Homocysteine Activates B Cells via Regulating PKM2-Dependent Metabolic Reprogramming

Abstract

The overactivation of immune cells plays an important role in the pathogenesis of hyperhomocysteinemia (HHcy)-accelerated atherosclerosis. Homocysteine (Hcy) activates B cell proliferation and Ab secretion; however, the underlying mechanisms for these effects remain largely unknown. Metabolic reprogramming is critical for lymphocyte activation and effector function. In this study, we showed that Hcy-activated B cells displayed an increase in both oxidative phosphorylation and glycolysis, with a tendency to shift toward the latter, as well as an accumulation of intermediates in the pentose phosphate pathway, to provide energy and biosynthetic substrates for cell growth and function. Mechanistically, Hcy increased both the protein expression and glycolytic enzyme activity of the pyruvate kinase muscle isozyme 2 (PKM2) in B cells, whereas the PKM2 inhibitor shikonin restored Hcy-induced metabolic changes, as well as B cell proliferation and Ab secretion both in vivo and in vitro, indicating that PKM2 plays a critical role in metabolic reprogramming in Hcy-activated B cells. Further investigation revealed that the Akt-mechanistic target of rapamycin signaling pathway was involved in this process, as the mechanistic target of rapamycin inhibitor rapamycin inhibited Hcy-induced changes in PKM2 enzyme activity and B cell activation. Notably, shikonin treatment effectively attenuated HHcy-accelerated atherosclerotic lesion formation in apolipoprotein E-deficient mice. In conclusion, our results demonstrate that PKM2 is required to support metabolic reprogramming for Hcy-induced B cell activation and function, and it might serve as a critical regulator in HHcy-accelerated initiation of atherosclerosis.

FIGURE 1.

HHcy induces B cell proliferation and Ab secretion both in vivo and in vitro. (A–E) C57BL/6J mice were fed a normal chow diet and given drinking water supplemented with or without 1.8 g/l Hcy for 2 wk. (A) Total cell numbers of splenic B cells purified from control or HHcy mice were counted. Plasma IgM (B) and IgG (C) levels were measured via ELISA. (D) IRF4 protein expression and quantification were analyzed via Western blot. β-Actin served as an internal control. (E) Gene expression of Aicda and postswitch transcripts (Iμ-Cγ3, Iμ-Cγ2b, Iμ-Cγ1) were measured via quantitative PCR in B cells. (F–K) Splenic B cells purified from C57BL/6J mice were cultured in vitro with or without 100 μM Hcy for the indicated times. (F) Purified B cells were labeled with CFSE prior to culture and cell proliferation was assessed by flow cytometry after 48 h. IgM (G) and IgG (H) levels in the culture supernatants after Hcy stimulation for 72 h were measured via ELISA. (I) After Hcy stimulation for 48 h, cells were labeled with CD138 and then analyzed via flow cytometry. (J) IRF4 protein expression and quantification in B cells at 24 h were analyzed via Western blot. (K) Gene expression of Aicda and postswitch transcripts were measured via quantitative PCR in B cells at 72 h. The data shown are representative [(A–D) and (F–I); upper panel in (J)] and cumulative [(E and K); lower panel in (J)] of at least three independent experiments [n = 3–5 mice in each group in (A–E)]. The data are presented as the mean ± SEM. *p < 0.05 compared with the control.

FIGURE 2.

Metabolic reprogramming in B cells induced by HHcy in vivo and in vitro. (A–C) Metabolic parameters of splenic B cells purified from control or HHcy C57BL/6J mice were analyzed using an extracellular flux analyzer. (D–H) Splenic B cells purified from C57BL/6J mice were cultured in vitro with or without 100 μM Hcy for 24 h. OCR and ECAR in B cells were determined via extracellular flux analysis. (A and D) The OCR over time was measured at a basal level and after the injection of oligomycin, FCCP, antimycin A, and rotenone. Basal OCR was determined before the addition of oligomycin, and maximal OCR was calculated by subtracting the nonmitochondrial OCR from the peak OCR following FCCP injection. SRC was calculated by subtracting basal OCR from max OCR. (B and E) The ECAR over time was measured at a basal level and after the injection of oligomycin, FCCP, antimycin A, and rotenone or the injection of glucose, oligomycin, and 2-DG. Basal ECAR was determined before the addition of oligomycin, and maximal ECAR was assessed after the addition of antimycin A and rotenone. Glycolysis was determined following the addition of glucose, and the glycolytic capacity was assessed after the addition of oligomycin. Glycolytic reserve was calculated by subtracting basal ECAR from max ECAR or by subtracting glycolysis from glycolytic capacity. (C and F) The OCR/ECAR ratio was calculated at the basal level. (G and H) B cells with or without Hcy treatment for 24 h were stained with 2-NBDG to assess glucose uptake via flow cytometry or imaging flow cytometry. Representative images of bright-field and 2-NBDG (green) are shown. The data shown are representative (A–G) and cumulative (H) of at least three independent experiments [n = 5 mice in each group in (A–C)]. The data are presented as the mean ± SEM. *p < 0.05 compared with the control. ns, not significant; Oligo, oligomycin.

FIGURE 3.

Distinct metabolomic profiles of Hcy-stimulated B cells. (A–C) Splenic B cells purified from C57BL/6J mice were cultured in vitro with or without 100 μM Hcy for 24 h. Metabolites were then extracted from three replicate B cell samples and analyzed using an LC-MS/MS system to determine the abundance of cellular metabolites. Metabolites were analyzed using a heat map generated from hierarchical clustering (A) and a principal component analysis (B) using MetaboAnalyst software. (C) Relative levels of metabolites in the glycolytic, pentose phosphate, and TCA cycle pathways are shown. The data shown are representative of three independent experiments. The data are presented as the mean ± SEM from triplicate samples. *p < 0.05 compared with the control. DHAP, dihydroxyacetone phosphate; FBP, fructose-1,6-bisphosphate; F6P, fructose-6-phosphate; GADP, glyceraldehyde-3-phosphate; G6P, glucose-6-phosphate; PEP, phosphoenolpyruvate; 3PG, 3-phosphoglycerate.

FIGURE 4.

Glucose metabolism is critical for Hcy-induced B cell activation. (A–D) Splenic B cells purified from C57BL/6J mice were pretreated with or without 0.5 mM or indicated doses of 2-DG for 30 min and were then cultured in the presence or absence of 100 μM Hcy for the indicated times. Proliferation of CFSE-labeled B cells (A) and CD138⁺ plasma cells (B) were identified via flow cytometry at 48 h. IgM (C) and IgG (D) levels in the culture supernatants were quantified via ELISA at 72 h. The data shown are representative of at least three independent experiments. The data are presented as the mean ± SEM. *p < 0.05 compared with the control. ^#p < 0.05 compared with the Hcy group.

FIGURE 5.

HHcy increases PKM2 expression and enzyme activity in B cells. (A) Western blot analysis with the indicated Abs was used to determine the protein expression of pyruvate kinase isoforms in B cells. Protein from muscle was loaded as a positive control for PKM1 expression. GAPDH served as an internal control. (B) Gene expression of total Pkm and pyruvate kinase isoforms in B cells was analyzed via RT-PCR, followed by digestion with NcoI (N), PstI (P), or an uncut control (U). Bands are detailed as follows: total Pkm1/2, uncut (511 bp); Pkm2, PstI-cleaved fragments (364 plus 98 plus 49 bp); and Pkm1, PstI-cleaved fragments (413 plus 98 bp) and NcoI-cleaved fragments (421 plus 84 plus 6 bp). Muscle was loaded as a positive control for Pkm1 expression. (C and D) Splenic B cells were purified from control or HHcy C57BL/6J mice. (E–G) Splenic B cells purified from C57BL/6J mice were cultured in vitro with or without 100 μM Hcy for 24 h in (E) and (F) or for the indicated times in (G). Cells were analyzed for PKM2 protein expression (C and E) via Western blotting and PKM2 enzyme activity (D and F) using an LDH-coupled enzyme assay. (G) Gene expression of Pkm2 was measured via quantitative PCR in B cells at the indicated times. The data shown are representative [(A–D); upper panel in (E)] and cumulative [lower panel in (E); (F and G)] of at least three independent experiments [n = 3–4 mice in each group in (C and D)]. The data are presented as the mean ± SEM. *p < 0.05 compared with the control.

FIGURE 6.

Akt-mTOR signaling pathway regulates PKM2 enzyme activity and mediates Hcy-induced B cell activation. (A and B) Western blot analysis and quantification of p-AKT, AKT, p-mTOR, and mTOR protein expression in B cells treated with 100 μM Hcy for 24 h. Eukaryotic translation initiation factor 5 served as an internal control. (C–H) Splenic B cells purified from C57BL/6J mice were preincubated with 10 nM rapamycin (Rapa) for 30 min, followed by stimulation with or without 100 μM Hcy for the indicated times. Western blot analysis of p-mTOR, mTOR, p-S6RP (C), PKM2 protein expression (D), PKM2 enzyme activity (E) at 24 h and cell proliferation (F) at 48 h were measured in B cells. IgM (G) and IgG (H) levels in culture supernatants at 72 h were measured via ELISA. The data shown are representative [upper panels in (A–D) and (E–H)] and cumulative [lower panels in (A–D)] of at least three independent experiments. The data are presented as the mean ± SEM. *p < 0.05 compared with the control. ^#p < 0.05 compared with the Hcy group. ns, not significant.

FIGURE 7.

Inhibition of PKM2 reverses Hcy-induced B cell proliferation and Ab secretion in vitro. (A) Schematic showing the rate-limiting step regulated by PKM2 in the glycolytic pathway and the inhibition of PKM2 by SKN. (B–I) Splenic B cells purified from C57BL/6J mice were preincubated with 0.25 μM or indicated doses of SKN for 30 min and then cultured with or without 100 μM Hcy for the indicated times. (J–N) Splenic B cells purified from C57BL/6J mice were transfected with scramble or PKM2 siRNA. They were then treated with or without 100 μM Hcy for 48 h. Measurements of PKM2 enzyme activity in B cells [at 24 h in (B) and at 48 h in (J)]. Cell lysates were used to measure PKM2 protein expression [at 24 h in (C) and 48 h in (K)] via Western blot analysis. (D and E) Basal and maximal OCR, SRC, glycolysis and glycolytic capacity at 24 h were analyzed via extracellular flux analysis. Cell proliferation (F), CD138⁺ plasma cells at 48 h (G and L), IgM [at 72 h in (H) and at 48 h in (M)], and IgG [at 72 h in (I) and at 48 h in (N)] levels in culture supernatants are shown. The data shown are representative [left panels in (C) and (K); (D–I) and (L)] and cumulative [right panels in (C) and (K); (B), (J), (M) and (N)] of at least three independent experiments. The data are presented as the mean ± SEM. *p < 0.05 compared with the control or scramble siRNA control. ^#p < 0.05 compared with the Hcy group or scramble siRNA plus Hcy group. ns, not significant.

FIGURE 8.

SKN inhibits B cell activation through regulating PKM2 and ameliorates HHcy-accelerated atherosclerosis. (A) Schematic flowchart of HHcy induction and SKN treatment in ApoE^−/− mice. HHcy was induced by giving mice drinking water supplemented with 1.8 g/l Hcy. Mice were i.p. injected with 1.2 mg/kg SKN every 3 d for the SKN treatment. Mice were divided in four groups: control (C), HHcy, SKN, and HHcy plus SKN. (B) Total numbers of splenic B cells purified from ApoE^−/− mice were counted. (C and D) Plasma IgM and IgG levels were measured via ELISA. (E) Splenic B cells from four groups were lysed and used to assess PKM2 enzyme activity using an LDH-coupled enzyme assay. (F and G) Time courses of OCR and ECAR in B cells are shown. Basal and maximal OCR and ECAR were analyzed via extracellular flux analysis. (H) Oil Red O staining of aortic roots (upper panel) isolated from control and HHcy mice with or without SKN treatment. Quantification of the mean atherosclerotic lesion area (lower panel) is shown. (I) Gene expression of Icam-1, Vcam-1, Tnf-α, Ifn-γ, Mcp-1, and Il-2 in thoracic aortas isolated from mice was measured via quantitative PCR. The data shown are representative (B–H) and cumulative (I) of at least two independent experiments (n = 3–5 mice in each group). The data are presented as the mean ± SEM. *p < 0.05 compared with the control. ^#p < 0.05 compared with the HHcy group.