5D Library › 酮体代谢、β-羟丁酸信号转导与间歇性禁食的生物能量学 · English

第四章 AMPK–mTORC1 开关与细胞自噬

细胞的回收系统

自噬(autophagy,字面意思是「自己吃自己」)是细胞把自身的一部分(老化的蛋白质、受损的线粒体)包进双层膜结构(自噬体),送到溶酶体降解回收的过程。大隅良典首先在酵母中观察到自噬,随后用遗传筛选找到了自噬所需的一批基因,奠定了这一领域的分子基础,并因此获得 2016 年诺贝尔生理学或医学奖 (Takeshige et al. 1992; Tsukada and Ohsumi 1993)。

自噬受营养状态的强烈调控。营养充足时它被抑制,营养缺乏时被激活。这一调控的核心是两个感受营养与能量的蛋白激酶:mTORC1 与 AMPK。

两个激酶,一个开关

  • mTORC1 感受氨基酸与生长因子(包括胰岛素)信号。它活跃时促进蛋白质合成与生长,同时磷酸化自噬起始激酶 ULK1 的抑制性位点,关闭自噬。
  • AMPK 感受能量不足(AMP 与 ADP 相对 ATP 升高)。它活跃时一方面磷酸化 ULK1 的激活位点,直接启动自噬;另一方面通过磷酸化 TSC2 和 raptor 抑制 mTORC1 (Inoki et al. 2003; Gwinn et al. 2008; Kim et al. 2011)。

于是 AMPK 抑制 mTORC1,mTORC1 也在一定程度上反过来抑制 AMPK 的作用。两个互相抑制的元件构成双负反馈,在系统生物学中,这样的回路在适当的参数下可以成为双稳态拨动开关:同样的输入水平,系统可以处在「开」或「关」两个稳定状态之一,取决于它从哪里来。霍尔采尔等人用数学模型和细胞实验论证了 mTORC1 与 AMPK 之间的双负反馈对自噬的精确开启有重要作用 (Holczer et al. 2019)。

注

这里有一个建模假设。 AMPK 抑制 mTORC1 的证据很充分;mTORC1 反过来抑制 AMPK 的作用较弱,机制也还有争论。下面的程序把相互抑制写得很陡,这是为了展示双稳态开关的性质而作的假设,不是对细胞中实际参数的估计。

推导

一个最小的 AMPK–mTORC1 拨动开关。营养信号 ss 从 0(禁食)到 1(进食)。先让营养缓慢升高,再缓慢降低,记录自噬起始的水平。

# A minimal AMPK-mTORC1 toggle switch (a modelling assumption, illustrative parameters).
# Nutrient signal s in [0, 1]: s = 1 fed (insulin and amino acids high), s = 0 fasted (energy stress).
#   dM/dt = 3 s / (1 + (A/0.5)^4) - M          mTORC1 (M): driven by nutrients, inhibited by AMPK
#   dA/dt = 3 (1 - s) / (1 + (M/0.5)^4) + 0.05 - A   AMPK (A): driven by energy stress, inhibited by mTORC1
# AMPK inhibits mTORC1 (via TSC2 and raptor); the reverse arm, mTORC1 dampening AMPK, is weaker
# and debated, so the steep mutual inhibition here is an assumption. ULK1, the autophagy initiator,
# is switched on by AMPK and off by mTORC1:  autophagy initiation = A/(A + 0.5) / (1 + (M/0.5)^2).
def settle(s, M, A, dt=0.01, T=60.0):
    for _ in range(int(T / dt)):
        dM = 3 * s / (1 + (A / 0.5) ** 4) - M
        dA = 3 * (1 - s) / (1 + (M / 0.5) ** 4) + 0.05 - A
        M += dM * dt; A += dA * dt
    return M, A

def flux(M, A):
    return A / (A + 0.5) / (1 + (M / 0.5) ** 2)

grid = [i / 10 for i in range(11)]
M, A = 0.0, 3.0                                   # start fully fasted
up = {}
for s in grid:                                    # slowly feed
    M, A = settle(s, M, A); up[s] = flux(M, A)
down = {}
for s in reversed(grid):                          # then slowly withdraw food
    M, A = settle(s, M, A); down[s] = flux(M, A)
print("nutrient signal s | autophagy initiation, rising nutrients | falling nutrients")
for s in grid:
    print(f"{s:17.1f} | {up[s]:38.2f} | {down[s]:17.2f}")
off = min(s for s in grid if up[s] < 0.2); on = max(s for s in grid if down[s] >= 0.2)
print(f"rising nutrients switch autophagy off at s = {off:.1f}; falling nutrients switch it back on only"
      f" at s = {on:.1f} (hysteresis)")
# nutrient signal s | autophagy initiation, rising nutrients | falling nutrients
#               0.0 |                                   0.86 |              0.86
#               0.1 |                                   0.85 |              0.85
#               0.2 |                                   0.83 |              0.83
#               0.3 |                                   0.81 |              0.81
#               0.4 |                                   0.79 |              0.03
#               0.5 |                                   0.76 |              0.01
#               0.6 |                                   0.71 |              0.01
#               0.7 |                                   0.60 |              0.01
#               0.8 |                                   0.00 |              0.00
#               0.9 |                                   0.00 |              0.00
#               1.0 |                                   0.00 |              0.00
# rising nutrients switch autophagy off at s = 0.8; falling nutrients switch it back on only at s = 0.3 (hysteresis)
模型中的滞后现象:营养信号升高时,自噬要到 s 介于 0.7 与 0.8 之间才关闭;营养信号降低时,要等到 s ≈ 0.3 才重新开启。

这个开关最有意思的性质是滞后:营养升高时,自噬一直维持到 ss 超过 0.7 才关闭;营养降低时,它并不在同一点重新开启,而是要等到 ss 降到 0.3 左右。也就是说,在这个模型中,一次进食就能把开关拨到「关」,但要把它拨回「开」,需要营养信号降得足够低、持续足够久。这为「进食频率可能比每餐吃多少更重要」提供了一个机制上的可能性,但只是可能性。

进食安排与开关

推导

把几种进食安排输入同一个开关。每餐把营养信号推高 0.8,餐间按时间常数 τ 衰减。程序以「小时」标注时间,但没有任何参数经过人体数据校准,只能比较不同安排之间的相对差别。最后一行把 τ 从 8 小时改为 6 小时,检验结论有多脆弱。

# Feeding schedules run through the toggle switch of the previous program. Each meal pushes the
# nutrient signal s up by 0.8 (capped at 1); between meals s decays with time constant tau (8 h).
# M and A relax with a 1-hour time constant. 'Hours on' counts hours per day with autophagy
# initiation above 0.5, averaged over days 3-7. The clock is labelled in hours, but NO parameter is
# calibrated to human data: compare schedules with each other, do not read off real hours.
import math

def simulate(meals_per_day, days=7, dt=0.02, fast_day=None, tau=8.0):
    M, A, s, on = 0.5, 0.5, 0.5, 0.0
    steps = int(24 / dt)
    for d in range(days):
        eaten = set()
        for i in range(steps):
            h = i * dt
            for m in meals_per_day:
                if (d != fast_day) and m not in eaten and h >= m:
                    s = min(1.0, s + 0.8); eaten.add(m)
            s -= s / tau * dt
            dM = 3 * s / (1 + (A / 0.5) ** 4) - M
            dA = 3 * (1 - s) / (1 + (M / 0.5) ** 4) + 0.05 - A
            M += dM * dt; A += dA * dt
            if d >= 2 and A / (A + 0.5) / (1 + (M / 0.5) ** 2) > 0.5:
                on += dt
    return on / (days - 2)

SCHED = {"grazing: 6 small meals 07-22 h": (7, 10, 13, 16, 19, 22),
         "three meals 07, 12, 19 h": (7, 12, 19),
         "three meals + late snack 22 h": (7, 12, 19, 22),
         "16:8, meals 12 and 20 h": (12, 20),
         "16:8 early, meals 08 and 16 h": (8, 16)}
print("schedule                           | model hours per day with autophagy initiation 'on'")
for name, meals in SCHED.items():
    print(f"{name:34s} | {simulate(meals):6.1f}")
print(f"{'three meals, one day a week fasted':34s} | {simulate((7, 12, 19), days=9, fast_day=4):6.1f}")
# How fragile is this? Let the nutrient signal fade faster (tau = 6 h instead of 8 h):
print("with tau = 6 h: three meals", f"{simulate((7, 12, 19), tau=6.0):.1f}", "h, 16:8", f"{simulate((12, 20), tau=6.0):.1f}",
      "h, grazing", f"{simulate((7, 10, 13, 16, 19, 22), tau=6.0):.1f}", "h")
# schedule                           | model hours per day with autophagy initiation 'on'
# grazing: 6 small meals 07-22 h     |    0.0
# three meals 07, 12, 19 h           |    0.0
# three meals + late snack 22 h      |    0.0
# 16:8, meals 12 and 20 h            |   11.9
# 16:8 early, meals 08 and 16 h      |   11.9
# three meals, one day a week fasted |    4.1
# with tau = 6 h: three meals 22.4 h, 16:8 24.0 h, grazing 0.0 h

在这组参数下,少量多餐与一日三餐(含夜宵)都使开关几乎一直处于「关」,16:8 的安排(两餐,夜间间隔 16 小时)每天有约 12 个模型小时处于「开」,每周一天不吃则把平均值提高到约 4 个模型小时。但最后一行显示,只要把营养信号的衰减稍微加快,一日三餐也会使开关一天中大部分时间处于「开」。结论完全取决于未经校准的参数。这正是本章想说明的:开关模型能告诉我们哪些量值得测量(营养信号衰减有多快、开关的阈值在哪里),却不能告诉我们人需要禁食多少小时才能「开启自噬」。

小鼠的 24 小时不是人的 24 小时

「禁食 16 小时(或 24、48 小时)开启自噬」的说法流传很广。它的证据主要来自小鼠。

证据

水岛昇等人培育了表达荧光标记 LC3(一种自噬体膜蛋白)的转基因小鼠,用来观察各组织中的自噬体。禁食 24 小时后,骨骼肌、心脏、肝脏等许多组织中的自噬体明显增加,各器官的时间进程不同(例如骨骼肌在 48 小时时进一步增加);而大脑中几乎没有观察到自噬体的增加 (Mizushima et al. 2004)。

把这些时间直接搬到人身上,忽略了一个基本事实:小鼠的代谢速度比人快得多。

推导

按克莱伯定律,静息代谢率大致与体重的 3/4 次方成正比,所以每千克体重的代谢率与体重的 −1/4 次方成正比 (Kleiber 1932)。比较 25 克的小鼠与 70 千克的人。

# Why a mouse's 24-hour fast is not a human's 24-hour fast. Kleiber's law: whole-body resting
# metabolic rate scales roughly as body mass^(3/4), so the rate PER KILOGRAM scales as mass^(-1/4).
# Masses: laboratory mouse 25 g, adult human 70 kg.
m_mouse, m_human = 0.025, 70.0
ratio = (m_human / m_mouse) ** 0.25
print(f"mass-specific metabolic rate, mouse / human: {ratio:.1f}x")
for h in (12, 24, 48):
    print(f"a {h:2d}-hour fast in a mouse ~ {h * ratio / 24:4.1f} days of a human's metabolic time"
          " (a naive scaling, see below)")
# Kleiber's formula itself: BMR (kcal/day) ~ 70 * M^0.75
for name, m in (("mouse", m_mouse), ("human", m_human)):
    bmr = 70 * m ** 0.75
    print(f"{name}: Kleiber estimate {bmr:7.1f} kcal/day = {bmr / m:5.0f} kcal per kg per day")
# Why the naive scaling is itself not a conversion table:
#  * different processes scale differently: liver glycogen in a fasted mouse is largely gone within
#    about 12-24 h, but autophagy in each tissue has its own kinetics (and the mouse brain showed
#    little induction even at 48 h in GFP-LC3 mice);
#  * mice are usually fasted during their active (dark) phase and are kept below thermoneutrality,
#    which raises their energy use further;
#  * in humans there is still no simple, non-invasive marker that times autophagic flux in tissues.
# mass-specific metabolic rate, mouse / human: 7.3x
# a 12-hour fast in a mouse ~  3.6 days of a human's metabolic time (a naive scaling, see below)
# a 24-hour fast in a mouse ~  7.3 days of a human's metabolic time (a naive scaling, see below)
# a 48-hour fast in a mouse ~ 14.5 days of a human's metabolic time (a naive scaling, see below)
# mouse: Kleiber estimate     4.4 kcal/day =   176 kcal per kg per day
# human: Kleiber estimate  1694.0 kcal/day =    24 kcal per kg per day

按这一标度律,小鼠每千克体重的代谢率约为人的 7 倍;在最粗略的意义上,小鼠禁食 24 小时消耗的「代谢时间」相当于人的约一周。但这本身也不是一张换算表。不同的生理过程按不同的方式标度;实验小鼠通常在它们活跃的黑夜禁食,并且饲养在低于热中性的温度下,这进一步提高了它们的能量消耗;而且,即使是小鼠,不同组织的自噬也有不同的时间进程。所以本书的结论是:动物实验中的自噬时间线不能直接等同于人类的具体小时数,也不能用克莱伯定律简单换算。

注

标志物不是过程本身。 在人体中测量自噬非常困难。自噬是一个「通量」:自噬体形成、与溶酶体融合、内容物被降解,这几步的速度共同决定了回收的多少。单个时间点上自噬体的数量(或 LC3 等蛋白的水平)多了,既可能是自噬增强,也可能是降解受阻、自噬体堆积。自噬研究的国际指南因此强调必须测量通量,而不只是标志物 (Klionsky et al. 2021)。在人体中,目前还没有一种简单、无创、能够在各组织中为自噬计时的标志物。一些人体研究在血液细胞中测到了自噬相关基因表达的变化,例如贾姆谢德等人发现早期限时进食提高了全血中 LC3A 的表达 (Jamshed et al. 2019),但血液细胞中基因的表达既不等于自噬通量,也不等于肌肉、大脑或肝脏中发生的事。

假说 · 尚未验证

进食频率假说(尚未确立)。如果营养感应确实具有滞后性,那么在总热量与营养组成相同的条件下,减少进食次数(拉长餐间间隔)对营养感应通路的影响,应当大于把同样的食物分成多次少量进食。可以检验的推论是:在人体肌肉活检中,用通量测定法(例如在活检组织中加入溶酶体抑制剂比较 LC3-II 的积累),两餐安排应当比六餐安排显示更高的自噬通量。如果两种安排没有差别,或者差别只出现在静态标志物上而不出现在通量上,这一假说就被削弱。

本章小结

AMPK 与 mTORC1 的相互抑制可以构成一个带滞后的开关:一次进食就能关闭自噬,重新开启则需要营养信号降得够低、持续够久。但开关模型的结论取决于未经校准的参数;小鼠的自噬时间线不能换算成人的小时数;人体中也缺乏能为自噬计时的简单标志物。「禁食多少小时开启自噬」在目前是一个没有答案的问题。下一章离开机制,看看人体随机对照试验说了什么。

参考文献

Gwinn, Dana M., David B. Shackelford, Daniel F. Egan, et al. 2008. “AMPK Phosphorylation of Raptor Mediates a Metabolic Checkpoint.” Molecular Cell 30 (2): 214–26.
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Inoki, Ken, Tianqing Zhu, and Kun-Liang Guan. 2003. “TSC2 Mediates Cellular Energy Response to Control Cell Growth and Survival.” Cell 115 (5): 577–90.
Jamshed, Humaira, Robbie A. Beyl, Deborah L. Della Manna, Eddy S. Yang, Eric Ravussin, and Courtney M. Peterson. 2019. “Early Time-Restricted Feeding Improves 24-Hour Glucose Levels and Affects Markers of the Circadian Clock, Aging, and Autophagy in Humans.” Nutrients 11 (6): 1234.
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Mizushima, Noboru, Akitsugu Yamamoto, Makoto Matsui, Tamotsu Yoshimori, and Yoshinori Ohsumi. 2004. “In Vivo Analysis of Autophagy in Response to Nutrient Starvation Using Transgenic Mice Expressing a Fluorescent Autophagosome Marker.” Molecular Biology of the Cell 15 (3): 1101–11.
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Fan, Y. (2026). Ketone Metabolism, β-Hydroxybutyrate Signalling and the Bioenergetics of Intermittent Fasting: From the Randle Cycle and Epigenetic Inhibition to Autophagy Kinetics, Read Beside Classical Chinese Fasting Texts [酮体代谢、β-羟丁酸信号转导与间歇性禁食的生物能量学] (Version 1.0.0). 5D Library. https://doi.org/10.5281/zenodo.23753263 · Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0).