Supplementary document for Reflective one-to-multi-polarization conversion metasurface - 6504161.pdf

supplementary materials of (i) discussion on transmission performance, (ii) derivations of Eqs. shown in manuscrip

Identification of Novel Pathways in Idelalisib Metabolism and Bioactivation

Idelalisib (ILB) is a selective phosphatidylinositol-3-kinase delta inhibitor approved for the treatment of hematological malignancies. However, ILB frequently causes hepatotoxicity, and the exact mechanism remains unclear. The current study profiled the metabolites of ILB in mouse liver, urine, and feces. The major metabolites found in the liver were oxidized metabolite GS-563117 (M1) and ILB-glutathione (GSH) adduct (M2). These metabolic pathways were confirmed by analysis of urine and feces from mice treated with ILB. Identification of ILB-GSH adduct (M2) suggests the formation of reactive metabolites of ILB. We also found that M1 can produce reactive metabolites and form M1-GSH adducts. The GSH-conjugates identified in mouse liver were also found in the incubations of ILB and M1 with human liver microsomes. Furthermore, we illustrated that CYP3A4 and 2C9 are the key enzymes contributing to the bioactivation pathway of ILB and M1. In summary, our work revealed that both ILB and its major metabolite M1 can undergo bioactivation to produce reactive metabolites in the liver. Further studies are required to determine whether these metabolic pathways contribute to ILB hepatotoxicity

Regulation of hepatic stellate cell proliferation and activation by glutamine metabolism

<div><p>Liver fibrosis is the excessive accumulation of extracellular matrix proteins, which is mainly caused by accumulation of activated hepatic stellate cells (HSCs). The mechanisms of activation and proliferation of HSCs, two key events after liver damage, have been studied for many years. Here we report a novel pathway to control HSCs by regulating glutamine metabolism. We demonstrated that the proliferation of HSCs is critically dependent on glutamine that is used to generate α-ketoglutarate (α-KG) and non-essential amino acid (NEAA). In addition, both culture- and in vivo-activated HSCs have increased glutamine utilization and increased expression of genes related to glutamine metabolism, including GLS (glutaminase), aspartate transaminase (GOT1) and glutamate dehydrogenase (GLUD1). Inhibition of these enzymes, as well as glutamine depletion, had a significant inhibitory effect on HSCs activation. In addition to providing energy expenditure, conversion of glutamine to proline is enhanced. The pool of free proline may also be increased via downregulation of POX expression. Hedgehog signaling plays an important role in the regulation of glutamine metabolism, as well as TGF-β1, c-Myc, and Ras signalings, via transcriptional upregulation and repression of key metabolic enzymes in this pathway. Finally, changes in glutamine metabolism were also found in mouse liver tissue following CCl4-induced acute injury. Conclusion: Glutamine metabolism plays an important role in regulating the proliferation and activation of HSCs. Strategies that are targeted at glutamine metabolism may represent a novel therapeutic approach to the treatment of liver fibrosis.</p></div

Glutamine is essential for HSCs proliferation.

<p>A&B: relative proliferation of LX2 cells (A,B) or primary HSCs (D) by BrdU staining or cell counting (C). Cells were plated in complete medium or Gln deficient medium with or without α-KG, NEAA, or the mixture of the two. BrdU-positive cells were quantified per optical section. Relative BrdU-positive cells for each group were analyzed. Error bars represent s.d. of triplicate samples from a representative experiment. *P < 0.05.</p

Gln metabolism influences transactivation of HSCs.

<p>A, primary HSCs were cultured for 7 days with or without glutamine. Autofluorescence was assessed. B &C, primary HSCs were cultured with or without Gln, Bptes, or AOAA. D, LX2 cells were treated with MDI. Relative changes of mRNA expression of Gln metabolism genes were analyzed by real-time PCR. Immunocytochemistry was used to assess the expression of col1A1 at protein level.</p

Gln metabolism enzyme inhibitors suppress HSCs proliferation.

<p>A&B, relative proliferation of LX2 cells (A) or primary HSCs (B) by BrdU staining assay. Cells were plated in complete medium and treated with Bptes (GLS inhibitor), EGCG (GLUD1 inhibitor) or AOAA (transaminase inhibitor) at different concentrations for 72 hrs.</p

Gln metabolism is reprogrammed during HSC transactivation.

<p>A, primary HSCs isolated from rats were cultured for 7 days. B, primary HSCs isolated from donor patients were cultured for 7 days. C, HSCs were isolated from control mice and mice with liver fibrosis induced by 8 weeks of CCl₄ treatment. Relative changes of mRNA expression of Gln metabolism genes were examined by RT-PCR.</p

Gln metabolism is reprogrammed by Hedgehog signaling, Ras, Myc and TGF-β1.

<p>Primary HSCs were treated with GDC-0449 (to inhibit SMO) (A), FTS (B) (Ras inhibitor), 10058-F4 (C) (Myc inhibitor). LX2 cells and MDI-pretreated LX2 cells were treated with TGF-β1. RT-PCR was used to analyze expression of glutamine metabolic enzyme genes.</p

Gln-Pro metabolism is reprogrammed in human fibrotic livers.

<p>Liver tissues were collected from donor patients. RT-PCR was used to analyze expression of glutamine metabolic enzyme genes (A). Proline metabolizing genes were also analyzed (B).</p

Gln metabolism is reprogrammed following acute liver injury.

<p>Mice received a single injection of CCl₄ and the expression of several glutamine metabolizing genes in liver tissue (A), HSCs (B) or hepatocytes (C) were examined 2 days later. POX expression was also analyzed (D).</p