《Cell》:二甲双胍降糖机制新发现—肠道微生物竟是重要参与者

久坐不动,高热量饮食的摄入,不规则作息…… 现代人不良的生活习惯大大增加了二型糖尿病的患病风险。而二甲双胍是目前治疗糖尿病的主要用药,但其副作用明显,据调查结果显示约25%患者会遭受二甲双胍相关的副作用,如虚弱、胃痛和恶心等等[1]。所以表明二甲双胍的降糖机制同时降低其副作用是目前成丞待解决的重要问题。


既以往的研究表明,二甲双胍通过抑制线粒体复合物1和激活AMP活化的激酶(AMPK)来降低高血糖症和较少肝葡萄糖的产生。 随后的研究验证了肝AMPK的作用,但结果提示二甲双胍可能通过肝AMPK非依赖性机制抑制糖酵解酶和胰高血糖素作用以及线粒体和细胞溶质氧化还原状态的变化以降低葡萄糖产量[2]。 因此,二甲双胍诱的降糖的潜在机制仍然存在争议。


近年来越来越多的研究表明了二甲双胍的降糖作用可能与时下的研究热门肠道菌群有密切关系,早在2015年12月《Nature》上发表的一项研究就率先表明二甲双胍使2型糖尿病患者肠道菌群发生了有利的变化(某些短链脂肪酸生成增加)。在今年10月19日的《Cell》子刊《Cell Metabolism》上,林国栋(Tom Lam)教授带领多伦多大学研究团队首次可以通过调节乳酸菌,影响肠道上段吸收葡萄糖的重要转运蛋白SGLT-1,从而控制血糖水平[3]

首先研究人员发现高脂饮食大鼠乳酸菌数量明显减少,SGLT-1表达下降,但在二甲双胍治疗后,乳酸菌数量及SGLT-1表达有所恢复。
研究者还将来自已经过二甲双胍治疗的脂喂养的大鼠小肠上段的肠道细菌移植到未经二甲双胍治疗的高脂喂养大鼠体内,结果效果显著,在移植后的第一天,就发现被移植大鼠肠道内的乳酸菌数量显著上升,SGLT-1的表达也得到恢复。

所以,肠道菌群确确实实是二甲双胍发挥降糖作用的‘幕后功臣’。而对于二甲双胍这种降糖机制的新发现,有利于研究者在发挥二甲双胍药效的同时降低其副作用,或者帮助鉴定和开发替代性的、更有效的糖尿病用药。同时,小肠上段微生物对营养代谢的作用目前仍然是科研界的盲区之一,因此,本研究的发现对解释小肠微生物作用的神秘面纱有重要的作用,或许将来可使更多的患者受益。


原文

Methods:


•Three-day HFD Fed Model

Rats were placed on a lard-oil enriched diet 1 day following intestinal and vascular cannulations, and maintained on this diet for 3 days, which results in hyperphagia and upper small intestinal lipid sensing defects


•Rat Pancreatic (Basal Insulin) Euglycemic Clamp Procedures

Rats were subject to a 4-6 hr fast before the clamp experiment. In a subgroup of rats, the day before the clamp experiment, rats were given an upper small intestinal infusion of metformin (200 mg/kg) or saline for 50 min (0.01 ml/min) to mimic a previous study that showed this dose and infusion rate restricts metformin exposure to the upper small intestine. For microbiota transplant studies, rats received 0.5 mL of diluted luminal contents via the upper small intestinal cannula. The following morning, the clamp procedure (t = 200 min total) was performed in unrestrained rats in vivo. For the clamp, at the onset of the experiment (t = 0 min), a primed intravenous infusion of [3-3H] glucose (Perkin Elmer; 40 mCi bolus; 0.4 mCi min-1) was started and continued throughout (t = 200 min) to allow for measurement of glucose kinetics using the tracer-dilution methodology. The clamp was started at t = 90 min, where somatostatin (3 mg kg-1 min-1; somatostatin-14 was used, which has been shown to have a very minor inhibitory effect on GLP-1 release when compared with somatostatin-28 was infused intravenously to inhibit endogenous insulin and glucagon secretion. At the same time, insulin was infused at a dose of 1.2 mU kg-1 min-1 for the pancreatic (basal-insulin) clamp, along with a variable 25% glucose infusion that was periodically adjusted to maintain euglycemia (from t = 120 to t = 200 min). At t = 150 min, the upper small intestinal infusion (2 mL min_1) was started and continued until the end of the experiment (t = 200 min). Plasma samples were obtained every 10 min to determine the specific activity of [3-3H] glucose and measure insulin levels.


•Microbiota Transplant Studies

For transplant studies, surgery in donor rats was performed one day prior to recipient rats, and followed the exact same protocol as above. Rats were placed on a HFD for 3 days and one day prior to sacrifice, donor rats received an upper small intestinal infusion of metformin (200 mg/kg) or saline. Metformin- and saline-treated rats were sacrificed the next morning following a 4-6 hr fast and upper small intestinal microbiota contents were removed from the upper small intestine (from 6 to 15 cm distal to the pyloric sphincter to target the microbiota in contact with the upper small intestine-targeted metformin infusion and in an area shown to regulate whole body glucose levels in response to intestinal glucose). Contents were then quickly homogenized with a rotor homogenizer, diluted 1:4 in saline, and 500ul of contents were slowly administered into 1 recipient rat (≈90% of experiments) or 2 recipient rats (≈10% of experiments) and followed by a saline flush of the intestinal catheter. Clamp experiments were performed on recipient rats the following day


Results:


•Upper Small Intestinal Glucose Sensing Lowers GP in Chow-Fed Rodents

•Activation of SGLT1 and GLP-1 Receptor Is Necessary for Upper Small Intestinal Glucose Sensing in Healthy Rodents

•High-Fat Feeding Disrupts Upper Small Intestinal Glucose Sensing in Parallel to a Reduction of SGLT1 Expression and Glucose-Stimulated GLP-1 Release: A Potential Target for Metformin Therapy

•Metformin Restores Upper Small Intestinal Glucose Sensing by Normalizing SGLT1 Expression

•Metformin Alters Upper Small Intestinal Microbiota Composition Upper Small Intestinal Microbiota Transplantation from

•Metformin-Pretreated Donors Restores Upper SmallIntestinal Glucose Sensing in HFD Recipients


Summary:


The gut microbiota alters energy homeostasis. In parallel, metformin regulates upper small intestinal sodium glucose cotransporter-1 (SGLT1), but whether changes of the microbiota or SGLT1-dependent pathways in the upper small intestine mediate metformin action is unknown. Here we report that upper small intestinal glucose sensing triggers an SGLT1-dependent pathway to lower glucose production in rodents. High-fat diet (HFD) feeding reduces glucose sensing and SGLT1 expression in the upper small intestine. Upper small intestinal metformin treatment restores SGLT1 expression and glucose sensing while shifting the upper small intestinal microbiota partly by increasing the abundance of Lactobacillus. Transplantation of upper small intestinal microbiota from metformin-treated HFD rats to the upper small intestine of untreated HFD rats also increases the upper small intestinal abundance of Lactobacillus and glucose sensing via an upregulation of SGLT1 expression. Thus, we demonstrate that metformin alters upper small intestinal microbiota and impacts a glucose-SGLT1-sensing glucoregulatory pathway.


Reference:

[1] Florez, H., Luo, J., Castillo-Florez, S., Mitsi, G., Hanna, J., Tamariz, L., Palacio, A., Nagendran, S., and Hagan, M. (2010). Impact of metformin-induced gastrointestinal symptoms on quality of life and adherence in patients with type 2 diabetes.  Postgrad. Med. 122, 112–120.

[2] Madiraju, A.K., Erion, D.M., Rahimi, Y., Zhang, X.M., Braddock, D.T., Albright,R.A., Prigaro, B.J., Wood, J.L., Bhanot, S., MacDonald, M.J., et al. (2014). Metformin suppresses gluconeogenesis by inhibiting mitochondrial glycerophosphate dehydrogenase. Nature 510, 542–546.

[3]http://www.cell.com/cell-metabolism/fulltext/S1550-4131(17)30571-5


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