The adenosine monophosphate–activated protein kinase—vacuolar adenosine triphosphatase–pH axis: A key regulator of the profibrogenic phenotype of human hepatic stellate cells

Nonalcoholic steatohepatitis (NASH) is the most severe form of nonalcoholic fatty liver disease (NAFLD), which to date has no approved drug treatments. There is an urgent need for better understanding of the genetic and molecular pathways that underlie NAFLD/NASH, and currently available preclinical models, be they in vivo or in vitro, do not fully represent key aspects of the human disease state. We have developed a human in vitro co‐culture NASH model using primary human hepatocytes, Kupffer cells and hepatic stellate cells, which are cultured together as microtissues in a perfused three‐dimensional microphysiological system (MPS). The microtissues were cultured in medium containing free fatty acids for at least 2 weeks, to induce a NASH‐like phenotype. The co‐culture microtissues within the MPS display a NASH‐like phenotype, showing key features of the disease including hepatic fat accumulation, the production of an inflammatory milieu, and the expression of profibrotic markers. Addition of lipopolysaccharide resulted in a more pro‐inflammatory milieu. In the model, obeticholic acid ameliorated the NASH phenotype. Microtissues were formed from both wild‐type and patatin‐like phospholipase domain containing 3 (PNPLA3) I148M mutant hepatic stellate cells. Stellate cells carrying the mutation enhanced the overall disease state of the model and in particular produced a more pro‐inflammatory milieu. Conclusion: The MPS model displays a phenotype akin to advanced NAFLD or NASH and has utility as a tool for exploring mechanisms underlying the disease. Furthermore, we demonstrate that in co‐culture the PNPLA3 I148M mutation alone can cause hepatic stellate cells to enhance the overall NASH disease phenotype.

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“…Cells were isolated as previously described. (26) In total, nine HSC donors were used throughout the study (Supporting Table S1).…”

Section: Methods and Materials Phh Culture And Treatmentmentioning

confidence: 99%

A Microphysiological System for Studying Nonalcoholic Steatohepatitis

et al. 2019

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“…Partial or isoform-specific loss of function in different tissues can cause rare congenital human diseases, such as distal renal tubular acidosis with sensorineural deafness (7), osteopetrosis (8,9), cutis laxa (10), and infertility (11). A pathogenic gain of V-ATPase function is implicated in cancer (12), liver cirrhosis (13), and osteoporosis (14). In addition, V-ATPases regulate the mTORC1, AMP-activated protein kinase, Notch-Delta, and Wnt signaling pathways (15).…”

Section: The Low-level Endo-lysosomal Signaling Lipid Phosphatidylinmentioning

confidence: 99%

Interaction of the late endo-lysosomal lipid PI(3,5)P2 with the Vph1 isoform of yeast V-ATPase increases its activity and cellular stress tolerance

Banerjee

Clapp

Tarsio

et al. 2019

Journal of Biological Chemistry

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“…In cells, Baf‐A1 usually targets to the lysosomes for regulating cellular acidification (Yamamoto et al, ). The acidification of lysosomes revealed by the incubation with acridine orange was completely inhibited when cells were treated with 0.1–1 μM bafilomycin A1, then intracellular pH robust increased (Marrone et al, ; Pejler, Hu Frisk, Sjostrom, Paivandy, & Ohrvik, ; Yoshimori, Yamamoto, Moriyama, Futai, & Tashiro, ). Generally, cell acidification is essential for cell proliferation, autophagy, and apoptosis.…”

Section: Introductionmentioning

confidence: 99%

Inhibition of silkworm vacuolar‐type ATPase activity by its inhibitor Bafilomycin A1 induces caspase‐dependent apoptosis in an embryonic cell line of silkworm

Tan

Wang

Liu

et al. 2018

Arch Insect Biochem Physiol

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Vacuolar‐type ATPase (V‐ATPase) is a type of hydrogen ion transporter located in the vesicular membrane‐like system, which mediates active transport and intracellular acidification in various compartments. In mammals, V‐ATPase has been reported to play a key role in cell proliferation and apoptosis. The studies of V‐ATPase in silkworm mainly focus on the acidification regulation of midgut and silk gland and immune resistance. However, there are few reports about the function of silkworm V‐ATPase on cell proliferation, autophagy, and apoptosis. Thus, the function of V‐ATPase in a cell line of Bombyx mori (BmE) was investigated by treating the cell line with bafilomycin A1, a specific inhibitor of V‐ATPase. Cell counting kit 8 (CCK8) and flow cytometry analysis showed that bafilomycin A1 treatment decreased the cell proliferation activity, affected the cell cycle progression and induced cell apoptosis. LysoTracker Red staining showed that the target of bafilomycin A1 is lysosome. The expression of all autophagy‐related genes ( BmATG5, BmATG6, and BmATG8) decreased, indicating that cell autophagy was inhibited. The analysis of the apoptosis pathway demonstrated that inhibiting the activity of V‐ATPase of BmE cells could promote mitochondria to release cytochrome C, inhibit the expression of BmIAP, and activate the caspase cascade to induce apoptosis. All these findings systematically illustrate the effects of V‐ATPase on the proliferation, autophagy, and apoptosis in BmE cells, and provide new ideas and a theoretical basis for further study on the function of V‐ATPase in BmE.

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The adenosine monophosphate–activated protein kinase—vacuolar adenosine triphosphatase–pH axis: A key regulator of the profibrogenic phenotype of human hepatic stellate cells

Abstract: The down-regulation of v-ATPase might represent a promising target for the development of antifibrotic strategies. (Hepatology 2018).

Cited by 14 publications

References 35 publications

A Microphysiological System for Studying Nonalcoholic Steatohepatitis

A Microphysiological System for Studying Nonalcoholic Steatohepatitis

Interaction of the late endo-lysosomal lipid PI(3,5)P2 with the Vph1 isoform of yeast V-ATPase increases its activity and cellular stress tolerance

Inhibition of silkworm vacuolar‐type ATPase activity by its inhibitor Bafilomycin A1 induces caspase‐dependent apoptosis in an embryonic cell line of silkworm

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