Betulinic Acid Stimulates Glucose Uptake through the Activation of PI3K and AMPK in 3T3-L1 Adipocytes

Jung Kyung Lee(이정경),Jae Eun Park(박재은),Ji Sook Han(한지숙) 한국생명과학회 2022 생명과학회지 Vol.32 No.10

제 2형 당뇨병은 고혈당을 특징으로 하는 만성 대사성 질환으로 인슐린 민감성및 저항성을 개선하여 세포속으로 포도당 흡수를 촉진시킴으로서 완화될 수 있다. 본 연구는 triterpenoid 화합물인 betulinic acid가 3T3-L1 지방세포에서 인슐린 신호전달체계를 개선하여 포도당 흡수를 촉진시키는지를 조사하고 그 작용기전을 규명하였다. Betulinic acid는 3T3-L1 지방세포에서 농도의존적으로 포도당 흡수를 유의하게 증가시켰으며, 이는 PM-GLUT4의 발현 증가와 관련이 있음을 관찰하였다. Betulinic acid는 인슐린 신호전달 경로에서 PI3K의 활성화 및 IRS-1tyr, Akt의 인산화를 대조군에 비해 유의하게 증가시켰다. 또한 AMPK의 활성화를 나타내는 pAMPK와 AMPK 하위인자인 pACC의 수준을 유의하게 증가시켰다. Betulinic acid에 의한 PI3K 및 AMPK 경로의 활성화를 증명하기 위해, PI3K 억제제(Wortmannin)와 AMPK의 억제제(Compound C)를 사용하여 이들 처리에 의한 포도당 흡수능과 PM-GLUT4의 발현을 측정한 결과 이들의 발현이 유의하게 저해되었다. 본 연구에서 betulinic acid는 3T3-L1 지방세포에서 PI3K 및 AMPK 경로의 활성화를 통해 세포막으로 포도당 수송체인 GLUT4 전위를 촉진시키고 포도당 흡수를 증가시킬 수 있음을 나타내었다. 이러한 결과는 betulinic acid가 인슐린 민감성을 증진시키고 고혈당을 완화하는데 도움이 될 수 있음을 시사한다. Hyperglycemia in type 2 diabetes can be alleviated by promoting cellular glucose uptake. Betulinic acid (3β,-3-hydroxy-lup-20(29)-en-28-oic acid) is a pentacyclic lupane-type triterpenoid compound. Although there have been studies on the antidiabetic activity of betulinic acid, studies on cellular glucose uptake are lacking. We investigated the effects of betulinic acid on glucose uptake and its mechanism of action in 3T3-L1 adipocytes. Betulinic acid significantly stimulated glucose uptake in 3T3-L1 adipocytes by increasing the phosphorylation of the insulin receptor substrate 1-tyrosine (IRS-1tyr) in the insulin signaling pathway, which in turn stimulated the activation of phosphoinositide 3-kinase (PI3K) and the phosphorylation of protein kinase B (Akt). The activation of PI3K and Akt by betulinic acid translocated glucose transporter 4 to the plasma membrane (PM-GLUT4), thereby increasing the expression of PM-GLUT4 and thus stimulating cellular glucose uptake. Betulinic acid also significantly increased the phosphorylation/activation of AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase. The activation of PI3K and AMPK by betulinic acid was confirmed using the PI3K inhibitor wortmannin and the AMPK inhibitor compound C. The increase in glucose uptake induced by betulinic acid was significantly decreased by wortmannin and compound C in the 3T3-L1 adipocytes. These results suggest that betulinic acid stimulates glucose uptake by activating PI3K and AMPK in 3T3-L1 adipocytes.

Betulinic acid 등의 세포독성 조절 연구

염영나,조현영,김현석,황명실,윤은경,이효민,김승희,양지선,양기화 식품의약품안전청 2001 식품의약품안전청 연보 Vol.5 No.-

과일이나 식물체 등애 천연적으로 존재하면서 돌연변이억제나 항산화효과 등, 암이나 성인병 등에 대한 예방효과를 보이는 것으로 알려진 phytocherilical을 중심으로 aminopept건ase 활성 억제 능력을 조사하여 암전툴과정과 혈관신생과정에서 발된이 증가하는 것으로 알려진 arnjnoepeptidase N APh억제제를 스크리닝하고 암전이 예방이나 암치료에 활용하고자 본 연구를 수행하게 되었다 Leucine andnopeptidase에 대한 식물유래성분(betulinic acid, curcvmin, indomethacin, ellagic acid quercetin, nocodazole, urso:ic acid, resveratrol, h)'pericin, caffeic acid)의 찰성억제능력을 조사하여 그중 촬성억제능력이 탁월한 curcundn, indomethacin, betulinlc 3c펀의 APN enzyme netics를 수헝 하였다. Curcumin의 andnopeptidase 띠 활성을 억제하는 농도와 혈관신생의 억제 농도가 일치하고 APN 단클론항체 처리한 HT1080 세포의 유세포측정결과 curcumin·라 APN 단클론항체가 경정적으로 APN예 결합됨을 확인함에 따라 curcumin이 암전이를 억제하는 기전이 APN 억제에 기인한 것으로 추정되는 결과이다. 한편 Betulinic ac,김는 APN 활성에는 영향을 미치지 못하고, 미토콘드리아의 막전 위를 떤화시키고 caspase근 활성을 증가시켜 apoptosis를 유발하였다. 포한 betulinic acid로 tlibe formation assay와 」'n ufuo CAM assay를 수행한 결과 혈관신생과정을 억제하는 것이 확인되었다 따라저 Betulinic 3fid와 curcumin은 서로 작용기전은 다르지만, 암전이 떼방이나 암치료에 활용가치가 높은 약물로 인식되었다. This study is to evaluate phytochemicals that have inhibitory activity on aminopeptidase N (APN) which is transmembrane metalloprotease to play functional role in matrix degradation and invasion by tumor cells. At first, we did screening of APN inhibitors among the phytochemicals with chemopreventive effect including betulinic acid, curcumin, indomethacin, ellagic acid, quercetin, nocadazole, ursolic acid, reveratrol, hypericin, and caffeic acid etc. by the fluorometric assay using L-alanine 4-methylcoumaryl-7-amide(Ala-AMC) as a substrate. Curcumin has inhibitory effect on APN activity as well as angiogenesis in a same condition(IC_(50) value=about 10μM). Curcumin on APN activity works as a noncompeptitive ingibitor. FCM analysis shows the competitive binding of curcumin and monoclonal antibody of APN to HT1080 cell. This results means that curcumin blocks the angiogenesis by the mechanism of APN inhibition. Betulinic acid which is working as a inhibitor of Leu aminopeptidase, dose not inhibit the aminopeptidase N activitiy. But betulinic acid blocks angiogenesis from the results of tube formation assay and in vivo CAM assay. The angiogenesis blocking mechanism of betulinic acid is supposed to induce apoptosis. These results suggest that curcumin and betulinic acid can be used as angiogenesis blocker and chemopreventive agent of cancer despite of different mechanism.

C2C12 근아세포에서 산화 스트레스에 의한 세포사멸 및 DNA 손상 유도에 대한 betulinic acid의 보호 효과

김다혜,홍수현,최영현 한국영양학회 2025 Journal of Nutrition and Health Vol.58 No.3

Purpose: Betulinic acid, a naturally occurring pentacyclic triterpenoid, has various pharmacological properties, including antioxidant, anti-inflammatory, and antitumor activities. On the other hand, its protective effects against oxidative injury in skeletal muscle cells are poorly understood. This study examined whether betulinic acid can attenuate oxidative stress-induced cytotoxicity in immortalized mouse myoblast C2C12 cells. Methods: C2C12 cells were treated with hydrogen peroxide (H2O2) to mimic an oxidative environment. The protective effects of betulinic acid against H2O2-induced cytotoxicity were investigated to determine if they were related to inhibition of apoptosis, DNA damage, and autophagy. The antioxidant activity of betulinic acid was assessed by examining its inhibitory effects on H2O2-induced reactive oxygen species (ROS) production. Results: Betulinic acid significantly attenuated H2O2-induced cytotoxicity in C2C12 cells, primarily by suppressing apoptosis. Betulinic acid also prevented the H2O2-induced increase in the Bax/Bcl-2 ratio, inhibited cytochrome c release into the cytoplasm, and suppressed caspase activation accompanied by fragmentation of poly (ADP-ribose) polymerase. In addition, betulinic acid markedly reduced H2O2-induced DNA damage, including the levels of DNA damage markers and oxidized nucleosides, and downregulated autophagy-related markers, inhibiting H2O2-induced autophagy. Betulinic acid effectively blocked H2O2-induced ROS generation, including mitochondrial superoxide, suggesting a role in maintaining mitochondrial homeostasis and preventing apoptotic signaling. Conclusion: Butulinic acid is a promising protective agent against oxidative damage in skeletal muscle cells. Nevertheless, further studies, including animal experiments, are needed to fully elucidate the mechanisms through which betulinic acid preserves mitochondrial function and biogenesis under oxidative stress.

호흡기 상피세포에서 MUC5AC와 MUC5B 발현에 대한 Betulinic Acid의 효과

김훈성,최윤석,이준혁,박나경,박창휘,이영하,김귀옥,송시연,배창훈,이승호,김용운,김용대 대한이비인후과학회 2014 대한이비인후과학회지 두경부외과학 Vol.57 No.8

Background and Objectives MUC5AC and MUC5B are representative secretory mucin genes in the human airway, whose expressions are increased by a variety of inflammatory mediators. Betulinic acid, a naturally occurring pentacyclic triterpenoid, is known to have an anti-inflammatory property. However, the effects of betulinic acid on mucin secretion of airway epithelial cells still have not been reported. Therefore, in this study, the effect of betulinic acid on inflammatory mediators-induced MUC5AC and MUC5B expressions was investigated in human airway epithelial cells. Materials and Method In the mucin-producing human NCI-H292 airway epithelial cells, the effects of betulinic acid on interleukin-1β (IL-1β)-, lipopolysaccharide (LPS)-, and phorbol myristate acetate (PMA)-induced MUC5AC and MUC5B expressions were analyzed by reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. Results Betulinic acid attenuated IL-1β-, LPS-, and PMA-induced MUC5B mRNA and glycoprotein expression in NCI-H292 cells. On the other hand, betulinic acid did not attenuate IL-1β-, and LPS-, but induced PMA-induced MUC5AC mRNA and glycoprotein expressions in NCI-H292 cells. Conclusion These results suggest that betulinic acid attenuates IL-1β-, LPS-, and PMA-induced MUC5B expression in the airway epithelial cells. Therefore, betulinic acid may modulate a control of mucus-hypersecretion in airway inflammatory diseases. Korean J Otorhinolaryngol-Head Neck Surg 2014;57(8):526-32

Betulinic Acid Inhibits LPS-Induced MMP-9 Expression by Suppressing NF-kB Activation in BV2 Microglial Cells

( Sung Soo Kim ),( Jae Won Lee ),( Yong Joon Choi ),( Sue Young Lee ),( Sang Soo Kang ),( Nam Ho Kim ),( Yong Soo Kwon ),( Hee Jae Lee ),( Wan Joo Chun ) 한국응용약물학회 2011 Biomolecules & Therapeutics(구 응용약물학회지) Vol.19 No.4

Aberrant activation of microglia has been reported to cause neuronal damages by releasing a variety of pro-infl ammatory cytokines. Besides where microglia become active, damages have been also observed in remote places, which is considered due to the migration of activated microglia. Therefore, an agent that could suppress abnormal activation of microglia and their subsequent migration might be valuable in activated microglia-related brain pathologies. The objective of the present study was to evaluate anti-infl ammatory effects of betulinic acid on lipopolysaccharide (LPS)-stimulated BV2 microglial cells. Pretreatment of betulinic acid signifi cantly attenuated LPS-induced NO production and protein expression of iNOS. Betulinic acid also signifi cantly suppressed LPS-induced release and expression of cytokines such as TNF-α and IL-1β. Furthermore, betulinic acid signifi cantly suppressed LPS-induced MMP-9 expression, which has been suggested to play an important role in the migration of activated microglia. In order to understand the possible mechanism by which betulinic acid suppresses LPS-induced cytokine production and migration of microglia, the role of NF-kB, a major pro-infl ammatory transcription factor, was examined. Betulinic acid signifi - cantly suppressed LPS-induced degradation of IKB, which retains NF-kB in the cytoplasm. Therefore, nuclear translocation of NF-kB upon LPS stimulation was signifi cantly suppressed with betulinic acid. Taken together, the present study for the fi rst time demonstrates that betulinic acid possesses anti-infl ammatory activity through the suppression of nuclear translocation of NF-kB in BV2 microglial cells.

인간 전립선 암세포에서 DNA 손상, 세포주기 교란, 세포 사멸 유도 및 침윤성 억제를 통한 베툴린산의 항암 활성

김소영,황보현,최영현 한국생명과학회 2024 생명과학회지 Vol.34 No.11

다양한 식물에서 발견되는 루핀형 펜타사이클릭 트리테르펜(lupine-type pentacyclic triterpene) 계열에 속하는 천연물인 베툴린산은 항암 활성을 포함하여 광범위한 약리학적 잠재력을 가지고 있는 것으로 알려져 있다. 그러나 인간 전립선 암세포에서 베툴린산의 항암 활성 관련 기전 연구는 거의 보고된 바 없다. 본 연구에서는 베툴린산이 전립선 암세포(PC-3, DU145 및 LNCaP 세포)의 증식을 억제하였지만, 세포주기 교란에 미치는 영향은 전립선 암세포의 종류에 따라 다른 양상을 보여주었다. 베툴린산 처리 농도 의존적으로 G1기 세포의 빈도는 감소되면서 S 및 G2/M기에 속하는 세포의 빈도를 증가시킨 PC-3 세포를 대상으로 이루어진 결과에 의하면, 베툴린산에 의한 세포 생존율의 감소는 DNA 손상 유도와 연관이 있었다. 아울러 베툴린산은 PCNA와 cyclins 및 Cdks의 발현을 모두 감소시켰지만, p21의 발현은 증가시켰으며, PC-3 세포는 p53 유전자가 결손된 세포주이기 때문에 p21의 발현 증가는 p53 비의존적인 현상임을 알 수 있었다. 그리고 베툴린산은 미토콘드리아에서 세포질로 cytochrome c의 유리와 Bax/Bcl-2의 발현 비율을 증가시키면서 caspase-3의 활성 증가에 따른 PARP의 분해를 통해 세포 사멸을 유도하였다. 아울러 베툴린산은 colony 형성, 세포의 이동성 및 침윤성을 모두 감소시켰으며, 이는 CD147, MMP-2 및 MMP-9의 발현 감소와 관련성이 있었다. 추가적으로 EMT 관련 단백질들의 발현을 변화시켰지만, 이에 관한 연구는 추가로 진행되어야 할 사항이다. 본 연구의 결과는 베툴린산이 전립선 암세포에서 DNA 손상, 세포주기 교란, 세포 사멸 유도 및 세포의 침윤성 억제를 통하여 항암 활성을 나타낸다는 것으로 보여주는 결과이며 베툴린산의 항암 활성을 밝히는 기초 자료로서 활용성이 높을 것으로 생각한다. Betulinic acid, a lupine-type pentacyclic triterpene, exhibits broad pharmacological potential, including anticancer properties. However, the mechanisms underlying its anticancer effects in human prostate cancer cells remain underexplored. This study investigated the effect of betulinic acid on the proliferation of prostate cancer cells. The results demonstrated that betulinic acid significantly inhibited the proliferation of prostate cancer cells (PC-3, DU145, and LNCaP cells), with cell cycle disruption effects varying by cell line. In PC-3 cells, the reduction in cell viability was found to be associated with the induction of DNA damage. Betulinic acid also decreased the expression of proliferating cell nuclear antigen, cyclins, and cyclin-dependent kinases (Cdks) while increasing the expression of Cdk inhibitor p21WAF1. Additionally, betulinic acid triggered apoptosis via poly(ADP-ribose) polymerase degradation, caspase-3 activation, cytosolic release of cytochrome c, and an increased Bax/Bcl-2 ratio. Moreover, betulinic acid reduced colony formation, cell motility, and invasiveness by downregulating CD147 and matrix metalloproteinases, as well as modulating the expression of key proteins involved in regulating epithelial–mesenchymal transition. Collectively, the results indicate that betulinic acid exerts anticancer activity through DNA damage induction, cell cycle disruption, apoptosis, and invasion inhibition in prostate cancer cells. This study provides valuable insights into the anticancer potential of betulinic acid.

인간 신세포암 세포에서 betulinic acid에 의한 세포 사멸, DNA 손상 및 G2/M기 세포주기 정지의 유도와 ROS 생성과의 연관성

김성옥(Sung Ok Kim),손변우(Byunwoo Son),이상협(Sang-Hyup Lee),홍수현(Su Hyun Hong),홍상훈(Sang Hoon Hong),최영현(Yung Hyun Choi) 한국생명과학회 2025 생명과학회지 Vol.35 No.2

Betulinic acid, a member of the lupin-like pentacyclic triterpene family found in a variety of plants, is known to have various physiological potentials, including anticancer activity. Although the effects of betulinic acid on the proliferation of human renal cell carcinoma (RCC) cells have been reported, the exact mechanism has not yet been specifically studied. In this study, we performed additional mechanistic studies on the anticancer activity of betulinic acid in RCC Caki-1 cells. Our results showed that betulinic acid significantly inhibited the cell viability of Caki-1 cells in a dose-dependent manner, which was closely related to the induction of apoptosis through the activation of caspase-3. In addition, the expression of the DNA strand break biomarker and the production of the oxidative DNA damage indicator were increased in cells exposed to betulinic acid. Betulinic acid also blocked the proliferation of Caki-1 cells in the G2/M phase of the cell cycle by decreasing the expression of cyclin A and cyclin B1 and increasing the expression of tumor suppressor p53 and cyclin-dependent kinase (Cdk) inhibitor p21WAF1/CIP1. Additionally, the increased p21 enhanced the binding affinity to Cdks. Furthermore, betulinic acid treatment contributed to mitochondrial dysfunction by increasing the ratio of Bax/Bcl-2 expression, which led to cytochrome c release from thr mitochondria to the cytosol. These anticancer activities of betulinic acid observed in Caki-1 cells were found to be associated with reactive oxygen species (ROS) production, and in particular, increased mitochondrial ROS production may act as an upstream regulator of the initiation of this anticancer activity.

산화 스트레스로 인한 HaCaT 인체 각질세포의 세포 손상에 대한 Betulinic Acid의 억제 효과

김다혜(Da Hye Kim),최영현(Yung Hyun Choi) 한국식품영양과학회 2024 한국식품영양과학회지 Vol.53 No.11

Oxidative stress influences the onset and progression of chronic diseases in various organs, including the skin. Betulinic acid, a member of the naturally occurring lupine-type pentacyclic triterpene family, has broad pharmacological potential, including antioxidant activity. However, the molecular mechanisms by which it can protect against oxidative damage in human keratinocytes have not been investigated thoroughly. Therefore, this study aimed to evaluate whether betulinic acid could protect against oxidative stress-mediated cellular damage mimicked by hydrogen peroxide (H₂O₂) in human keratinocyte HaCaT cells. The results demonstrated that betulinic acid suppressed H₂O₂-induced cytotoxicity while suppressing generation of reactive oxygen species. Betulinic acid also significantly inhibited H₂O₂-induced autophagy, which was associated with the down-regulation of the expression of key autophagy inducers. In addition, betulinic acid maintained mitochondrial homeostasis by reducing cytochrome c release from mitochondria to the cytosol and the loss of mitochondrial membrane potential in H₂O₂-treated cells. Moreover, in the presence of betulinic acid, the H₂O₂ㄹ-induced increase in the Bcl-2 associated X protein/B-cell lymphoma-2 (Bax/Bcl-2) expression ratio, caspase-3 activity, and cleavage of poly (ADP-ribose) polymerase were effectively attenuated, thereby offsetting the induction of apoptosis. Furthermore, betulinic acid pretreatment significantly abolished H2O2-induced endoplasmic reticulum (ER) stress by suppressing ER stress-regulating proteins and cytosolic calcium (Ca²⁺) overload. Therefore, our results demon- strated that betulinic acid could protect against mitochondrial impairment and Ca²⁺-mediated ER stress by minimizing oxidative stress, thereby inhibiting H₂O₂-induced cellular injury in HaCaT keratinocytes.

호흡기 상피세포에서 MUC5AC와 MUC5B 발현에 대한 Betulinic Acid의 효과

김훈성 ( Hoon Sung Kim ),최윤석 ( Yoon Seok Choi ),이준혁 ( Jun Hyeok Lee ),박나경 ( Na Kyung Park ),박창휘 ( Chang Hwi Park ),이영하 ( Young Ha Lee ),김귀옥 ( Gui Ok Kim ),송시연 ( Si Young Song ),배창훈 ( Chang Hoon Bae ),이승호 영남대학교 약품개발연구소 2014 영남대학교 약품개발연구소 연구업적집 Vol.24 No.0

Background and Objectives MUC5AC and MUC5B are representative secretory mucin genes in the human airway. MUC5AC and MUC5B expression are increased by a variety of inflammatory mediators. Betulinic acid, a naturally occurring pentacyclic triterpenoid, is known to have an anti-inflammatory property. However, the effects of betulinic acid on mucin secretion of airway epithelial cells still have not been reported. Therefore, in this study, the effect of betulinic acid on inflammatory mediators-induced MUC5AC and MUC5B expression was investigated in human airway epithelial cells. Materials and Method In the mucin-producing human NCI-H292 airway epithelial cells, the effects of betulinic acid on interleukin-1β (IL-1β)-, lipopolysaccharide (LPS)-, and phorbol myristate acetate (PMA)-induced MUC5AC and MUC5B expression were analyzed by reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. Results Betulinic acid attenuated IL-1β-, LPS- and PMA-induced MUC5B mRNA and glycoprotein expression in NCI-H292 cells. Betulinic acid did not attenuate IL-1β-, LPS- and PMA-induced MUC5AC mRNA and glycoprotein expression in NCI-H292 cells. Conclusion These results suggest that betulinic acid attenuates IL-1β-, LPS- and PMA-induced MUC5B expression in airway epithelial cells. Therefore, betulinic acid may modulate a control of mucus-hypersecretion in airway inflammatory disease. Korean J Otorhinolaryngol-Head Neck Surg 2014;57(8):526-32

HL-60 세포에서 $TNF-{\alpha}$에 의한 MCP-1 발현에 미치는 Betulinic Acid의 효과

김경찬,이추희,Kim, Kyung-Chan,Lee, Chu-Hee 대한약학회 2008 약학회지 Vol.52 No.1

Betulinic acid, a naturally occurring pentacyclic triterpenoid, is found in abundance in the outer bark of white birch (Betula alba). In this study, we investigated if betulinic acid affects cytokine expression from activated macrophage cells. ELISA result showed that stimulation of HL-60 cells with proinflammatory cytokine such as $TNF-{\alpha}$ resulted in MCP-1 release into culture medium. In addition, transcriptional upregulation of MCP-1 in response to $TNF-{\alpha}$ was observed by RT-PCR analysis. However, incubation of HL-60 cells with betulinic acid prior to $TNF-{\alpha}$ treatment abrogated MCP-1 expression in transcription and translational level. Consistent with a number of studies which reported requirement of ERK activation for $TNF-{\alpha}$ expression, Western blot analysis showed that $TNF-{\alpha}-induced$ ERK activation was suppressed by pretreatment of HL-60 cells with betulinic acid. Taken together, our data indicate that betulinic acid exerts its anti-inflammatory effect through inhibition of $TNF-{\alpha}-induced$ ERK activation which is required for the subsequent MCP-1 release.