Background & Aims

Cirrhosis results from accumulation of myofibroblasts derived from quiescent hepatic stellate cells (Q-HSCs); it regresses when myofibroblastic HSCs are depleted. Hedgehog signaling promotes transdifferentiation of HSCs by activating Yes-associated protein 1 (YAP1 or YAP) and inducing aerobic glycolysis. However, increased aerobic glycolysis alone cannot meet the high metabolic demands of myofibroblastic HSCs. Determining the metabolic processes of these cells could lead to strategies to prevent progressive liver fibrosis, so we investigated whether glutaminolysis (conversion of glutamine to alpha-ketoglutarate) sustains energy metabolism and permits anabolism when Q-HSCs become myofibroblastic, and whether this is controlled by hedgehog signaling to YAP.

Methods

Primary HSCs were isolated from C57BL/6 or Smo^flox/flox mice; we also performed studies with rat and human myofibroblastic HSCs. We measured changes of glutaminolytic genes during culture-induced primary HSC transdifferentiation. Glutaminolysis was disrupted in cells by glutamine deprivation or pathway inhibitors (bis-2-[5-phenylacetamido-1,2,4-thiadiazol-2-yl] ethyl sulfide, CB-839, epigallocatechin gallate, and aminooxyacetic acid), and effects on mitochondrial respiration, cell growth and migration, and fibrogenesis were measured. Hedgehog signaling to YAP was disrupted in cells by adenovirus expression of Cre-recombinase or by small hairpin RNA knockdown of YAP. Hedgehog and YAP activity were inhibited by incubation of cells with cyclopamine or verteporfin, and effects on glutaminolysis were measured. Acute and chronic liver fibrosis were induced in mice by intraperitoneal injection of CCl₄ or methionine choline-deficient diet. Some mice were then given injections of bis-2-[5-phenylacetamido-1,2,4-thiadiazol-2-yl] ethyl sulfide to inhibit glutaminolysis, and myofibroblast accumulation was measured. We also performed messenger RNA and immunohistochemical analyses of percutaneous liver biopsies from healthy human and 4 patients with no fibrosis, 6 patients with mild fibrosis, and 3 patients with severe fibrosis.

Results

Expression of genes that regulate glutaminolysis increased during transdifferentiation of primary Q-HSCs into myofibroblastic HSCs, and inhibition of glutaminolysis disrupted transdifferentiation. Blocking glutaminolysis in myofibroblastic HSCs suppressed mitochondrial respiration, cell growth and migration, and fibrogenesis; replenishing glutaminolysis metabolites to these cells restored these activities. Knockout of the hedgehog signaling intermediate smoothened or knockdown of YAP inhibited expression of glutaminase, the rate-limiting enzyme in glutaminolysis. Hedgehog and YAP inhibitors blocked glutaminolysis and suppressed myofibroblastic activities in HSCs. In livers of patients and of mice with acute or chronic fibrosis, glutaminolysis was induced in myofibroblastic HSCs. In mice with liver fibrosis, inhibition of glutaminase blocked accumulation of myofibroblasts and fibrosis progression.

Conclusions

Glutaminolysis controls accumulation of myofibroblast HSCs in mice and might be a therapeutic target for cirrhosis.

中文翻译：

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刺猬YAP信号通路调节谷氨酰胺分解来控制肝星状细胞的激活。

背景与目标

肝硬化是由静止的肝星状细胞（Q-HSC）衍生的成肌纤维细胞积累引起的；当肌纤维母细胞HSC耗尽时，它会消退。刺猬信号通过激活Yes相关蛋白1（YAP1或YAP）并诱导有氧糖酵解来促进HSC的转分化。然而，仅增加有氧糖酵解不能满足肌成纤维细胞HSC的高代谢需求。确定这些细胞的代谢过程可能导致预防进行性肝纤维化的策略，因此我们研究了谷氨酰胺分解（谷氨酰胺向α-酮戊二酸的转化）是否能维持能量代谢，并在Q-HSC变成肌纤维母细胞时允许合成代谢，以及这是否受到控制。向YAP发送刺猬信号。

方法

从C57BL / 6或Smo ^{flox / flox}中分离出主要的HSC老鼠; 我们还对大鼠和人的肌纤维母细胞HSC进行了研究。我们在培养诱导的原代HSC转分化过程中测量了谷氨酰胺分解基因的变化。谷氨酰胺剥夺或途径抑制剂（双-2- [5- [5-苯基乙酰胺基-1,2,4-噻二唑-2-基]乙硫]，CB-839，表没食子儿茶素没食子酸酯和氨基氧乙酸）破坏了谷氨酰胺分解作用。测量线粒体呼吸，细胞生长和迁移以及纤维发生。通过腺病毒表达Cre重组酶或通过YAP的小发夹RNA敲低，刺破了向YAP传递的刺猬信号。通过将细胞与环巴胺或维替泊芬一起孵育可抑制刺猬和YAP活性，并测量其对谷氨酰胺分解的影响。腹腔注射CCl诱发小鼠急性和慢性肝纤维化₄或蛋氨酸胆碱缺乏饮食。然后给一些小鼠注射双-2- [5- [5-苯基乙酰胺基-1,2,4-噻二唑-2-基]乙基硫化物，以抑制谷氨酰胺分解，并测量成肌纤维细胞的积累。我们还对健康人和4例无纤维化的患者，6例轻度纤维化的患者和3例重度纤维化的患者的经皮肝活检进行了信使RNA和免疫组织化学分析。

结果

在初级Q-HSC分化为肌纤维母细胞HSC的过程中，调节谷氨酰胺分解的基因的表达增加，抑制谷氨酰胺分解会破坏转分化。阻断肌纤维母细胞HSC中的谷氨酰胺分解抑制线粒体呼吸，细胞生长和迁移以及纤维生成。向这些细胞补充谷氨酰胺分解代谢物恢复了这些活性。敲除刺猬信号转导中间体使YAP变平滑或敲低，抑制了谷氨酰胺酶的表达，谷氨酰胺酶是谷氨酰胺分解中的限速酶。Hedgehog和YAP抑制剂可阻止HSC中的谷氨酰胺分解并抑制肌成纤维细胞活性。在患者或患有急性或慢性纤维化的小鼠的肝脏中，肌成纤维母细胞HSC诱导了谷氨酰胺分解。在患有肝纤维化的小鼠中，

结论

谷氨酰胺分解控制小鼠中成纤维细胞HSC的积累，并且可能是肝硬化的治疗靶标。