Mn porphyrin regulation of aerobic glycolysis: implications on the activation of diabetogenic immune cells

Abstract

Aims: The immune system is critical for protection against infections and cancer, but requires scrupulous regulation to limit self-reactivity and autoimmunity. Our group has utilized a manganese porphyrin catalytic antioxidant (MnTE-2-PyP(5+), MnP) as a potential immunoregulatory therapy for type 1 diabetes. MnP has previously been shown to modulate diabetogenic immune responses through decreases in proinflammatory cytokine production from antigen-presenting cells and T cells and to reduce diabetes onset in nonobese diabetic mice. However, it is unclear whether or not MnP treatment can act beyond the reported inflammatory mediators. Therefore, the hypothesis that MnP may be affecting the redox-dependent bioenergetics of diabetogenic splenocytes was investigated.

Results: MnP treatment enhanced glucose oxidation, reduced fatty acid oxidation, but only slightly decreased overall oxidative phosphorylation. These alterations occurred because of increased tricarboxylic acid cycle aconitase enzyme efficiency and were not due to changes in mitochondrial abundance. MnP treatment also displayed decreased aerobic glycolysis, which promotes activated immune cell proliferation, as demonstrated by reduced lactate production and glucose transporter 1 (Glut1) levels and inactivation of key signaling molecules, such as mammalian target of rapamycin, c-myc, and glucose-6-phosphate dehydrogenase.

Innovation: This work highlights the importance of redox signaling by demonstrating that modulation of reactive oxygen species can supplant complex downstream regulation, thus affecting metabolic programming toward aerobic glycolysis.

Conclusion: MnP treatment promotes metabolic quiescence, impeding diabetogenic autoimmune responses by restricting the metabolic pathways for energy production and affecting anabolic processes necessary for cell proliferation.

FIG. 1.

Stopping redox modulation leads to finite autoreactive immune cell control. BDC-2.5.TCR.Tg mice were treated for 7 days i.p. with MnP or HBSS at 10 mg/kg. (A) On day 8, splenocytes were harvested and adoptively transferred intravenously into NOD.scid recipients. Recipients were monitored by glucosuria and blood glucose and considered diabetic after two consecutive blood glucose readings of >300 mg/dL. n=3 transfers/group, p<0.0001. (B) On day 8, splenocytes were harvested for in vitro stimulation with 2.5 mimotope±MnP. At 48–96 h, supernatants were collected and used in an IFN-γ ELISA. Data show the average of independent experiments performed in triplicate from n=5 mice/group, *p<0.05. BDC, Barbara Davis Center; ELISA, enzyme-linked immunosorbent assay; HBSS, Hank's balanced salt solution; i.p., intraperitoneal; NOD, nonobese diabetic; Tg, transgenic; IFN, interferon.

FIG. 2.

Respiration is not significantly reduced following in vivo redox modulation. BDC-2.5.TCR.Tg mice were treated for 7 days i.p. with MnP or HBSS at 10 mg/kg. On day 8, splenocytes were harvested and analyzed in an Oroboros Respirometer. Dulbecco's minimal essential medium was used as an incubation media with 20 mM glucose as a substrate for oxidation. Oxygen consumption was measured as pmol/(s*Mill). (A) Respiration (O₂ flow per cell; open squares=control, open triangles=MnP) was determined at basal conditions and after the addition of mitochondrial inhibitors, oligomycin (Oligo) and rotenone (Rot), and additive amounts of the mitochondrial uncoupler FCCP. Total oxygen concentration also displayed (nmol/ml O₂; closed squares=control, closed triangles=MnP). Graph representative of typical Oroboros respirometry measurements. (B) Data show the average of independent experiments performed in triplicate from n=5 mice/group, *p<0.05, **p<0.005. FCCP, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone; pmol/(s*Mill), pmol/second/million cells.

FIG. 3.

Glucose oxidation efficiency is enhanced after redox modulation. BDC-2.5.TCR.Tg mice were treated for 7 days i.p. with MnP or HBSS at 10 mg/kg. On day 8, spleens were harvested. (A) Isolated splenocytes were given D-[6-¹⁴C] glucose and 2.5 μM cold glucose, incubated for 1 h, and ¹⁴CO₂ was measured. (B) Isolated splenocytes were given [1-¹⁴C] palmitic acid, incubated for 1 h, and ¹⁴CO₂ was measured. (C) Spleens were weighed, homogenized, boiled, and used in an ATP determination assay. ATP (nM) was quantified per mg of spleen. Data show the average of independent experiments performed in triplicate from n=3 mice/group, *p<0.05, **p<0.005. ATP, adenosine triphosphate.

FIG. 4.

MnP treatment does not significantly alter the amount of mitochondria. BDC-2.5.TCR.Tg mice were treated for 7 days i.p. with MnP or HBSS at 10 mg/kg. On day 8, splenocytes were harvested for protein lysates. (A) Whole cell lysates were probed for MitoOXPHOS antibody cocktail by western blot. Actin was probed as a loading control. Each complex is indicated based on its molecular weight. Data are representative of 3 independent experiments. (B) Densitometry was quantified for each complex by normalizing control and MnP-treated cells to actin. Expression of complex proteins quantified from 3 independent experiments. (C) Mitochondrial biogenesis mRNA levels were measured by qRT-PCR. The fold change of control samples were set arbitrarily to 1 and compared to MnP treatment. All samples were normalized to the endogenous GAPDH control. Data show the average of independent experiments performed in triplicate from n=4–5 mice/group, *p<0.05. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

FIG. 5.

Redox modulation sustains aconitase activity. BDC-2.5.TCR.Tg mice were treated for 7 days i.p. with MnP or HBSS at 10 mg/kg. On day 8, spleens were harvested. Spleens were homogenized and diluted to a concentration of 1000 μg/ml based on BCA assay. Aconitase activity was measured kinetically over 15 min. Median activity of independent experiments performed in triplicate from n=3 mice/group,*p<0.05.

FIG. 6.

MnP treatment decreases lactate production. BDC-2.5.TCR.Tg mice were treated for 7 days i.p. with MnP or HBSS at 10 mg/kg. On day 8, spleens were harvested. Spleens were weighed, homogenized, and used in a lactate assay. Lactate (nmol) was quantified per mg of spleen. Data show the average of independent experiments performed in triplicate from n=3 mice/group,*p<0.05.

FIG. 7.

Redox modulation leads to decreased aerobic glycolysis and PPP signaling. (A) Signaling important for the collaboration of glycolysis, cell cycle, protein synthesis, and PPP. (B) BDC-2.5.TCR.Tg mice were treated for 7 days i.p. with MnP or HBSS at 10 mg/kg. On day 8, splenocytes were harvested for protein lysates. Whole cell lysates were probed for Glut-1; c-Myc; phospho-mTOR and mTOR; phospho-p70s6K and phospho-PHAS; and G6PD by western blot. Actin was probed as a loading control for all panels. Data show representative blots of independent experiments from n=3–4 mice/group. G6PD, glucose-6-phosphate dehydrogenase; mTOR, mammalian target of rapamycin; PPP, pentose phosphate pathway; PHAS-1 (4E-BP1), eukaryotic initiation factor 4E-binding protein.