High-mobility group box 1 is dispensable for autophagy, mitochondrial quality control, and organ function in vivo

Abstract

In vitro studies have demonstrated a critical role for high-mobility group box 1 (HMGB1) in autophagy and the autophagic clearance of dysfunctional mitochondria, resulting in severe mitochondrial fragmentation and profound disturbances of mitochondrial respiration in HMGB1-deficient cells. Here, we investigated the effects of HMGB1 deficiency on autophagy and mitochondrial function in vivo, using conditional Hmgb1 ablation in the liver and heart. Unexpectedly, deletion of Hmgb1 in hepatocytes or cardiomyocytes, two cell types with abundant mitochondria, did not alter mitochondrial structure or function, organ function, or long-term survival. Moreover, hepatic autophagy and mitophagy occurred normally in the absence of Hmgb1, and absence of Hmgb1 did not significantly affect baseline and glucocorticoid-induced hepatic gene expression. Collectively, our findings suggest that HMGB1 is dispensable for autophagy, mitochondrial quality control, the regulation of gene expression, and organ function in the adult organism.

Figure 1. Mice with hepatocyte-specific deletion of Hmgb1 develop normally and do not display significant alterations in hepatic gene expression

(A) Growth curves of Hmgb1^f/f and Hmgb1^Δhep mice do not diverge (n≥6 mice per group and time point). (B) Quantification of Hmgb1 deletion via qPCR (n=4 per group) and western blot analysis on whole liver extracts reveals efficient reduction of Hmgb1 levels in Hmgb1^Δhep livers (−90% and −72%, respectively, both p<0.001). (C) Immunohistochemical staining demonstrates HMGB1 deletion in hepatocytes, but not in non-parenchymal cells (arrows) in Hmgb1^Δhep livers. (D) Microarray heatmap of Hmgb1^f/f and Hmgb1^Δhep livers, showing significant changes of Hmgb1 and only 6 additional probesets in a total of >30,000 probesets in Hmgb1^Δhep livers. (E) qPCR shows similar hepatic mRNA levels of albumin, AFP and inflammatory and proliferative genes in Hmgb1^f/f and Hmgb1^Δhep mice (n=3/group). (F–G) Serum ALT levels (F) and hepatic triglyceride content (G) show no differences in liver injury and steatosis in 8-week-old mice (n=5/group). (H) H&E-, Oil-red-O-, PAS-, and TUNEL-stained sections of livers from 8-week old mice reveal no differences in liver architecture, fat and glycogen content or apoptosis in the absence of hepatocyte HMGB1. Scale bars = 20 μm (C) and 50 μm (H). Data are represented as mean ± SD. ** p <0.01; n.s. = non-significant. See also Figure S1 and Table S1.

Figure 2. Liver-specific Hmgb1 deletion does not affect mitochondrial function

(A–B) Blood glucose before and after 24h of starvation (A) and in response to glucagon administration (16 ng/g i.p. following 6h of starvation) (B) demonstrate normal regulation of glucose homeostasis in the absence of hepatocyte HMGB1. (C–D) Enzymatic activity analysis of key respiratory chain enzymes (C), and histochemical activity staining for respiratory chain complexes II (COX) and IV (SDH) activities (D) reveal no differences between Hmgb1^f/f (n=5) and Hmgb1^Δhep (n=5) mice. (E–F) Quantification of whole liver ATP levels (E) and ATP/ADP ratio (F) as end points of energy substrate generation demonstrate completely preserved mitochondrial function and ATP generation in Hmgb1^Δhep livers (n=5/group). (G) Whole body metabolic assessment of 4 month-old male Hmgb1^f/f and Hmgb1^Δhep mice (n=4 per group) reveals similar activity levels, energy expenditure and respiratory exchange rates in mice from both groups. (H) Real-time assessment of oxidative phosphorylation using an extracellular flux analyzer after sequential addition of oligomycin (2 μM), FCCP (1 μM), 2-DG (100 mM), and rotenone (1 μM) demonstrates similar basal OXPHOS, glycolysis and reserve capacity, and a similar glutamine-induced increase of OCR in isolated primary hepatocytes from Hmgb1^Δhep and Hmgb1^f/f mice (n=5 isolations each). Scale bars = 100 μm. Data are represented as mean ± SD. n.s. = non-significant.

Figure 3. Mitochondrial structure and mitophagy are preserved in mice with a hepatocyte-specific Hmgb1 deletion

(A) Electron microscopy of liver sections shows normal mitochondrial ultrastructure in Hmgb1^Δhep livers. (B–D) Primary hepatocytes from Hmgb1^f/f LC3-GFP-positive (n=3) and Hmgb1^Δhep LC3-GFP-positive double-transgenic mice (n=3) loaded with MitoTracker Red (100 nM) both show regularly shaped mitochondria, with only few GFP-positive puncta under baseline conditions (B). Stimulation of the cells with glucagon (2 μM for 90 min) or rotenone (1 μM for 6 h) results in a robust induction of GFP-positive organelles (lysosomes) (C) that partially colocalize with MitoTracker-stained mitochondria (inlays) (D). (E–F) 24h-starvation of Hmgb1^f/f LC3-GFP-positive (n=3) and Hmgb1^Δhep (n=3) LC3-GFP-positive double-transgenic mice results in similar patterns of LC3-GFP expression in the liver, as assessed by confocal microscopy (E) and similar increases in LC3-GFP cleavage (F). Scale bars = 2 μm (A), 10 μm (B) and 50 μm (E). Data are represented as mean ± SD. n.s. = nonsignificant. See also Figure S2.

Figure 4. Cardiomyocyte-specific deletion of Hmgb1 does not affect mitochondrial structure and organ function

(A) Growth curves of Hmgb1^f/f and Hmgb1 ^CM mice do not diverge (n=5 mice per group and time point). (B–C) Efficient deletion of Hmgb1 from Hmgb1^ΔCM hearts is demonstrated by qPCR and western blot (B), and immunohistochemistry (C) (n=4/group). (D) Electron microscopy demonstrates comparable mitochondrial morphology in Hmgb1^f/f and Hmgb1^ΔCM cardiomyocytes. (E) Quantitative assessment of cardiac ATP levels and ATP/ADP ratios and respiratory chain enzyme activities reveal normal mitochondrial function in the absence of cardiomyocyte HMGB1. (F) Histochemical activity staining for respiratory chain complexes II (COX) and IV (SDH) activities reveals no differences between Hmgb1^f/f and Hmgb1^ΔCM mice. (G) Echocardiographic assessment of cardiac architecture and function reveals normal ejection fraction, fractional shortening, heart rate and relative wall thickness in 4 month-old Hmgb1^ΔCM mice (n=5/group). (H–I) qPCR (H) and western blot analysis (I) demonstrate similar Hspb1 mRNA and protein expression in the heart irrespective of the HMGB1 status (n=4/group). Scale bars = 50 μm (C), 2 μm (D) and 100 μm (F). Data are represented as mean ± SD. * p <0.05; ** p <0.01; n.s. = non-significant. See also Figure S3.