一般的な食品添加物カラギーナンは、ヒト腸内分泌 L 細胞によるプログルカゴン発現と GLP-1 分泌を阻害します
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公開日:2024 年 5 月 16 日
一般的な食品添加物カラギーナンは、ヒト腸内分泌 L 細胞によるプログルカゴン発現と GLP-1 分泌を阻害します
https://www.nature.com/articles/s41387-024-00284-4
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栄養と糖尿病 音量 14、記事番号: 28 ( 2024 ) この記事を引用する
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培養ヒト L 細胞 (NCI-H716) によるプログルカゴン mRNA の発現と GLP-1 分泌は、加工食品に一般的に使用される添加物である λ-カラギーナンへの曝露後に阻害されました。カラギーナンは、α-1,3 結合とβ-1,4 結合が交互に結合した硫酸化または非硫酸化ガラクトース残基で構成され、内因性硫酸化グリコサミノグリカンに似ています。しかし、カラギーナンには珍しいα-1,3-ガラクトシド結合があり、これは人間の細胞に本来備わっているものではなく、免疫応答に関係しています。カラギーナンへの曝露は予想どおり炎症を引き起こし、カラギーナンは耐糖能を損ない、インスリン抵抗性の原因となります。培養ヒト L 細胞からグルコースと血清を一晩除去し、その後高グルコース、10% FBS、および λ-カラギーナン (1 μg/ml) に 10 分、1 時間、および 24 時間曝露すると、プログルカゴンとGLP-1の分泌は、カラギーナンに曝露されなかった対照細胞と比較して、有意に減少した。マウス L 細胞 (STC-1) によるプログルカゴンの mRNA 発現も大幅に減少しており、ヒト細胞での発見を裏付けています。共培養ヒト腸上皮細胞 (LS174T) をカラギーナン処理 L 細胞の使用済み培地に曝露すると、24 時間で GLUT-2 の mRNA 発現が低下しました。これらの発見は、カラギーナンを含む加工食品の摂取がGLP-1の産生を損ない、GLP-1受容体アゴニストの効果を打ち消し、腸上皮細胞に対する二次的な影響を誘発する可能性を示唆しています。
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導入
グルカゴン様ペプチド (GLP)-1 は、遠位小腸および結腸の腸内分泌 L 細胞によって合成および分泌されるインクレチンです。生成と分泌は、パルミチン酸、オレイン酸、肉加水分解物などの腸内腔内の栄養素と非栄養素の両方に応じて調節されます[ 1、2、3、4 ]。 GLP-1 は、グルコース刺激による膵島からのインスリン分泌を促進し、胃内容排出を遅らせ、満腹感を促進します [ 2 ]。これらの効果は食物摂取量の減少と体重減少につながり、糖尿病や肥満の治療におけるGLP-1受容体アゴニストの臨床使用増加の基礎となります。
この短い通信では、ヒト L 細胞を一般的な食品添加物であるカラギーナンに曝露すると GLP-1 産生が阻害されるという予期せぬ発見を紹介します。カラギーナンは、カゼインや他の生理活性分子と結合する能力があるため、加工食品の食感を改善するために広く使用されている天然産物ですが、栄養価はありません。米国の成人の予想一日摂取量は 100 mg/日を超える可能性が高く、18 ～ 40 mg/kg/日の間であると推定されています[ 5、6 ]。食品安全規制当局は、乳児用粉ミルクやさまざまな食品へのカラギーナンの使用に対する懸念から、カラギーナンの安全性について繰り返し取り組んできました [ 7 ]。紅藻由来のカラギーナンは、炎症を引き起こすことが予測でき、抗炎症薬の有効性をテストしたり、炎症性メディエーターを特定したりするために使用できるため、数十年にわたって科学実験室で使用されてきました。カラギーナンの 3 つの主要な形態 (カッパ、ラムダ、およびイオタ) は、交互の α-1,3 および β-1,4-グリコシド結合によって結合された異なる硫酸化二糖で構成されています。 α-gal結合はヒトの細胞によって作られるものではなく、ヒト以外の臓器移植の拒絶反応、ダニ咬傷疾患、肉アレルギーなどの自然免疫反応に関連しています[ 8 ]。実験では、カラギーナンへの曝露が活性酸素種への影響とトール様受容体 (TLR)-4 との相互作用により、NF-κB の核移行を引き起こすことが示されています [ 9 ]。耐糖能不耐症とインスリン抵抗性は、経口カラギーナンに曝露されたマウスで発生し、細胞内インスリンシグナル伝達の混乱と炎症経路の活性化に起因していました [ 10 ]。他の実験では、カラギナンが、N-アセチルガラクトサミン-4-スルファターゼ（アリールスルファターゼB、ARSB）、コンドロイチン4-硫酸、およびガレクチン-3とインスリン受容体との相互作用に影響を与えるため、インスリン受容体の応答性を阻害することが示されました[ 11、12 ]。 。以前の実験では、カラギーナンはマウスの体重やカラギーナンを含まない食餌を与えた前糖尿病患者の体重に対して独立した影響を及ぼさず[ 13、14 ]、インクレチンとマウスの腸内分泌細胞への影響は試験されなかった。
GLP-1 産生に対するカラギーナン曝露の影響を検討するために、培養ヒト L 細胞、マウス腸内分泌細胞、およびヒト腸上皮細胞 (IEC) と共培養したヒト L 細胞で実験を実施しました。 IECに対するヒトL細胞分泌の影響は、GLP-1の影響を受けることが報告されているGLUT2発現の測定によって検討された[ 15 ]。
材料と方法
Cell lines were procured and grown under the recommended conditions, including 37 °C and 5% CO2. The human intestinal L-cell line NCI-H716 (CCL-251, ATCC, Manassas, VA, USA) was originally isolated from the ascites fluid of a patient with colorectal adenocarcinoma [2]. LS 174 T cells (CL-188, ATCC) are colonic epithelial cells isolated from a patient with colorectal cancer. STC-1 cells (CRL-3254, ATCC) are an intestinal neuroendocrine cell line isolated from a mouse intestinal tumor [16].
The human and mouse L-cells were incubated overnight in low glucose medium without serum, then exposed to. high glucose (25 mM) DMEM with 10% FBS with or without λ-carrageenan (1 μg/ml; Sigma Aldrich, St. Louis MO, USA) for 10 minutes, 1 h, or 24 h. Spent media and cell homogenates were collected. GLP-1 protein levels in the spent media were measured by ELISA (Immuno-Biological Laboratories IBL-America, Minneapolis, MN, USA; #27784 ). mRNA levels of proglucagon, the precursor of GLP-1, were determined by QRT-PCR, using established procedures with the following primers:
proglucagon (human): left: 5′-CGTTCCCTTCAAGACACAGAGG-3′;
right: 5′-ACGCCTGGAGTCCAGATACTTG -3′;
proglucagon (mouse): left: 5′-CCTTCAAGACACAGAGGAGAACC-3′; and
right: 5′-CTGTAGTCGCTGGTGAATGTGC-3′.
The effect of carrageenan exposure on GLUT2 expression by the human intestinal epithelial cells was tested in an in vitro co-culture model. NCI-H716 human intestinal L-cells were grown on transwell inserts on top of a monolayer of human intestinal epithelial cells (IEC) (LS174T). The L-cells were treated with λ-carrageenan (1 μg/ml) for 10 min, 1 h, and 24 h, and the impact on mRNA expression of GLUT2 by the IEC was measured using the primers:
GLUT2 (human; NM_000340): left-5′-ATGTCAGTGGGACTTGTGCTGC-3′ and
right - 5′-AACTCAGCCACCATGAACCAGG-3′.
Statistics were performed with Microsoft Excel and PRIZM 9.5 software (GraphPad, Boston, MA, USA) using unpaired t-tests, two-tailed with unequal variance for all of the comparisons. Mean values and standard deviations of at least triplicate experiments are presented in the figures. P ≤ 0.05 is considered statistically significant.
Results
Following overnight deprivation, human L-cells were incubated with high glucose, serum, and carrageenan. The mRNA expression of proglucagon declined over time from 10 min to 24 h in the carrageenan-exposed cells, to 0.44-fold the level in the time-matched controls, which were exposed to high glucose and serum, but no carrageenan (p = NS at 10 min, p = 0.004 at 1 h; p = 0.0005 at 24 h; unpaired t-test, two-tailed, unequal variance; n = 3) (Fig. 1a). In the carrageenan-treated cells, mRNA expression was significantly less at 1 h and at 24 h than at 10 min (p = 0.014, p = 0.010), whereas there was no significant decline in the untreated control cells between 10 min and 24 h. Consistent with the decline in proglucagon expression, GLP-1 secretion by the L-cells into the media was significantly less than the time-matched control values following exposure to carrageenan (Fig. 1b). The secreted GLP-1 levels in the spent media following carrageenan vs. control were: 12.3 ± 0.7 vs. 17.8 ± 0.2 pmol/mg protein at 10 min (p = 0.003); 15.1 ± 1.2 vs. 27.9 ± 1.8 pmol/mg protein at 1 h (p = 0.001); and 7.3 ± 0.8 vs. 12.8 ± 0.6 pmol/mg protein at 24 h (p = 0.001).
Fig. 1: Carrageenan reduces GLP-1 production in enteroendocrine cells.
a Following exposure to carrageenan (1 µg/ml) for 10 min, 1 h, and 24 h, mRNA expression of proglucagon (GLP-1) was measured in the human L-cell line. mRNA expression was significantly less at 1 h and 24 h following carrageenan than in the untreated controls (p = 0.004 and p = 0.0005, respectively; n = 3). In the carrageenan-treated cells, expression was 0.4-fold at 1 h and at 24 h, compared to the value at 10 min (p = 0.014, p = 0.010; n = 3), whereas there was no significant decline in the control cells. b GLP-1 secretion from the human L-cells was significantly less at all time points in the L-cells exposed to carrageenan than in the cells exposed to media without carrageenan (p = 0.003 at 10 min, p = 0.001 at 1 h, p = 0.001 at 24 h; n = 3). The secreted GLP-1 declined from 12.3 ± 0.7 at 10 min to 7.3 ± 0.8 pg/mg protein at 24 h following carrageenan exposure (p = 0.001, n = 3). All comparisons are by unpaired t-tests, two-tailed with unequal variance.
Experiments were performed in mouse enteroendocrine cells to support the findings in the human cells, Proglucagon expression was also significantly less at 24 h following exposure to carrageenan (p < 0.01, unpaired t-test, two-tailed, unequal variance, n = 3), relative to the control with no carrageenan exposure (0.44 ± 0.09 fold-change compared to control value of 1.0 ± 0.18).
To test if products of the carrageenan-treated L-cells could affect transcription in the IEC, the mRNA expression of GLUT-2 was measured in co-cultured IEC. A monolayer of LS174T IEC was exposed to the spent media from carrageenan-treated L-cells grown on inserts, as depicted (Fig. 2a). Glucose Transporter (GLUT)2 expression in the IEC declined significantly (p = 0.03 at 24 h, unpaired t-test, two-tailed, unequal variance, n = 3) (Fig. 2b). In contrast, direct carrageenan treatment of the LS174T cells had no effect on the expression of GLUT2 (p > 0.05) (Fig. 2c).
Fig. 2: Impact of L-cell secretion on GLUT2 expression by co-cultured intestinal epithelial cells.
a The schematic drawing shows the two-layer culture in which L-cells in filters were directly exposed to carrageenan. The product of the L-cells affecting the IEC may be GLP-1 or another secretory product. b GLUT-2 expression in the IEC at 1 h was 0.64-fold, and at 24 h, was 0.40-fold the value at 10 min, following co-culture with human L-cells exposed to carrageenan, compared to the value at 10 min (p = 0.002, p = 0.002, n = 3). At the same time, no-carrageenan control was significantly greater at 24 h (p = 0.03; n = 3). c Direct exposure of the IEC to carrageenan did not affect the mRNA expression of GLUT-2. All comparisons are by unpaired t-tests, two-tailed with unequal variance.
These data indicate that carrageenan exposure inhibited the expression and secretion of GLP-1 by human intestinal L-cells. Mouse L-cell expression of proglucagon was also reduced. Also, spent media from the carrageenan-treated human L-cells inhibited the GLUT2 expression in co-cultured IEC, indicating the potential for significant paracrine effects of L-cell products, including GLP-1, on the surrounding IEC. GLUT-2 was not modified by direct exposure to carrageenan.
Discussion
Current interest in the effects of incretin-based therapy, including GLP-1 receptor agonists and dipeptidyl peptidase-4 inhibitors, is intense due to effects on weight loss, as well as diabetes treatment. The experiments presented in this report suggest that consumption of carrageenan in processed foods may act to reduce the endogenous secretion of GLP-1 and the effectiveness of these therapeutic agents. Previous work indicated no significant effect on weight in carrageenan-treated mice or in individuals on a carrageenan-elimination diet for 12 weeks [13, 14]. However, these study findings suggest that carrageenan exposure may affect weight over the long-term by the impact on GLP-1 production and by inhibition of response to treatment by GLP-1 receptor agonists, thereby limiting weight loss.
Effects of carrageenan on transcription may be attributable to its inhibition of the enzyme N-acetylgalactosamine-4-sulfatase (Arylsulfatase B; ARSB) [17]. ARSB mediates transcriptional events through effects on chondroitin 4-sulfate, galectin-3, and SHP2, and these effects may contribute to the current findings [18]. Additional effects of carrageenan may be mediated by secondary effects of GLP-1 or other L-cell secretory products by paracrine effects on neighboring cells, affecting transporters such as GLUT2. GLUT-2, by effects on glucose transport, may participate in the regulation of responses to ambient glucose concentration [19]. Further studies are required to define the interactions and possible feedback mechanisms involving these vital mediators of glucose homeostasis.
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Funding
Funding
Studies were supported in part by a grant to JKT from American Diabetes Association (ADA-1-16-ICTS-114). The facilities and resources of the VA also supported the research. The content is solely the responsibility of the authors and does not necessarily represent the official views of the VA or the ADA.
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Authors and Affiliations
Department of Medicine, University of Illinois at Chicago, Chicago, IL, USA
Sumit Bhattacharyya & Joanne K. Tobacman
Research, Jesse Brown VA Medical Center, Chicago, IL, USA
Sumit Bhattacharyya & Joanne K. Tobacman
Department of Clinical & Translational Sciences, Marshall University, Huntington, WV, USA
Alip Borthakur
Contributions
JKT, SB, and AB planned the studies; AB provided cell lines; SB performed assays; JKT drafted the paper.
Corresponding author
Correspondence to Joanne K. Tobacman.
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Bhattacharyya, S., Borthakur, A. & Tobacman, J.K. Common food additive carrageenan inhibits proglucagon expression and GLP-1 secretion by human enteroendocrine L-cells. Nutr. Diabetes 14, 28 (2024). https://doi.org/10.1038/s41387-024-00284-4
Received07 August 2023
Revised20 April 2024
Accepted25 April 2024
Published16 May 2024
DOIhttps://doi.org/10.1038/s41387-024-00284-4
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