Single-cell Transcriptome Profiling Reveals Hedgehog Critically Regulates Hepatocyte Resiliency
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As the largest organ in the body, liver is responsible for maintaining systemic metabolic homeostasis, and also plays an important role in immune functions. Nutrients absorbed through the gastrointestinal tract are processed by the liver and are either stored or distributed to other organs to provide energy. When these metabolic processes become unbalanced, fat builds up in the liver and hepatic steatosis may ensue. Current global estimates indicate non-alcoholic fatty liver disease (NAFLD) afflicts at least 25% of adults worldwide and is the leading cause of cirrhosis and liver cancer. It remains challenging to individualize treatment for NAFLD because its prevalence and severity vary considerably among patients depending on their age, gender, inheritable traits, and even environmental factors. For the past several decades, substantial resources have been devoted to studying this disease and our understanding of its pathogenesis has advanced. Nevertheless, there are currently no effective therapies approved specifically for preventing or treating non-alcoholic steatohepatitis (NASH; the inflammatory liver injury that develops when fatty hepatocytes experience lipotoxicity) or cirrhosis (the end result of maladaptive efforts to repair NASH that replace lipotoxic hepatocytes with scar).Hence, it is critical to improve understanding of the mechanisms that cause hepatic steatosis (NAFL) to progress to steatohepatitis (NASH), as well as the mechanisms that determine whether NASH resolves, stabilizes or progresses to cirrhosis. In the work presented here I focused on Hedgehog, a lipid-sensing developmental morphogenic pathway, and uncovered how its regulation mediates multiple hepatocyte functions in adult livers, including metabolic homeostasis and the regenerative response to liver injury. These findings not only clarify why hepatocytes lose their resiliency as NAFLD advances to cirrhosis, but also reveal novel therapeutic targets that we might manipulate to restore hepatocyte function, improve recovery from NASH, and prevent NAFLD-related cirrhosis and liver cancer. Hedgehog signaling plays a key role in tissue patterning during embryo development. Until recently, activity of this pathway was presumed to be silenced in healthy adults because dysregulated gain-of-function mutations and epigenetic activation of this pathway has been implicated in hepatocellular carcinoma and many other cancer types. To thoroughly investigate the role of Hedgehog in adult liver, we utilized a viral vector-based approach to study whether disruption of Hedgehog in healthy mature hepatocytes evokes any phenotype of interest. We discovered that mice with Hedgehog-deficient hepatocytes rapidly developed NAFLD within a week as characterized by hepatic accumulation of fatty acids, triglyceride and cholesterol, providing strong evidence that Hedgehog is active and broadly regulates lipid metabolism in adult liver. Using single-cell transcriptomic profiling, we demonstrated that Hedgehog activity was spatially restricted to hepatocytes localized in the mid-lobular area. By manipulating Hedgehog activity in hepatocytes of intact mice and challenging them with an acute intraportal injection of insulin, we demonstrated that disruption of Hedgehog induced steatohepatitis, and caused hepatic and systemic insulin resistance by stimulating the mTOR pathway. Further in silico analyses and in vitro studies revealed that Hedgehog pathway disruption evoked mitochondrial dysfunction and induced pre-mature aging. Importantly, analysis of human bulk RNA-seq datasets revealed an inverse relationship between Hedgehog activity and the severity of liver fibrosis, such that activity of this pathway declined progressively as lipotoxicity, and fibrosis severity worsened in NAFLD. These results suggest Hedgehog controls hepatocyte resiliency in adult livers. Loss of hepatocyte Hedgehog activity induces metabolic stress, disrupts energy homeostasis, and promotes NAFLD and its metabolic sequela. NAFLD encompasses a broad spectrum of fatty liver damage because both lipid-related injury (lipotoxicity) and responses to prevent and repair injury are quite dynamic. During the early stage of NAFLD (known as NAFL or hepatic steatosis), most people are asymptomatic. However, when excessive fat accumulation in hepatocytes is accompanied by recurrent bouts of lipotoxicity, inflammation and wound healing, hepatocyte death increases, marking the transition to NASH. When lipotoxicity persists and repair responses are maladaptive chronically, functional hepatocytes cannot be replaced and scarring (fibrosis) progresses; cirrhosis eventually results and risk for liver cancer is increased. Because our initial studies showed that Hedgehog is critical for maintaining liver metabolic homeostasis and thus, regulates susceptibility to NAFL and NASH, we also studied whether hepatocyte Hedgehog activity might be important for the liver to respond appropriately to a regenerative challenge and thereby, impact susceptibility to cirrhosis and/or liver cancer... Using single-cell technology, we first studied how individual hepatocytes with normal Hedgehog activity respond to liver injury to achieve robust regeneration. We discovered that when challenged to regenerate, mature hepatocytes functionally diversify. Some continue to fulfill liver specific functions, while others undergo dedifferentiation to give rise to a fetal-like progenitor population that can be further subdivided into hepatic, biliary and bipotent lineages. Work by us and others indicates that adult-to-fetal reprogramming is a reversible pre-requisite for effective liver regeneration and suggest that changes in Hedgehog signaling may be involved in the process. Remarkably, when we disrupted Hedgehog signaling in hepatocytes, formation of both hepatic and bipotent proliferative progenitors was abrogated; the dedifferentiation process became skewed towards biliary cells and liver regeneration was inhibited significantly. Together, these results demonstrate that hepatocyte Hedgehog activity is critical for effective liver repair and that failure to activate this signaling impairs hepatocyte regeneration by interrupting hepatic lineage specification during the adult-to-fetal reprogramming. Together, these studies reveal that Hedgehog has fundamental roles in regulating liver homeostasis and regeneration. My research uncovers hepatic and systemic responses that are orchestrated by Hedgehog. In particular, the single-cell transcriptome data provide a powerful resource that will enable discovery of unique hepatocyte states in both normal and diseased livers, including NAFLD. Importantly, the new data also have implications for pathology in other organs that become damaged due to metabolic stress.
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