Effects of Cholesterol on Autophagy and Apoptosis in HepG2 cells

학위논문 (석사)-- 서울대학교 대학원 : 식품영양학과, 2016. 8. 권영혜.Background & Aims: Non-alcoholic fatty liver disease (NAFLD) is one of the most prevalent liver diseases. Recent studies have shown that cholesterol is accumulated in liver during the progression of NAFLD. Thus, increasing attention has been drawn to the role of cholesterol in the pathogenesis of the disease. Autophagy, a cellular degradative pathway, is an important self-defense mechanism to maintain cellular homeostasis in response to various stresses including lipid stimulation. In this context, this study was undertaken to determine cholesterol lipotoxicity in hepatocytes using cholesterol overloaded HepG2 cells. Precisely, whether dysregulated cholesterol homeostasis alters autophagic activity and contributes to apoptosis was investigated. Methods: Human hepatocarcinoma cell line, HepG2 cells were treated with cholesterol with either 25 or 50 µg/mL of concentrations, which are considered to deposit cholesterol as much as the amount found in human and experimental NAFLD model. Additional treatment using 10µM chloroquine was conducted in some experiments to inhibit lysosomal activity and degradation of autophagosome. Intracellular cholesterol level was measured by enzymatic colorimetric methods and the cell viability was determined through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. The relative levels of several autophagy or apoptosis related proteins were analyzed by Western blotting. Production of reactive oxygen species (ROS) was assessed by observing emission of green fluorescence following oxidation of 2,7-dichlorofluorescein diacetate. Lipid peroxide levels were determined by measuring cellular thiobarbituric acid reactive substances (TBARS) levels. Results: Intracellular total cholesterol level increased in cells treated with 50 µg/mL cholesterol after 24 hours compared with vehicle treated cells. MTT assays showed dose-dependent cholesterol toxicity and microscopic observations showed reduction of cell populations compared with vehicle control cells when 25 or 50 µg/mL of cholesterol were treated. Detection of cleavage of non-erythroid spectrin and caspase-3 revealed that cholesterol treatment prominently induced the apoptosis in HepG2 cells. Meanwhile, regarding autophagy pathway, treatment of cholesterol induced the increases of LC3-II and p62 protein levels dose-dependently. Beclin-1 remained unchanged. Moreover, in a LC3-II turnover assay, cells treated with chloroquine plus cholesterol showed less LC3-II degradation than those with chloroquine alone. Cholesterol also resulted in concomitant increase of ubiquitinated protein levels. Thus, these data collectively indicated that cholesterol treatment reduced autophagic flux. To clarify whether defects in autophagy is the primary cause of cholesterol induced caspase-3 activation, cell viability was measured using MTT assay after chloroquine or/and cholesterol treatment. As a result, 10 µM chloroquine treatment induced 10.7% viability loss while 50 µg/mL cholesterol treatment 44.6% viability loss, despite similar level of autophagy inhibition. Thus, it can be inferred that blockage of autophagic flux is not enough to cause systemic cell death, unless other toxic mechanisms are involved. Production of ROS and induction of lipid peroxides were observed after 50 µg/mL of cholesterol were treated, suggesting cholesterol accumulation induced oxidative stress. Activation of c-Jun N-terminal kinase (JNK) was also detected following the cholesterol loading. Furthermore, mitochondrial dysfunction which is strongly associated with oxidative stress was confirmed by detection of cleavage of caspase-9. Increased phosphorylation level of adenosine monophosphate-activated kinase (AMPK) was also observed, which indicates occurrence of ATP depletion. Conclusion: These findings demonstrate that excessive hepatic cholesterol induces blockage of autophagic flux, oxidative stress and the subsequent mitochondrial dysfunction. Although reduced autophagic activity per se was insufficient to cause cholesterol induced hepatocyte apoptosis entirely, it seems to aggravate oxidative stress due to attenuated removal of dysfunctional mitochondria. Overall, the present study demonstrates that cholesterol, besides free fatty acids, may contribute to lipotoxicity occurring in the progression of NAFLD.I. Introduction 1 1. Cholesterol-mediated lipotoxicity in NAFLD development 1 1.1. Cholesterol metabolism in the liver 1 1.2. Cholesterol accumulation and NAFLD 2 1.3. Mechanisms of cholesterol toxicity 4 2. Autophagy as a house keeping system 7 2.1. The roles and the process of autophagy pathway 7 2.2. Markers for monitoring autophagy 10 3. Aims of the study 14 Ⅱ. Materials and Methods 15 1. Cell culture 15 2. Cholesterol overloading 15 3. Light microscopy 16 4. Total cholesterol mass quantification 16 5. Lipid peroxidation measurement 17 6. Detection of Reactive Oxygen Species Generation 17 7. Cell viability determination 18 8. Total protein extraction 18 9. Protein solubility fractionation 19 10. Western blotting 19 11. Statistical analysis 20 Ⅲ. Results 21 1. Incubation with cholesterol increases cellular cholesterol content in HepG2 cells 21 2. Cholesterol overloading suppresses cell viability and induces apoptosis in HepG2 cells 23 3. Cholesterol overloading results in increases of autophagic markers in HepG2 cells 28 4. Autophagic flux is reduced in cholesterol overloaded HepG2 cells 31 5. Attenuation of autophagic flux by cholesterol is insufficient to trigger systemic cell death in HepG2 cells 33 6. Cholesterol overloading produces intracellular ROS and induces lipid peroxidation in HepG2 cells 35 7. Defective mitochondria is involved in cholesterol-induced cell death in HepG2 cells 38 Ⅳ. Discussion 40 Ⅴ. References 46 국문 초록 52Maste

흡연 청소년의 금연시도에 미치는 영향요인 탐색: 매일흡연자와 비(非)매일흡연자 비교

학위논문 (석사)-- 서울대학교 보건대학원 : 보건학과 보건정책관리전공, 2016. 8. 유승현.담배는 건강을 위협하는 대표적인 발암물질임에 따라 흡연은 주요한 건강이슈이다. 흡연의 시작 시기는 대부분 청소년기이며 우리나라의 청소년 흡연율은 2015년도에는 역대 최저치를 기록하였으나 성인흡연율과 달리 뚜렷한 하락세를 보이지는 않았었다. 따라서 청소년의 흡연문제에 있어 흡연예방이 금연보다 더 강조되고 있지만 청소년 흡연율의 지속적인 하락을 위해서는 이미 흡연을 시작한 자들에 대한 효과적인 금연중재도 필요할 것이다. 최근 해외연구에서는 흡연자를 흡연빈도에 따라 매일흡연자와 비매일흡연자로 구분하고 있으며 청소년의 흡연은 성인의 흡연과 달리 비매일흡연 비율이 높은 특징을 보인다. 또한 비매일흡연자는 매일흡연자와 흡연량, 흡연시작시기, 흡연인식 등에서 차이를 보이고 있지만 이에 관한 국내의 연구는 상대적으로 적은 실정이다. 따라서 흡연유형에 따른 흡연자 집단의 구분이 필요할 것이다. 본 연구에서는 2014년도와 2015년도의 청소년건강행태온라인조사 자료를 활용하여 총 11,430명의 청소년 흡연자를 매일흡연(5,695명)과 비매일흡연(5,735명)으로 구분하고, 두 집단 간의 사회인구학적특성, 개인건강행동특성, 주변인흡연특성, 흡연환경특성에 따른 차이를 알아보았다. 또한 다수의 연구에서 금연성공의 예측변인으로 밝혀진 금연시도를 종속변수로 각 집단의 금연시도 영향요인을 탐색·비교하였다. 통계프로그램은 SAS9.4를 이용하였으며 카이제곱검정과 로지스틱회귀분석으로 분석하였다. 연구결과 매일흡연자와 비매일흡연자는 사회인구학적특성, 개인건강행동특성, 주변인흡연특성, 흡연환경특성에 따라 두 집단 간의 유의미한 차이를 보였다. 매일흡연자와 비매일흡연자의 금연시도 비율은 차이가 없었으나 금연시도의 표면적 이유는 집단 간에 차이를 보였다. 본 연구에서 흡연시작시기, 흡연량, 주관적 건강, 건강생활습관, 주된 흡연장소, 구매방법이 비매일흡연자의 금연시도 관련요인으로 나타났으며, 주관적 체형, 문제음주, 가정내 간접흡연, 홍보매체는 매일흡연자의 금연시도 관련요인이었다. 금연교육과 전자담배경험은 두 집단에 공통된 금연시도 관련요인이었다. 또한 친구흡연은 두 집단에서 유의한 영향요인이었으나 모든 특성을 투입한 최종모델에서는 유의미한 결과를 보이지 않았다. 본 연구결과를 종합해보면 매일흡연자와 비매일흡연자는 금연시도 영향요인이 달랐다. 따라서 청소년 금연중재에 각 집단별 특성을 고려한 정책과 교육이 필요하다고 사료된다.Ⅰ. 서론 1 1. 연구의 필요성 1 2. 연구의 목적 5 3. 연구의 가설 5 Ⅱ. 문헌고찰 6 1. 매일흡연자와 비매일흡연자 6 2. 금연시도 영향요인 9 3. 금연관련 영향요인 11 Ⅲ. 연구방법 17 1. 연구설계 17 2. 용어의 정의 18 3. 연구자료 및 대상 19 4. 변수의 정의 21 5. 분석방법 26 Ⅳ. 연구결과 27 1. 매일흡연자와 비매일흡연자의 특성 27 2. 매일흡연자와 비매일흡연자의 금연시도 34 Ⅴ. 논의 55 1. 연구결과 고찰 55 2. 연구의 제언 및 제한점 64 Ⅵ. 결론 68 참고 문헌 70 설문지 90 Abstract 98Maste

The care cascade for hepatitis C virus and prognosis of chronic hepatitis C patients treated with antiviral agents in a tertiary hospital

Background Some studies have analyzed the frequency of HCV RNA testing and actual treatment among anti-HCV positive patients in Korea, which has a low prevalence of HCV infection. This study aimed to analyze the diagnosis process, treatment results, and prognosis according to care cascade in patients who are anti-HCV positive. Methods Three thousand two hundred fifty-three anti-HCV positive patients presented to a tertiary hospital between January 2005 and December 2020. The number of patients who underwent HCV RNA testing, treatment, and proportion of sustained virologic response (SVR) according to the type of antivirals was investigated. We investigated the cumulative incidence of hepatocellular carcinoma (HCC) and liver cirrhosis. Results Of a total of 3,253 people, 1,177 (36.2%) underwent HCV RNA testing and 858 (72.9%) were positive for HCV RNA. 494 (57.6%) of HCV RNA positive patients received antiviral treatment, and 443 (89.7%) of initiated hepatitis C treatment experienced SVR. Of the 421 treated patients, 16 (14.2%) developed HCC. The cumulative incidence of HCC at 15 years was significantly different according to the presence of liver cirrhosis (10/83, 29.5% vs. 6/338, 10.8%, p < 0.001). The cumulative incidences of HCC or liver cirrhosis did not show significant differences according to the presence of SVR12 (14/388, 13.2% vs. 2/33, 52.5%, p = 0.084, 21/319, 15.0%, vs. 3/22, 28.7%, p = 0.051). Conclusions Owing to the introduction of direct-acting antivirals, high SVR12 was achieved, but the proportion of anti-HCV positive patients who received HCV RNA testing and treatment was not high. HCC surveillance after SVR12 is recommended for chronic hepatitis C patients with cirrhosis.ope

Glycated albumin and the risk of micro- and macrovascular complications in subjects with type 1 diabetes

BACKGROUND: We investigated the relationship between the glycemic indices glycated albumin (GA) and glycated hemoglobin (HbA1c) and the progression of diabetic vascular complications [diabetic nephropathy (DN) and carotid artery atherosclerosis (CAA)] in subjects with type 1 diabetes (T1D). METHODS: A total of 154 participants with a median follow-up of 2.8 years were enrolled in this retrospective longitudinal study. We recruited T1D subjects who had regularly measured urine albumin-creatinine ratios and estimated glomerular filtration rates, as well as tested HbA1c and GA levels consecutively every 3 or 6 months. A subgroup of 54 subjects was measured repeated carotid intima-media thickness (IMT). RESULTS: We classified subjects into the DN progression (Group I; n = 30) with either deteriorated stages of chronic kidney disease (n = 18) or albuminuria progression (n = 17), and the non-progression (Group II; n = 124). In multiple logistic regression analyses, baseline albuminuria (odds ratio [OR] = 2.64, 95 % confidence interval [CI] = 1.03-6.74), mean GA levels (OR = 2.03, 95 % CI = 1.27-3.26) were significantly associated with progression of DN. However, there was no association with mean HbA1c (OR = 0.98, 95 % CI = 0.62-1.54). In a subgroup analysis for follow-up measurements of carotid IMT, age was independently associated with the presence of plaque and the mean IMT. However glycemic indices were not significantly associated with CAA. CONCLUSIONS: Mean GA levels were more closely associated with DN progression than mean HbA1c in subjects with T1D. However, they were not associated with the CAA.ope

The renal tubular damage marker urinary N-acetyl-β-D-glucosaminidase may be more closely associated with early detection of atherosclerosis than the glomerular damage marker albuminuria in patients with type 2 diabetes

BACKGROUND: To determine the association between urinary N-acetyl-β-D-glucosaminidase (NAG), a marker of renal tubulopathy, and carotid intima-media thickness (IMT) and plaques in patients with type 2 diabetes mellitus (T2D) and to compare the predictive value of NAG versus albuminuria, a marker of renal glomerulopathy. METHODS: A total of 343 participants were enrolled in this retrospective cross-sectional study. We recruited participants with T2D who were tested for blood glucose parameters, urinary NAG, and urinary albumin-to-creatinine ratio (ACR) and had been checked for carotid ultrasonography. RESULTS: We classified participants into a below-median urinary NAG group (Group I; n = 172) or an above-median group (Group II; n = 171). Mean, maximum, and mean of maximum carotid IMT and the proportion of patients with carotid plaques were significantly higher in Group II compared with Group I. In multiple linear regression analyses, high urinary NAG (Group II) was significantly associated with carotid IMT, independently of urinary ACR and other confounding factors. In terms of carotid plaques, both urinary NAG and ACR were significantly higher in participants with carotid plaques than in those without carotid plaques. After adjustment for confounding factors, both urinary NAG and ACR were significantly associated with the presence of carotid plaques. CONCLUSIONS: Elevated urinary NAG, a marker of renal tubular damage, was related to increased carotid IMT and the presence of carotid plaques in patients with T2D. Urinary NAG may be a more sensitive biomarker than urinary albumin for early detection of atherosclerosis.ope

Glycated Albumin Levels in Patients with Type 2 Diabetes Increase Relative to HbA1c with Time

We recently reported that glycated albumin (GA) is increased in subjects with longer duration of diabetes and with decreased insulin secretory function. Based on this, we investigated whether GA increases with time relative to glycated hemoglobin (HbA1c) and the association between GA and beta-cell function. We analyzed 340 type 2 diabetes patients whose serum GA and HbA1c levels had been repeatedly measured over 4 years. We assessed the pattern of changes with time in glycemic indices (GA, HbA1c, and GA/HbA1c ratio) and their relationship with beta-cell function. In all patients, glycemic indices decreased and maintained low levels around 15 and 27 months. However, from 39 months to 51 months, GA significantly increased but HbA1c tended to increase without statistical significance. We defined ΔGA/HbA1c as the difference between the nadir point (at 15 to 27 months) and the end point (at 39 to 51 months) and found that ΔGA/HbA1c was positively correlated with diabetes duration and negatively related to beta-cell function. In multivariable linear regression analyses, ΔGA/HbA1c was independently associated with diabetes duration. In conclusion, this study demonstrated that serum GA levels increase relative to HbA1c levels with time.ope

Morning Spot Urine Glucose-to-Creatinine Ratios Predict Overnight Urinary Glucose Excretion in Patients With Type 2 Diabetes

Background : With the advent of sodium glucose co-transporter 2 inhibitors to control glucose and treat diabetes, laboratory data aided by either timed or spot glucose levels in the urine could be used as an alternative marker of drug response. The aim of this study was to assess the agreement between overnight urinary glucose excretion (UGE) and morning spot urinary glucose-to-creatinine ratio (UGCR). Methods : In this prospective cross-sectional study, we enrolled a total of 215 participants with either normal glucose tolerance (NGT), pre-diabetes, or type 2 diabetes mellitus (T2DM). To exclude external factors such as food intake and physical activity, urine samples collected overnight at an 8-hr interval and the first-voided morning spot urine were collected and compared. Results : The median values of overnight 8-hr UGE in participants with NGT (N=14), pre-diabetes (N=41), and T2DM (N=160) were 35.0 mg, 35.6 mg, and 653.4 mg, respectively. In participants with T2DM, the median values of overnight 8-hr UGCR and first-voided morning spot UGCR (M-UGCR) were 1.37 mg/mg and 0.16 mg/mg, respectively. Quantitative analyses using an intraclass correlation coefficient (ICC) demonstrated a good reliability of measurement of the overnight 8-hr UGCR and M-UGCR (ICC=0.943, P<0.001). The M-UGCR was also significantly related to the overnight 8-hr UGE (r=0.828, P<0.001). Conclusions : M-UGCR and overnight 8-hr UGCR showed good agreement, suggesting that M-UGCR be used as a simple index for estimating overnight amounts of UGE in patients with T2DM.ope

Short Term Isocaloric Ketogenic Diet Modulates NLRP3 Inflammasome Via B-hydroxybutyrate and Fibroblast Growth Factor 21

A ketogenic diet (KD) is known to have beneficial health effects. Various types of KD interventions have been applied to manage metabolic syndrome based on modification of diet parameters such as duration of intervention, macronutrient components, and total calories. Nevertheless, the beneficial health impact of isocaloric KD is largely unknown, especially in healthy subjects. The present study investigated the acute effects of a 3-day isocaloric KD. In this non-randomized intervention study, we recruited 15 healthy volunteers aged 24-38 years (7 men and 8 women) and placed them on an isocaloric KD restricting intake of carbohydrates but not energy (75% fat, 20% protein, 5% carbohydrate) for 3 days. Biochemical profiles and laboratory measurements were performed. Peripheral blood monocular cells were cultured, and measured cell stimulated cytokines. After short-term isocaloric KD, subjects lost body weight and serum free fatty acid levels were increased. These results accompanied elevated serum β-hydroxybutyrate (BHB) concentration and fibroblast growth factor 21 (FGF21) levels and improved insulin sensitivity. Regarding the direct effect of BHB on inflammasome activation, interleukin-1β (IL-1β) and tumor necrosis factor-α secretion in response to adenosine triphosphate or palmitate stimulation in human macrophages decreased significantly after isocaloric KD. In ex-vivo experiments with macrophages, both FGF21 and BHB further reduced IL-1β secretion compared to either BHB or FGF21 alone. The inhibitory effect of FGF21 on IL-1β secretion was blunted with bafilomycin treatment, which blocked autophagy flux. In conclusion, isocaloric KD for 3 days is a promising approach to improve metabolic and inflammatory status. Clinical trial registration: clinicaltrials.gov (NCT02964572).ope

Next-generation sequencing reveals somatic mutations that confer exceptional response to everolimus

BACKGROUND: Given the modest responses to everolimus, a mTOR inhibitor, in multiple tumor types, there is a pressing need to identify predictive biomarkers for this drug. Using targeted ultra-deep sequencing, we aimed to explore genomic alterations that confer extreme sensitivity to everolimus. RESULTS: We collected formalin-fixed paraffin-embedded tumor/normal pairs from 39 patients (22 with exceptional clinical benefit, 17 with no clinical benefit) who were treated with everolimus across various tumor types (13 gastric cancers, 15 renal cell carcinomas, 2 thyroid cancers, 2 head and neck cancer, and 7 sarcomas). Ion AmpliSeqTM Comprehensive Cancer Panel was used to identify alterations across all exons of 409 target genes. Tumors were sequenced to a median coverage of 552x. Cancer genomes are characterized by 219 somatic single-nucleotide variants (181 missense, 9 nonsense, 7 splice-site) and 22 frameshift insertions/deletions, with a median of 2.1 mutations per Mb (0 to 12.4 mutations per Mb). Overall, genomic alterations with activating effect on mTOR signaling were identified in 10 of 22 (45%) patients with clinical benefit and these include MTOR, TSC1, TSC2, NF1, PIK3CA and PIK3CG mutations. Recurrently mutated genes in chromatin remodeling genes (BAP1; n = 2, 12%) and receptor tyrosine kinase signaling (FGFR4; n = 2, 12%) were noted only in patients without clinical benefit. CONCLUSIONS: Regardless of different cancer types, mTOR-pathway-activating mutations confer sensitivity to everolimus. Targeted sequencing of mTOR pathway genes facilitates identification of potential candidates for mTOR inhibitors.ope

SGLT2 Inhibition Modulates NLRP3 Inflammasome Activity via Ketones and Insulin in Diabetes With Cardiovascular Disease

Sodium-glucose cotransporter 2 (SGLT2) inhibitors reduce cardiovascular events in humans with type 2 diabetes (T2D); however, the underlying mechanism remains unclear. Activation of the NLR family, pyrin domain-containing 3 (NLRP3) inflammasome and subsequent interleukin (IL)-1β release induces atherosclerosis and heart failure. Here we show the effect of SGLT2 inhibitor empagliflozin on NLRP3 inflammasome activity. Patients with T2D and high cardiovascular risk receive SGLT2 inhibitor or sulfonylurea for 30 days, with NLRP3 inflammasome activation analyzed in macrophages. While the SGLT2 inhibitor's glucose-lowering capacity is similar to sulfonylurea, it shows a greater reduction in IL-1β secretion compared to sulfonylurea accompanied by increased serum β-hydroxybutyrate (BHB) and decreased serum insulin. Ex vivo experiments with macrophages verify the inhibitory effects of high BHB and low insulin levels on NLRP3 inflammasome activation. In conclusion, SGLT2 inhibitor attenuates NLRP3 inflammasome activation, which might help to explain its cardioprotective effects.ope