisms in the liver remain unknown. Here we found that Plin5 expression was increased in steatotic livers. Using Plin5 knockout mice, we found that Plin5 deficiency resulted in reduced hepatic lipid content and smaller-sized LDs, which was due to the elevated lipolysis rate and fatty acid utilization. Plin5-deficient hepatocytes showed increased mitochondria proliferation, which could be explained by the increased expression and activity of PPARα stimulated by the increased NEFA levels. Meanwhile, Plin5-deficient livers also exhibited enhanced mitochondrial oxidative capacity. We also found that Plin5 deficiency induces lipotoxic injury in hepatocytes, attributed to lipid peroxidation. Mechanistically, we found that Plin5 blocks adipose triglyceride lipase (ATGL)-mediated lipolysis by competitively binding to comparative gene identification-58 (CGI-58) and disrupting the interaction between CGI-58 and ATGL. CONCLUSION: Plin5 is an important protective factor against hepatic lipotoxicity induced by NEFAs generated from lipolysis. This provides an important new insight into the regulation of hepatic lipid storage and relation between lipid storage and lipotoxicity.