Lysophsphatidylcholine as an important mediator of free fatty acid-induced insulin resistance in hepatocytes

강미라 성균관대학교 일반대학원 2010 국내박사

Insulin resistance is a very basic condition in metabolic syndrome and type 2 diabetes and also a risk factor for cardiovascular disease. Free fatty acids (FFAs) are considered as an important link because insulin resistant subjects show abnormal lipid metabolism of increased FFA concentrations. It’s well established that FFA causes insulin resistance. FFAs are metabolized to ceramide, diacylglycerol (DAG) or lysophosphatidylcholine (LPC) in vitro. However, it is not clear which is the effector for the insulin resistance. LPC is the most abundant lysophospholipid in blood and mainly produced by phospholipase A2 (PLA2). According to our previous study (unpublished data), we found that LPC, not ceramide is an important mediator linking FFA and insulin resistance in rat L6 skeletal muscle cells. To determine the effect of de novo synthesized LPC on insulin resistance in hepatocytes, we investigated the mechanisms of palmitic acid-induced insulin resistance in hepatocytes in the presence or absence of Ca2+-independent phospholipase A2 (iPLA2) inhibitors. iPLA2 inhibitors ameliorated the suppression of Akt Ser473 phosphorylation under the PA treatment, in addition, decreased PA-induced JNK phosphorylation, suggesting important roles of iPLA2 and LPC in PA-induced insulin resistance in hepatocytes. Ceramide synthesis inhibitors didn’t affect JNK phosphorylation which is central in FFA-induced insulin resistance. Stable hepa1c1c7 cell transfectants expressing αi2 G203T were resistant to FFA-induced insulin resistance as well. A dominant-negative αi mutation has attenuated the phosphorylation of JNK and IRS-1 Ser307 activation, and ultimately restored Akt Ser473 phosphorylation. Pertussis toxin (PTX), a specific inhibitor of Gi type of G-protein, also reversed the attenuation of Akt Ser307 phosphorylation by PA. Collectively, GPCR/Gαi might be a receptor to mediate PA-induced insulin resistance. This study suggests the involvement of LPC in the FFA-induced insulin resistance and potential therapeutic values of PLA2 inhibitiors or GPCR/Gαi inhibitors in insulin resistance. 인슐린 저항성은 대사증후군과 제2형 당뇨병 발병의 근원이 되는 요소로서 심혈관질환의 위험인자로 여겨지고 있다. 인슐린저항성을 갖는 사람에서 유리지방산이 상승되는 이상 지질 대사를 보이기 때문에 유리지방산은 인슐린 저항성에서 매우 중요하다. 유리지방산이 인슐린 저항성을 일으킨 다는 것이 잘 알려져 있긴 하지만, 유리지방산 중 어느 대사물이 인슐린 저항성에 영향을 줄 것인지는 아직까지 확실하지 않은 상태이다. 유리지방산은 체내에서ceramide, diacylglycerol (DAG) 또는 lysophosphatidylcholine (LPC) 로 바뀔 수 있는데, 최근 들어 이러한 인지질 (phospholipid) 들이 인슐린 저항성을 매개하는 것으로 여겨지고 있다. LPC는 혈중 내에서 가장 풍부한 lysophospholipid로서 주로 phospholipase A2 (PLA2) 에 의해 생성된다. 과거 우리 실험실의 결과에 따르면 (unpublished data), 쥐의 L6 근육세포에서는 ceramide 가 아니라 LPC 가 유리지방산과 인슐린 저항성을 연결하는 중요한 매개체임이 밝혀졌다. 세포 내부에서 생기는 LPC 가 간세포의 인슐린 저항성에 어떤 영향을 미치는지 확인하기 위해 Ca2+-independent phospholipase A2 (iPLA2) 억제제 처리 유무에 따라 palmitic acid (PA) 에 의한 인슐린 저항성이 어떻게 변하는지 연구하였다. iPLA2 억제제는 PA 처리에 의해 감소된Akt Ser473 phosphorylation 을 회복시켰으며, PA에 의해 유발된JNK phosphorylation 을 감소시켰다. 이는 간 세포에서 발생하는 PA 에 의한 인슐린 저항성에서 iPLA2 와 LPC 가 중요한 역할을 한다는 것을 의미한다. 반면, ceramide 합성억제제 처리 시에는 JNK phosphorylation에서 이러한 변화가 관찰되지 않아 ceramide 가 PA-induced insulin resistance 에서 직접적인 effector 가 아님을 시사한다. αi2 G203T 를 발현하는 안정된hepa1c1c7 돌연변이 세포는 유리지방산에 의한 인슐린 저항성이 잘 생기지 않았다. αi2 G203T 돌연변이에 의해 JNK and IRS-1 activation 이 감소되고 궁극적으로는 Akt Ser307 phosphorylation 회복되어 인슐린 감수성이 회복되었다. Pertussis toxin (PTX) 는 Gi type 의G-protein 에 대한 특이도가 있는 억제제로서, PA 처리 시 약화된 Akt Ser307 phosphorylation 을 회복시켰다. 종합적으로 보면, GPCR/Gi2α 는 PA 에 의한 인슐린저항성을 매개하는 수용체일 가능성이 있다. 이 연구에 의하면, LPC는 유리지방산에 의한 인슐린 저항성에 관여하며, PLA2 억제제나GPCR/Gi2α억제제가 향후 인슐린저항성에 대한 치료에 이용될 수 있음을 시사한다.

Studies on the molecular and biochemical mechanisms of candesartan, an angiotensin-II-receptor blocker, to ameliorate obesity-associated insulin resistance and hepatosteatosis

이진욱 가천대학교 대학원 2023 국내박사

Insulin resistance is a pathophysiological state of metabolic syndrome and the crucial defect leading to type 2 diabetes. Insulin resistance is strongly linked to intracellular Ca2+ overload in which excess Ca2+ inhibits insulin action by preventing membrane localization of AKT through Ca2+-phosphoinositides formation. However, whether targeting intracellular Ca2+ overload is beneficial for insulin sensitivity is still unclear. Here, we show that candesartan and azilsartan among several angiotensin-II-receptor blockers (ARBs), a class of antihypertensive agents, attenuated intracellular Ca2+ overload by inhibiting store-operated Ca2+ entry (SOCE) in palmitic acid (PA)-treated HepG2 cells, which ameliorated the impaired insulin-stimulated phosphorylation of AKT and its critical downstream substrates by promoting insulin-stimulated membrane localization of the AKT. Furthermore, candesartan administration ameliorates obesity-induced insulin resistance, hepatic steatosis, and tissue inflammation. Meanwhile, its administration normalized intracellular Ca2+ homeostasis by inhibiting SOCE, which leads to rescuing the impaired insulin signaling pathway in the liver and muscle via promoting postprandial membrane localization of AKT. Thus, these findings identify an effective therapeutic strategy by which approved drugs can regulate insulin resistance by attenuating intracellular Ca2+ overload, and underscore the contribution of dysregulated intracellular Ca2+ homeostasis to the pathophysiology of insulin resistance

인슐린 저항성 특이 골격근육 유래의 분비 단백질체 발굴 및 체계적인 연구 : Systematic Study on Skeletal Muscle Secretome under Insulin Resistance

윤종혁 포항공과대학교 일반대학원 2012 국내박사

Skeletal muscle plays major function in the regulation of energy metabolism in the whole body. Secretomic studies to identify secretory proteins from metabolic cells/tissues have detected several candidate cytokines but skeletal muscle-derived cytokines have not been studied seriously yet. This study sought to provide lists of skeletal muscle-derived secretory proteins that have the potential to play a role in insulin resistance and to characterize insulin resistance through systematic analysis of the secretomes from different causes of insulin resistance. By novel label-free methods for secretomics using G-statistics, this study found 33 insulin-modulated skeletal muscle secretory proteins. After establishment of cell-based insulin resistant condition by TNF-alpha and palmitate, subsequent studies provided the lists of insulin resistant-modulated skeletal muscle secretory proteins using reliable quantitative methods: 28 TNF-alpha-modulated secretory proteins and 42 palmitate-modulated secretory proteins. This study found candidate myokines by clear verification. In insulin-treated secretome study, MMP-2 and PAI-1 were found that are highly related with metabolic disease especially with atherosclerotic complications. In TNF-alpha-treated secretome study, clusterin, nucleobindin-2, IGFBP-4 and DJ-1 were found. Clusterin and nucleobindin-2 are novel factors to explain roles skeletal muscle in anti-atherogenic responses and food intake, relatively. Annexin A1 was screened from palmitate-treated secretome study, which able to explain insulin resistance of skeletal muscle by autocrine pathway. From individual secretome analysis, several of novel skeletal muscle-derived insulin resistant-modulated secretory proteins that have not yet been reported as the proteins related with metabolic disease were discovered: 26 of insulin-modulated, 23 of TNF-alpha modulated and 23 of palmitate-modulated secretory proteins. In systematic analysis, TNF-alpha secretome study combined analysis with transcriptome of skeletal muscle of diabetic fatty rat suggested TNF-alpha-mediated inflammation is an important cause of diabetic skeletal muscle phenotypes. In systematic study of the secretomes from three different insulin resistant conditions, 7 proteins were common and 3 proteins of these were commonly modulated by the given insulin resistant conditions (Alpha-enolase, Nucleobindin-1, Metalloproteinase inhibitor 2). Those are identified as the common insulin resistance-dependent secretory proteins. Systematic bioinformatics analysis found common terms in biological process and molecular function of functional annotation that become foothold to further characterization of insulin resistance of skeletal muscle. This study revealed that insulin, TNF-alpha and palmitate-treated skeletal muscle secrete a variety of cytokines disparately and the extracellular environment affects cytokine secretion by skeletal muscle tissue. Therefore, the lists of identified proteins provides useful information for designing further metabolic disease studies, such as studies to identify potential communication mediators involving endo-/para-/auto-crines, as well as biomarkers for irregular states like obesity induced insulin resistance.

LGALS3BP-Induced Hepatic Insulin Resistance via IRS1 Serine Phosphorylation Mediated by the TLR2-IKKα/β-NFκB Pathway

성민정 전남대학교 2025 국내박사

좌식 생활습관과 고칼로리 식단의 증가로 인해 전 세계적으로 제2형 당뇨병(T2D)과 대사기능연관 지방간질환(MASLD)과 같은 대사 질환의 유병률을 급증시키고 있다. 특히, 만성 염증은 MASLD의 주요 특징 중 하나인 인슐린 저항성을 유발하여 고혈당과 이상지질혈증을 초래하며, 이는 궁극적으로 심혈관 질환 및 간기능 장애와 같은 합병증의 위험을 증가시킨다. 이러한 염증 과정에서 Galectin-3-binding protein (LGALS3BP)은 면역 조절 및 대사 염증과 연관된 당단백질로, 간기능 이상과의 연관성이 보고되었으나, 그 구체적인 분자 기전은 명확히 규명되지 않았다. 본 연구는 대사성 염증 환경에서 간 인슐린 저항성에 미치는 LGALS3BP의 기능적 역할 및 그 기전을 규명하고자 하였다. 연구 결과, 비만을 동반한 당뇨병 환자 및 MASLD 환자의 간조직, 고지방식이(HFD) 유도 대사 질환 모델의 간 조직 및 혈청, 그리고 지방독성에 노출된 간세포주에서 LGALS3BP의 발현이 유의하게 증가함을 확인하였다. 반면, LGALS3BP 결손(KO) 마우스는 HFD 환경에서 전신 인슐린 감수성이 향상되었으며, 포도당 내성 개선, 혈당 및 인슐린 농도 감소, 그리고 간 인슐린 신호 전달 강화가 관찰되었다. 또한, LGALS3BP KO 마우스는 만성 HFD 조건에서 염증 신호전달 경로 및 염증성 사이토카인 유전자 발현이 감소하여 대사 염증이 완화되었으나, 간 내 지질 축적 및 대식세포 수 등에는 유의한 변화가 관찰되지 않았다. LGALS3BP 결핍은 인슐린 신호 전달을 강화하고 포도당 신생성 유전자 발현을 억제한 반면, 재조합 LGALS3BP는 직접적으로 인슐린 수용체 신호 전달을 저해하고, 포도당 생성을 증가시키는 것으로 나타났다. RNA-시퀀싱 분석 결과, LGALS3BP는 Toll-like receptor (TLR) 및 핵 인자 카파 B(NFκB) 신호전달 경로를 활성화하여 대사 기능 장애 및 염증을 유도하는 주요 매개체로 작용함을 확인하였다. 더욱이, LGALS3BP가 TLR2와 직접적으로 상호작용하여 염증 반응을 더욱 활성화하며, 이 과정에서 IKKα/β-NFκB 경로를 통해 IRS1 Ser307의 인산화를 증가시킴으로써 간 인슐린 저항성을 촉진하는 것으로 확인되었다. 결론적으로, 본 연구는 LGALS3BP가 TLR2 매개 IKKα/β-NFκB 신호 전달을 활성화하고 IRS1 Ser307의 인산화를 촉진함으로써 염증성 간 인슐린 저항성을 유도하는 핵심 조절자로 작용함을 규명하였다. 본 연구 결과는 LGALS3BP 표적하는 것이 염증을 효과적으로 제어함으로써 혈당 조절뿐만 아니라 간 손상 및 섬유화 진행 억제까지 포함하는 MASLD와 같은 만성 대사 질환의 포괄적인 치료를 위한 새로운 전략이 될 수 있음을 시사한다. The global prevalence of metabolic disorders, including type 2 diabetes (T2D) and metabolic dysfunction-associated steatotic liver disease (MASLD), is rapidly increasing due to sedentary lifestyles and high-calorie diets. Chronic inflammation is a key driver of insulin resistance, a hallmark of MASLD, which leads to hyperglycemia, dyslipidemia, and an increased risk of cardiovascular disease and liver dysfunction. Galectin-3-binding protein (LGALS3BP) is a glycoprotein involved in immune modulation and metabolic inflammation; however, its specific functional role and underlying molecular mechanisms in hepatic metabolic dysfunction have remained unclear. This study aims to elucidate the functional role of LGALS3BP in hepatic insulin resistance during metabolic inflammation. LGALS3BP-knockout (KO) mice and hepatic cell lines were used to examine LGALS3BP’s role in insulin resistance and inflammation. High-fat diet (HFD)- fed wild-type (WT) and KO mice were assessed for glucose metabolism and insulin signaling. Additionally, the direct effects of recombinant LGALS3BP on insulin signaling and inflammation were evaluated. RNA sequencing was performed to identify enriched pathways, and AlphaFold3 modeling combined with immunoprecipitation assays was employed to analyze LGALS3BP-Toll- like receptor (TLR2) interactions. LGALS3BP expression was significantly elevated in the livers of patients with obesity and diabetes, in HFD-induced metabolic disease models, and in hepatocytes subjected to lipotoxicity. LGALS3BP-deficient mice exhibited improved systemic insulin sensitivity under HFD conditions, enhancing glucose tolerance, reducing serum glucose and insulin levels, and increasing hepatic insulin signaling. Furthermore, LGALS3BP KO mice displayed attenuated hepatic inflammation under chronic HFD conditions, with downregulated inflammatory signaling pathways and lower expression of pro-inflammatory cytokine genes, while hepatic lipid accumulation and macrophage populations remained unaffected. LGALS3BP deficiency enhanced insulin signaling and suppressed gluconeogenic gene expression, whereas recombinant LGALS3BP directly impaired insulin receptor signaling and increased glucose production. Mechanistically, RNA sequencing identified LGALS3BP-mediated activation of the TLR and nuclear factor kappa B (NFκB) signaling pathways as a critical mediator of metabolic dysfunction and inflammation. Moreover, LGALS3BP was found to directly interact with TLR2, thereby exacerbating inflammatory responses by activating the IKKα/β-NFκB pathway, which in turn increased IRS1 Ser307 phosphorylation, a key inhibitory modification that promotes insulin resistance. In conclusion, this study identifies LGALS3BP as a critical regulator mediating inflammatory hepatic insulin resistance by activating TLR2- mediated IKKα/β-NFκB signaling, leading to IRS1 Ser307 phosphorylation. These findings suggest that targeting LGALS3BP presents a novel therapeutic strategy for chronic metabolic diseases like MASLD. This approach could extend beyond glycemic control to comprehensively treat the disease by effectively controlling inflammation, thereby suppressing liver damage and even fibrosis progression.

Adipose tissue-derived exosome affects insulin resistance by obesity in muscle cells

김유정 전남대학교 2021 국내석사

비만은 꾸준히 증가하고 있는 사회보건적 문제로 이에 따른 합병증인 제2형 당뇨병의 발병률과 사망률 또한 증가하고 있다. 과도한 지방은 인슐린 표적 조직(지방 조직, 간 및 근육)에 미토콘드리아 기능이상, 산화스트레스, 소포체스트레스, 염증, 지질 독성 등을 일으키고 이는 인슐린 저항성으로 연결된다. 특히 인슐린 작용에 의한 포도당 대사가 일어나는 근육에서의 인슐린 저항성은 전신적인 고혈당 증세를 일으킬 수 있다. 최근 지방 조직의 세포 외 소포가 인슐린 저항성 발달에 관여하는 것으로 나타났다. exosome은 세포 외 소포 중 하나로 공여자 세포의 특성을 공유하며 세포 간 소통에 중요한 역할을 한다. 이 연구의 목적은 지방 조직에서 분리한 exosome이 근육 세포주인 C2C12 세포에서 인슐린 신호전달을 변경하여 인슐린 저항성을 유도하는지 확인하는 것이다. 우리는 분화 된 3T3-L1 세포의 배양액이 분화된 C2C12 세포에서 인슐린 저항성을 유도하는 반면, 분화 된 3T3-L1에 exosome 억제제를 처리하여 얻은 배양액은 인슐린 저항성을 완화시키는 것을 발견하였다. 우리는 고지방식이를 통해 비만이 유도된 마우스의 부고환 지방을 분리, 배양하여 exosome을 분리하였다. 분리한 exosome의 크기, 표지를 확인하여 정상적인 exosome임을 확인하고 실험을 진행하였다. 고지방식이로 비만이 유도 된 생쥐의 부고환 지방에서 정상 생쥐의 부고환 지방에 비해 exosome 분비가 증가하였으며, 이 비율을 실험에 적용하였다. 고지방식이로 유도 된 비만 마우스의 지방 조직에서 추출한 exosome은 정상 식이를 한 마우스의 지방조직에서 추출한 exosome에 비해 C2C12 세포에서 직접적인 인슐린 저항성을 일으켰다. 또한 인슐린 저항성에 영향을 미칠 수 있는 근세포 지질도 측정한 결과 고지방식이로 유도 된 비만 마우스의 지방 조직에서 추출한 exosome은 C2C12 세포에서 중성 지방 축적을 증가 시켰다. 결론적으로, 비만 상태에서 지방 세포 유래 exosome이 근육 조직에서 인슐린 저항성과 지방 독성을 유발함을 시사한다. 이번 연구결과는 지방세포 유래 exosome이 인슐린 저항성 유발에 담당하는 역할과 제2형 당뇨병과 같은 비만과 대사 장애를 겨냥한 치료제 개발을 위한 잠재적 전략을 평가하였다. Obesity is a social health problem, and a steady increase in the incidence and mortality rates of obesity-associated disorders, such as type 2 diabetes mellitus (T2DM), has been reported. Several studies have reported that excess fat causes mitochondrial dysfunction, oxidative stress, endoplasmic reticulum stress, inflammation, and lipotoxicity in insulin target tissues (adipose tissue, liver, and muscles), which leads to insulin resistance. Specifically, insulin resistance in muscles, where insulin -mediated glucose metabolism occurs, can cause systemic hyperglycemia. Furthermore, adipose tissue-derived extracellular vesicles contribute towards the development of insulin resistance. Exosomes, a subtype of extracellular vesicles, have been reported to play a crucial role in intercellular communications by transporting donor cell attributes. This study aimed to determine whether adipose tissue-derived exosomes (Ad-exosomes) alter insulin signaling in the C2C12 muscle cell line and develop insulin resistance under obese conditions. We found that the culture medium of differentiated 3T3-L1 cells induced insulin resistance in differentiated C2C12 cells, whereas that from the exosome release inhibitor-treated differentiated 3T3-L1 cells improved insulin resistance. We isolated exosomes by sorting and incubating epididymal fat (eWAT) from high-fat diet (HFD)-fed obese mice, and confirmed them for the exosomal size and surface markers. The eWAT from HFD-fed obese mice was found to secrete more exosomes than normal mice-derived eWAT, and this ratio was used for experiments. Considerable insulin resistance was induced in C2C12 cells upon treatment with Ad-exosomes from HFD-fed obese mice compared to that upon treatment with Ad-exosomes from normal diet-fed mice. As myocellular lipids can influence insulin resistance, Ad-exosomes from HFD-fed obese mice were found to intensify the accumulation of triglycerides in C2C12 cells. Overall, we demonstrated that obesity-associated Ad-exosomes can develop insulin resistance and lipotoxicity in muscles. Our study assessed the role of Ad-exosomes in stimulating insulin resistance and proposes their use for development of potential therapeutic strategies targeting obesity and metabolic disorders such as T2DM.

Effect of IGBP mixture on obesity and adipose-derived exosome-induced insulin resistance

박수정 경희대학교 대학원 2021 국내박사

IGBP ameliorate insulin resistance induced by adipose-derived exosome in C2C12 cells Soo-Jeung Park Department of Medical Nutrition, Graduate School of East-West Medical Science, Kyung Hee University, Yong-in, 17104, Republic of Korea ABSTRACT Skeletal muscle insulin resistance is associated with various metabolic conditions, including obesity and hyperglycemia. In the present study, we investigated the effect of Indian gooseberry and barley sprout mix (IGBP) on insulin resistance induced by adipose-derived exosomes in C2C12 cells. We fed a 60% high-fat diet to the experimental animals for 12-weeks to induce obesity and separated their adipose tissue for exosome isolation. We confirmed exosome biosynthesis by isolating exosomes from adipose tissue-conditioned media, and treated differentiated C2C12 cells with these exosomes to determine their effect on insulin resistance. Our results confirmed the expression of CD63 and Alix, the exosome biosynthesis markers, in the adipose-derived exosomes. Our microRNA expression analysis showed that miR-27a expression increased in the exosomes of the obese control group. Increased expression of miR-27a targeted and suppressed PPARγ. This led to the decreased expression of GLUT4 by controlling IRS1, PI3K, PDK, Akt, and AS160 phosphorylation, thereby increasing insulin resistance. IGBP decreased miR-27a expression and increased PPARγ protein expression. These results influenced the phosphorylation of IRS1, PI3K, PDK, Akt, and AS160. Moreover, IGBP increased intracellular glucose influx by increasing GLUT4 expression. Therefore, our result showed that the IGBP helps inreducing insulin resistance in muscle cells. Significantly, the meaningful aspect of this study that was based on the insulin resistance-inducing effects of adipose tissue-derived exosomes. Based on our results, we suggest that IGBP could protect against obesity-induced muscle insulin resistance and may be used as a useful supplement for preventing hyperglycemia. Keywords: Phyllanthus emblica, barley sprout, insulin resistance, exosome Ellagic acid and saponarin present in IGBP ameliorate insulin resistance developed in response to exposure of C2C12 cells to adipose-derived exosomes Soo-Jeung Park Department of Medical Nutrition, Graduate School of East-West Medical Science, Kyung Hee University, Yong-in, 17104, Republic of Korea ABSTRACT An obesity-induced state increases the incidence of diabetes, and obesity adipose-derived exosomes have been found to increase the muscle insulin resistance by circulating throughout the body. In the present study, we quantitated ellagic acid and saponarin in Indian gooseberry (IG), barley sprout (BP), and IGBP, and we investigated the effect of ellagic acid, saponarin, and a mixture of the two (ES) on insulin resistance developed in response to the exposure of C2C12 cells to adipose-derived exosomes. In IGBP, the concentration of ellagic acid and saponarin was 10 ± 20% mg/g and 2.27 ± 20% mg/g, respectively. Based on the concentration of IGBP (50, 100, and 200 μg/mL) in our previous study, the C2C12 cells were treated with ES at a ratio of 4.4:1, followed by immediate stimulation with 30 μg of isolated exosomes for insulin resistance induction for 48 h. microRNA expression analysis revealed that miR-27a expression increased in the exosomes of the obese adipose tissues, thereby reducing the PPARγ expression. This resulted in a decreased glucose transporter 4 (GLUT4) expression via the regulation of IRS1, PI3K, PDK, AKT, and AS160 phosphorylation, finally resulting in increased insulin resistance. ES decreased miR-27a expression and increased PPARγ expression, and both of these phenomenon influenced the phosphorylation of IRS1, PI3K, PDK, AKT, and AS160 and were confirmed to increase the influx of glucose into cells by increasing GLUT4 expression. Therefore, ES helps reduce insulin resistance in skeletal muscle cells. In conclusion, we suggest that the effect of IGBP on insulin resistance in C2C12 cells can be attributed to the presence of ellagic acid and saponarin. Keywords: ellagic acid, saponarin, insulin resistance, exosome Anti-obese effect of IGBP on differentiated 3T3-L1 cell and high-fat diet-induced C57BL/6J mice Soo-Jeung Park Department of Medical Nutrition, Graduate School of East-West Medical Science, Kyung Hee University, Yong-in, 17104, Republic of Korea ABSTRACT The destruction of energy homeostasis due to fat accumulation leads to serious chronic diseases such as obesity. Obesity is a disease that has been found to affect diseases such as inflammation, high blood pressure, and diabetes, including insulin resistance. We investigated the anti-obese effect of indian gooseberry (Phyllanthus emblica, IG) and barley sprout (BP) mixture on differentiated 3T3-L1 cell and high-fat diet-induced C57BL/6J mice. Natural materials, IG and BP, are active against obesity and diabetes by controlling lipid metabolism, respectively. In vitro study, we differentiated 3T3-L1 cells and performed experiments corresponding to each mechanism, such as adipogenesis, lipogenesis, and lipolysis. We performed Oil red O staining, glycerol release, triglyceride contents and cAMP level measure, and western blot analysis. in vivo study, the mice were randomly divided into eight groups: normal control group, obesity-induced control group (60% high fat diet, HFD), positive control group (60% HFD + metformin 250mg/kg b.w.), IG 400 group (60% HFD + IG 400 mg/kg b.w.), BP 400 group (60% HFD + BP 400 mg/kg b.w.), IGBP groups (60% HFD + IGBP 100, 200, and 400 mg/kg b.w.). After 15 weeks, we performed a micro-CT imaging analysis, and mice were sacrificed. We measured the levels of serum triglyceride, total cholesterol, LDL/HDL cholesterol, fatty acid, and fecal triglyceride, and total cholesterol. We confirmed the cAMP level and lipid size in the white adipose tissue. We confirmed the expression of proteins related to adipogenesis, lipogenesis, lipolysis, and energy metabolism by a Western blot. The results revealed that IGBP mixture inhibited the expression of proteins related to adipogenesis and lipogenesis mechanism and regulated the expression of proteins related to lipolysis in vitro. These results reduced TG production and led to increased cAMP levels and glycerol release. Moreover, the IGBP mixture reduced weight gain, white adipose tissue (WAT) weight, serum lipid profiles, the adipose mass of systemic, subcutaneous, and visceral, and lipid size of WAT in high-fat diet-induced C57BL/6J mice. Furthermore, the IGBP mixture increased the cAMP level of adipose tissue and lipid profiles of feces and regulated the expression of proteins related to adipogenesis, lipogenesis, lipolysis, and energy metabolism in vivo study. Overall, these results suggest that the IGBP mixture may improve obesity by controlling adipogenesis, lipogenesis, lipolysis, and energy metabolism, and may be a useful substance for reducing body fat. Keywords: Phyllanthus emblica, barley sprout, obesity

Identification of Biogenic Amines, ADMA and 2-AAA as Biomarkers of Obesity and Diabetes

이효정 충북대학교 2021 국내박사

비만과 관련된 인슐린 저항성은 제2형 당뇨병과 밀접한 연관성이 있다. 제2형 당뇨병은 많은 합병증을 동반하는 심각한 만성질환이며 완치가 어려운 질환 중 하나이다. 따라서 제2형 당뇨병은 예방이 최우선이며, 조기에 발견하고 치료하는 것이 매우 중요하다. 비만과 관련된 당뇨병의 새로운 지표를 발굴하기 위해 임상자료를 기반으로 관련된 인자들을 분석해보았다. 첫번째로, 한국 소아 청소년 코호트 (KoCAS, n=430)와 지역 사회 기반 코호트 (KoGES, n=2,485)의 임상 시료를 이용하여 대사체 분석을 수행하였다. 67개의 대사체들이 청소년과 성인의 인슐린 저항성과 관련이 있었다. Asymmetric dimethylarginine (ADMA)는 청소년 비만 (p<0.001) 및 성인 당뇨병 (p=0.007)과 높은 연관성을 보였으며 비만 중재 프로그램을 통해서 역전되었다. 비만 관련 인슐린 저항성을 유도한 근육 세포와 마우스 모델에서 높은 ADMA 수치는 인슐린 저항성과 관련이 있음을 확인하였다. 근육세포에서의 ADMA 처리는 ADMA의 분해효소인 DDAH의 활성과mRNA 수치를 억제하였고, 인슐린 신호 관련 인자들의 손상을 유발하였다. 인슐린수용체, AKT, GLUT4의 인산화는 ADMA 처리했을 때 감소했고, PTP1B는 증가하였다. 또한 근육세포에서의 ADMA 처리는 포도당 흡수를 억제하였다. 두 번째로, 인슐린 저항성과 지방 형성에 관련된 대사체를 확인하기 위해, 분화전과 후의 지방세포에서 대사체 분석을 수행하였다. 변화를 보인 대사체 중에서 2-aminoadipic acid (2-AAA)는 분화 후의 지방세포에서만 검출되었다. KoCAS 소아 청소년 코호트 자료 분석을 통해서, 2-AAA는 baseline과 2년 후의 추적연구자료에서 비만 관련 요인들인 BMI, BMI z-score, 체지방량, 지방 비율, 허리둘레, 중성지방, 인슐린 및 HOMA-IR과 양의 상관관계가 있음을 확인하였다. 특히 baseline에서 BMI z-score, HOMA-IR과 2-AAA는 가장 높은 연관성을 보였으며, BMI z-score는 2년 후의 추적연구분석에서도 높은 연관성을 나타내었다. 두 개의 독립적인 비만 중재 프로그램 자료 분석을 통해서, 2-AAA는 BMI z-score와 HOMA-IR과 상관관계가 있음을 확인하였다. 또한, 비만 관련 인슐린 저항성을 유도한 세포 및 마우스 모델에서 2-AAA 수치가 대조군과 비교하여 증가하였다. 대표적인 인슐린 민감성 세포들인 간, 골격근, 지방세포에서 2-AAA 처리는 인슐린 신호 관련 인자들의 손상과 비정상적인 포도당 신생합성을 유발하였다. 본 연구에서 아미노산 대사산물인 ADMA와 2-AAA가 비만성 인슐린 저항성과 관련이 있음을 발견하였다. 우리는 ADMA와 중증 비만 및 당뇨병 위험과 관련이 있음을 확인하였고, 2-AAA는 비만 및 비만 중재 효과와 관련이 있음을 확인하였다. 이 결과는 ADMA와 2-AAA의 양을 개선시키는 것이 비만이나 당뇨병의 치료전략으로 활용될 수 있음을 시사한다. Obesity-induced insulin resistance is an important clinical feature of diabetes, which is a serious chronic disease with complications. Therefore, early diagnosis and intervention strategies for diabetes are important. To determine the novel factors related to obesity and diabetes, we investigated metabolic factors based on clinical data. In the first part of study, targeted metabolomics analysis was performed with samples from Korean Children-Adolescent Cohort Study (KoCAS)(N=430) and Korean Genome and Epidemiology Study (KoGES, n=2,485) to unravel the metabolic factors of obesity-induced insulin resistance. Sixty-seven metabolites were identified to be significantly related to insulin resistance in adolescents and adult population. Asymmetric dimethylarginine (ADMA), which is a biogenic amine, showed high association with adolescent obesity (P<0.001) and adult diabetes (P=0.007) and its levels were reversed after obesity intervention program. Functional studies in cellular and mouse models of obesity-mediated insulin resistance proved that ADMA accumulation regulates insulin resistance in skeletal muscle. Treatment of excessive ADMA suppressed activity and mRNA level dimethylarginine dimethylaminohydrolase (DDAH), an ADMA-degrading enzyme and induced impairment of insulin signaling-related factors in insulin resistance-induced muscle cells. ADMA treatment decreased phosphorylation of insulin receptor (IR), AKT, and GLUT4 but increased protein-tyrosine phosphatase 1B (PTP1B), leading to inhibition of glucose uptake in myotube cells. For the second part, metabolite profiling was performed in differentiated adipocytes to identify a novel metabolic determinants of insulin resistance and adipogenic differentiation. Among the metabolites, 2-aminoadipic acid (2-AAA) was associated with adipogenic differentiation. In addition, circulatory 2-AAA was positively correlated with obesity-related factors such as BMI, BMI z-score, fat mass, fat percentage, waist circumference, triglycerides, insulin, and HOMA-IR at baseline and after 2 years in the children cohort study (KoCAS). Especially, increased BMI z-score and HOMA-IR showed highest association with baseline 2-AAA levels. BMI z-score also showed the highest association with baseline 2-AAA levels in the 2-years follow-up study. Analyses of 2 independent obesity intervention programs showed that during the intervention period, 2-AAA levels were positively associated with BMI z-score and HOMA-IR after adjusting for confounders. In addition, 2-AAA levels were elevated by obesity-induced insulin resistance in cellular and mouse models. Treatment of excessive 2-AAA induced impairment of insulin signaling and abnormal gluconeogenesis in insulin-sensitive cells such as liver cells, skeletal muscle cells, and adipose cells. In conclusion, this study suggests that the amino acid metabolites, ADMA and 2-AAA are associated with obesogenic insulin resistance. We show an association of ADMA with the risk of severe obesity and diabetes and the relationship of 2-AAA to the effects of obesity intervention. In addition, our data suggests that enhancing ADMA and 2-AAA levels could be developed as a strategy to treat obesity or diabetes.

Anti-Diabetic Effects and Mechanisms of HM-chromanone Isolated from Portulaca oleracea L.

Park, Jae Eun 부산대학교 2022 국내박사

Diabetes mellitus is a chronic metabolic disease, in which blood glucose rises due to decreased insulin secretion, insulin resistance, or a combination of these two factors. In currently used drug therapy, most of them have restrictions on their use due to side effects, and there is a need to develop a substance for preventing diabetes from natural products without side effects. Portulaca oleracea L. is an annual plant, which is used as a medicinal herb. It has shown some effectiveness as an anti-diabetes candidate. However, it is not clear which component plays the role in P. oleracea. Thus, it was isolated (E)-5-hydroxy-7-methoxy-3-(2′-hydroxybenzyl)-4-chromanone (HM-chromanone) from P. oleracea by bioassay-guided fractionation and HPLC. Anti-diabetic effects and mechanisms of HM-chromanone were investigated, which consisted of insulin sensitivity and insulin resistance improvement effects and mechanisms in cellular system, hyperglycemia and dyslipidemia improvement effects in diabetic animal models, and INS-1 pancreatic β cell protective effect in cellular system and diabetic animal models. HM-chromanone increased glucose uptake through the activation of PI3K/AKT and CaMKKβ-AMPK signaling pathway, and promoted glycogen synthesis by increasing GS activation via the induction of GSK3 α/β phosphorylation in L6 skeletal muscle cells. HM-chromanone decreased PTP1B, JNK, and IKKβ, which are insulin resistance inducers induced by palmitate, and then upregulated the phosphorylation of IRS-1tyr, and restored the PI3K and AKT pathway activation in palmitate-induced insulin-resistant L6 skeletal muscle cells. In addition, HM-chromanone reduced hyperglycemia by improving insulin resistance-related genes in skeletal muscle, and improved dyslipidemia by regulating genes related to lipogenesis in liver of diabetic mice. HM-chromanone also dose-dependently improved fasting blood glucose levels and alleviated pancreatic islet cell damage in STZ-induced diabetic mice. Furthermore, HMC reduced ROS levels and increased the activities of antioxidant enzymes, thus protecting the pancreatic islet cells and consequently increasing insulin secretion and decreasing hyperglycemia. HM-chromanone promoted insulin secretion in INS-1 pancreatic β cells pretreated with high glucose and suppressed hyperglycemia-induced apoptosis. Therefore, these results suggest that HM-chromanone could have the potential as a useful material for the prevention and treatment of diabetes mellitus by investigating the improvement effect and mechanism of action of HM-chromanone.

옥덩굴의 RAW 264.7 대식세포와 3T3-L1 지방세포에서 비만에 의해 유도된 염증 및 인슐린 저항성 개선 효과

마난다 비카쉬 목포대학교 대학원 2018 국내석사

Obesity and metabolic syndrome are leading health issues in the world. Chronic obesity induces inflammation, and leads to the development of insulin resistance in adipocytes and ultimately metabolic syndrome (Type 2 diabetes, hypertension, cardiovascular diseases). Hence, the drugs that could target both obesity and obesity-induced insulin resistance can prevent or treat metabolic disorders without side effects of weight gain. Medicinal plants are currently being investigated for such bio-active compounds. However, only few such compounds have been reported yet. Marine plants have been extensively studied for their potential anti-obesity and anti-diabetic activity. Caulerpa okamurae is a green seaweed, eaten as food particularly in Japan, China, South Korea and Australia. Our previous results suggested that the ethanol extract of C. okamurae (COE) significantly inhibits lipid accumulation by inhibition of peroxisome proliferator-activated receptor-γ (PPARγ) and CCAAT/enhancer binding protein alpha (C/EBPα) expressions in 3T3-L1 adipocytes. Also, these results showed that COE decreased the body weight gain, adipose tissue weight, and plasma and hepatic lipid profiles in high fat diet-induced mice. In this research, we investigated the molecular mechanism for anti-adipogenic effect of COE in 3T3-L1 adipocytes and we further examined if COE can inhibit inflammation, regulate glucose metabolism and increase insulin sensitivity using cell culture models of RAW 264.7 macrophages and 3T3-L1 adipocytes. After adipogenic differentiation of 3T3-L1 adipocytes, COE treated group showed overexpression of β-catenin, which was accompanied by reduction in glycogen synthase kinase (GSK3β) expression, compared to control group. In LPS-induced RAW 264.7 macrophages, COE significantly downregulated nitric oxide (NO) production, protein expression of nitric oxide synthase 2 (NOS2), and nuclear translocation of nuclear factor-κB (NF-κB). In co-culture model, COE significantly reduced NO production and protein expression of NOS2 and insulin receptor substrate 1 phosphorylated at serine 307 [p-IRS1 (Ser307)]. Also, COE significantly improved glucose uptake levels by upregulating the protein expression of glucose transporter type-4 (GLUT-4) and adiponectin. In addition, mRNA expressions of pro-inflammatory cytokines, such as TNF-α, interleukin-6 (IL-6) and monocyte chemoattractant protein (MCP-1), were significantly inhibited with COE treatment in co-culture. In TNF-α–induced insulin resistance model of 3T3-L1 adipocytes, COE significantly improved glucose uptake with or without insulin. COE significantly increased the protein expression of insulin receptor substrate 1 phosphorylated at tyrosine 632 [p-IRS1 (Tyr632)], GLUT-4, phospho- 5' adenosine monophosphate-activated protein kinase alpha (p-AMPKα) and phospho-glycogen synthase kinase 3 beta (p-GSK3β) in presence of insulin. These findings suggest that COE inhibits lipid accumulation in 3T3-L1 adipocytes through the activation of Wnt/β-catenin pathway. On the other hand, the results suggest that COE improves glucose metabolism by inhibiting inflammatory interaction in co-culture of RAW 264.7 macrophages and 3T3-L1 adipocytes, and recovers insulin sensitivity in TNF-α-induced insulin resistance in 3T3-L1 adipocytes through possible inhibition of GSK3β or activation of AMPKα. Thus, COE may provide as potential nutritional supplement or alternative medicine to prevent and/or cure obesity and obesity-induced metabolic disorders in the future. 비만과 대사 증후군은 세계적으로 건강 문제로 대두되고 있다. 만성 비만은 염증을 유도하고 지방 세포 및 대사 증후군 (제2 형 당뇨병, 고혈압, 심혈관 질환)에서 인슐린 저항성의 발달로 이어진다. 따라서 비만과 비만에 의해 유도된 인슐린 저항성을 목표로 할 수 있는 약물은 체중 증가의 부작용없이 대사 장애를 예방하거나 치료할 수 있어야한다. 현재 약용 식물을 이용하여 생체활성 화합물에 대해 조사되고 있지만, 이런 화합물에 대해서는 아직까지 보고 된 바가 거의 없다. 우리는 해양식물이 잠재적으로 항비만 및 항당뇨 활성에 대해 광범위하게 연구되고 있는 점에 관심을 갖고, 그 중에서 아열대 해조류로 일본, 중국, 한국 및 호주에 분포되어 있으며, 일본에서는 식용으로 사용되고 있는 Caulerpa okamurae를 이용하여 연구하였다. 우리는 이전의 연구결과에서 C. okamurae (COE)의 에탄올 추출물이 3T3-L1 지방세포에서 peroxisome proliferator-activated receptor-γ (PPARγ)와 CCAAT/enhancer binding protein alpha (C/EBPα) 억제에 의해 지방 축적이 억제되는 것을 확인하였다. 또한, COE가 고지방식이 유도 마우스에서 체중 증가 및 지방 조직 중량, 혈장, 간 지질 프로파일을 감소시켰다. 이러한 결과를 토대로 본 연구에서는 3T3-L1 지방세포와 RAW 264.7 대식세포, 공동 배양 모델을 이용하여 아열대 해조류 옥덩굴 (Caulerpa okamurae) 에탄올 추출물 (COE)의 비만 및 비만으로부터 발생되는 만성 염증과 인슐린 저항성 개선 효과에 대해 분자 메커니즘적으로 연구 및 당대사를 조절, 인슐린 감수성에 미치는 영향을 검토하였다. 그 결과, 첫 번째로 3T3-L1 지방세포에서 COE 처리 군은 대조군에 비해 glycogen synthase kinase (GSK3β)의 발현이 감소하였고, 동시에 β- catenin는 과다 발현 하는 것을 확인하였다. 두 번째로 LPS로 유도된 RAW 264.7 대식세포에서 COE는 nitric oxide (NO) 생산과 nitric oxide synthase 2 (NOS2)의 발현, nuclear factor-κB (NF-κB)의 nuclear translation을 감소시켰다. 세 번째로 공동 배양모델에서 COE는 NOS2의 단백질 발현과 NO의 생성, phospho-insulin receptor substrate 1의 serine 307 [p-IRS1 (Ser307)]를 인산화를 유의적으로 감소 시켰으며, glucose transportoer-4 (GLUT-4) 및 adiponectin의 단백 발현을 효과적으로 조절함으로써 당 섭취능을 증강시키는 것으로 확인되었다. 또한, TNF-α, interleukin-6 (IL-6) 및 monocyte chemoattractant protein (MCP-1)과 같은 염증성 사이토카인의 mRNA 발현은 공동 배양에서 COE 처리로 유의적으로 억제되었다. 마지막으로 TNF-α로 유도된 인슐린 저항성 모델인 3T3-L1 지방 세포에서 COE는 인슐린 유무에 상관없이 유의적으로 당 섭취능을 개선시켰으며, phospho-insulin receptor substrate 1의 tyrosine 632 [p-IRS1 (Tyr632)], GLUT-4, phospho- 5' adenosine monophosphate-activated protein kinase alpha (p-AMPKα) 및 phospho-glycogen synthase kinase 3 beta((p-GSK3β) 의 단백질 발현을 유의적으로 증가시켰다. 이러한 결과는 COE가 Wnt/β-catenin 신호전달의 활성화를 통해 3T3-L1 지방세포에서 지방축적을 억제하는 것을 나타내고 있다. 한편, COE는 RAW 264.7 대식세포와 3T3-L1 지방세포의 공동 배양에서 염증의 상호 작용을 억제함으로써 당대사를 향상시키고, TNF-α로 유도된 인슐린 저항성 모델인 3T3-L1 지방 세포에서 GSK3β의 억제와 AMPKα의 활성화를 통해서 인슐린 감수성을 회복시켰다. 따라서 COE는 비만 및 비만으로부터 유래되는 만성염증과 인슐린저항성을 치료하는데 있어 효과적인 약물이자 2형 당뇨 치료약물의 부작용으로 잘 알려진 체중증가를 해소시킬 수 있는 잠재적인 후보약물로서의 가치가 매우 크다고 사료된다.

TAZ 활성제로 알려진, TM-25659가 고지방 식이로 유도된 인슐린 저항성 마우스에 미치는 효과

정종갑 아주대학교 2016 국내박사

2-butyl-5-methyl-6-(pyridine-3-yl)-3-[2'-(1H-tetrazole-5-yl)-biphenyl-4-ylmethyl]-3H-imidazo[4,5-b]pyridine] (TM-25659), known as the TAZ activator, inhibits adipocyte differentiation by interacting with peroxisome proliferator-activated receptor gamma (PPAR-γ). TM-25659 was previously shown to decrease weight gain in a high fat (HF) diet induced obesity (DIO) mouse model. However, the fundamental mechanisms underlying the effects of TM-25659 remain unknown. Therefore, we investigated the molecular mechanisms underlying the contribution of TM-25659 on palmitate (PA)-induced insulin resistance in both C2 myotubes and HepG2 cells. TM-25659 improved the PA-induced insulin resistance in C2 myotubes. TM-25659 induced expression of FGF21 mRNA and protein and secretion of FGF21 in C2 myotubes via activation of GCN2 pathways (GCN2-phosphor-eIF2α-ATF4 and FGF21). This effect of TM-25659 was diminished by FGF21 siRNA. Furthermore, TM-25659 improved the PA-induced insulin resistance in HepG2 cells. TM-25659 induced activation of AMPK in HepG2 cells. This beneficial effect of TM-25659 was diminished by AMPK inhibitor, compound C. Additionally, we studied the effects of TM-25659 on HF-diet induced insulin resistance in C57BL/6J mice. C57BL/6J mice were fed a HF-diet for 6 weeks. The HF group was randomly divided into two groups for the next 8 weeks: HF and HF+TM-25659. The HF+TM-25659-treated mice showed improvements in their obesity, fasting blood glucose and insulin levels, serum lipid levels, glucose homeostasis, insulin sensitivity, insulin resistance, and inflammation, but food intake was not affected. TM-25659 increased expressions of both FGF21 mRNA and protein on HF-diet induced insulin resistance in C57BL/6J mice muscle. Also, TM-25659 induced activation of AMPK protein on HF-diet induced insulin resistance in C57BL/6J mice liver. These data indicate TM-25659 may be beneficial for treating insulin resistance by inducing FGF21 and AMPK activation in models of PA-induced insulin resistance and HF-diet induced insulin resistance.