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. Author manuscript; available in PMC: 2015 Mar 4.
Published in final edited form as: Cell Metab. 2014 Mar 4;19(3):539–547. doi: 10.1016/j.cmet.2014.01.014

High Mobility Group Box 1 is Dispensable for Autophagy, Mitochondrial Quality Control and Organ Function in Vivo

Peter Huebener 1,*, Geum-Youn Gwak 1,2,*, Jean-Philippe Pradere 1, Catarina M Quinzii 3, Richard Friedman 4, Chyuan-Sheng Lin 5, Chad M Trent 6,7, Ingmar Mederacke 1, Enpeng Zhao 8, Dianne H Dapito 7, Yuxi Lin 7, Ira J Goldberg 6,7, Mark J Czaja 8, Robert F Schwabe 1,7
PMCID: PMC4099361  NIHMSID: NIHMS562524  PMID: 24606906

SUMMARY

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.

INTRODUCTION

High mobility group box 1 (HMGB1) is an evolutionarily conserved non-histone nucleoprotein with abundant expression in most mammalian cells (Mosevitsky et al., 1989; Muller et al., 2004). Through interactions with the minor groove of DNA, HMGB1 may facilitate the assembly of nucleoprotein complexes such as the transcription machinery, nuclear hormone receptors, and V(D)J recombinases (Agresti and Bianchi, 2003; Ueda and Yoshida, 2010). Mice carrying a global deletion of Hmgb1 die from hypoglycemia shortly after birth (Calogero et al., 1999), which has precluded further analysis of HMGB1 functions in vivo.

Recently, it was demonstrated that immortalized mouse embryonic fibroblasts (MEFs) from Hmgb1-deficient mice as well as mesenchymal and epithelial cell lines with shRNA-mediated knockdown of Hmgb1 exhibit defective mitochondrial respiration and ATP synthesis due to the accumulation of dysfunctional mitochondria (Tang et al., 2011). This phenotype was ascribed to defective autophagic clearance of mitochondria resulting from decreased expression of heat shock protein beta-1 (HSPB1), a transcriptional target of HMGB1 that mediates interactions between mitochondria and the cytoskeleton during mitophagy (Tang et al., 2011). Collectively, these studies attributed HMGB1 with a pivotal role in the regulation of mitochondrial quality and the maintenance of cellular energy homeostasis in vitro. Here, we generated mice with conditional HMGB1 ablation, allowing us for the first time to characterize functions of HMGB1 in vivo. In the current study, we analyzed how Hmgb1 deficiency affects mitophagy, mitochondrial function, gene expression, and organ function in the liver and the heart, two metabolically highly active organs with abundant mitochondria.

RESULTS

Characterization of mice with conditional Hmgb1 deletion

In order to circumvent the early postnatal lethality of the global Hmgb1 knockout (Calogero et al., 1999), and to investigate the role of HMGB1 in tissue-specific contexts, we generated a transgenic mouse in which exons 2–4, encoding 156 out of the 215 amino acids of Hmgb1, are flanked by loxP-sites (Hmgb1f/f mice, Figures S1A and S1B). Germline deletion of Hmgb1 by our targeting strategy, using EIIA-Cre (Lakso et al., 1996), resulted in lethality of all Hmgb1−/− newborn mice, thus reproducing the phenotype of the global knockout (Figure S1C). Moreover, HMGB1 deletion was highly efficient at both the mRNA and protein level, as demonstrated by a 98% and 92% reduction of expression, respectively, in Mx1-Cre transgenic mice (Figures S1D and S1E). Together, these results confirm efficient and functional deletion by our targeting approach, and lay the basis for investigating the role of Hmgb1 in mitochondrial functionality and quality control in vivo by conditional ablation strategies in this study.

To better understand the contribution of Hmgb1 to mitochondrial function and energy homeostasis in vivo, we first investigated the function of HMGB1 in the liver, an organ with abundant mitochondria and a key role in glucose homeostasis. For this purpose, we crossed Hmgb1f/f with albumin-Cre transgenic mice (Hmgb1Δhep). Hmgb1Δhep mice exhibited normal survival and were indistinguishable from Hmgb1f/f littermates, without differences in growth and development up to the age of 40 weeks (Figure 1A). Quantitative real-time PCR (qPCR) and western blot analysis demonstrated efficient hepatic Hmgb1 deletion with 90% and 72% reduction of mRNA and protein levels, respectively (Figure 1B), and HMGB1 immunohistochemistry confirmed highly efficient deletion in hepatocytes, but not in non-parenchymal liver cells (Figure 1C). Microarray analysis of gene expression in Hmgb1Δhep livers showed significant differences in the expression levels of only six probesets besides Hmgb1 out of >30,000 probesets tested, indicating that HMGB1 is not a major regulator of gene transcription in the normal adult liver (Figure 1D). Of note, expression of the putative HMGB1 downstream molecule HSPB1 was unaltered in the absence of HMGB1 in hepatocytes, as assessed by microarray analysis and confirmed by quantitative real time PCR (qPCR) (Figure S1F). HSPB1 protein levels were undetectable by western blot in Hmgb1f/f and Hmgb1Δhep livers (Figure S1F). Notably, we also did not observe a role for HMGB1 in glucocorticoid receptor-induced hepatic gene expression (Figure S1G), a pathway that had been postulated to be dysregulated in the absence of HMGB1 (Calogero et al., 1999). Moreover, we did not observe signs of hepatocellular injury, liver dysfunction, hepatic steatosis, inflammation, fibrosis or altered proliferation as determined by serum liver function tests, TUNEL, Sirius Red and Oil-red-O staining, as well as qPCR for Alb, Afp, Col1a1, mKi67, Tnf or Ccl2, in Hmgb1Δhep mice (Figures 1E–H, S1H and Table S1).

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

Figure 1

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(A) Growth curves of Hmgb1f/f and Hmgb1Δhep 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 Hmgb1Δhep 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 Hmgb1Δhep livers. (D) Microarray heatmap of Hmgb1f/f and Hmgb1Δhep livers, showing significant changes of Hmgb1 and only 6 additional probesets in a total of >30,000 probesets in Hmgb1Δhep livers. (E) qPCR shows similar hepatic mRNA levels of albumin, AFP and inflammatory and proliferative genes in Hmgb1f/f and Hmgb1Δhep 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.

Functionally, Hmgb1Δhep mice displayed normal resting blood glucose and responded to 24h of starvation in the same manner as their Hmgb1f/f littermates (Figure 2A). Conversely, challenging fasted Hmgb1Δhep mice with glucagon led to a similar transient increase of blood sugar when compared to Hmgb1f/f littermates, thus excluding any overt disturbance of glucose homeostasis or glucagon receptor functionality in the absence of hepatocyte HMGB1 (Figure 2B). Moreover, we observed no differences in gluconeogenesis between Hmgb1f/f and Hmgb1Δhep mice, as determined by similar blood glucose and gluconeogenic gene expression after starvation, as well as a comparable transient increase in blood sugar following pyruvate administration (Figure S1I–J and Figure 2A).

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

Figure 2

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(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 Hmgb1f/f (n=5) and Hmgb1Δhep (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 Hmgb1Δhep livers (n=5/group). (G) Whole body metabolic assessment of 4 month-old male Hmgb1f/f and Hmgb1Δhep 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 Hmgb1Δhep and Hmgb1f/f mice (n=5 isolations each). Scale bars = 100 μm. Data are represented as mean ± SD. n.s. = non-significant.

HMGB1 does not regulate mitochondrial function and autophagy

To accurately assess mitochondrial function in the livers of Hmgb1Δhep mice, we next performed enzymatic analysis of respiratory chain enzyme activity. This analysis did not reveal differences in the activity levels of complexes IV, I+III, II+III and II as well as citrate synthase in whole liver extracts (Figure 2C). Accordingly, histochemical activity stainings of complex IV (cytochrome c oxidase, COX) and complex II (succinic dehydrogenase, SDH) in fresh frozen liver sections were comparable in both groups (Figure 2D), as were whole tissue levels of ATP and ATP/ADP ratio, two indicators of energy substrate generation (Figure 2E and F). In addition, whole body metabolic assessment did not reveal significant differences in food intake, total movement, energy expenditure or respiratory exchange ratio between both groups (Figure 2G). In summary, our data showed no indication for impaired respiratory chain activities, oxidative phosphorylation (OXPHOS) and ATP generation in the normal adult liver despite efficient deletion of HMGB1.

To investigate mitochondrial function under cellular stress, we next employed an extracellular flux analyzer, allowing for real-time assessment of OXPHOS and highly sensitive detection of mitochondrial dysfunction (Qian and Van Houten, 2010), in isolated primary hepatocytes from Hmgb1f/f and Hmgb1Δhep mice. The oxygen consumption rate (OCR) as an indicator of cellular respiration was analyzed after serial addition of oligomycin, p-trifluoromethoxy carbonylcyanide phenyl hydrazone (FCCP), 2-deoxyglucose (2-DG) and rotenone, allowing for detailed assessment of mitochondrial respiration. We did not observe differences in basal OXPHOS, ATP production, maximal respiration, or reserve capacity between both groups using glucose as the energy substrate (Figure 2H). In addition, we observed a similar increase in OCR in hepatocytes from Hmgb1f/f and Hmgb1Δhep mice using glutamine to force mitochondrial respiration (Figure 2H). Together, these data exclude a critical involvement of HMGB1 in the maintenance of mitochondrial function even under cellular stress.

In light of the close relationship between mitochondrial morphology and function (Chan, 2006) and the observed effect of Hmgb1 deficiency on mitochondrial morphology in vitro (Tang et al., 2011), we next visualized mitochondria in the livers and in isolated primary hepatocytes from Hmgb1Δhep mice. Hepatic mitochondrial morphology - as assessed by electron microscopy of liver sections (Figure 3A) and in isolated primary hepatocytes stained with MitoTracker Red (Figure 3B) or tetramethylrhodamine (not shown) – and size (1.33±0.27 μm in Hmgb1f/f vs. 1.34±0.40 μm Hmgb1Δhep mice) did not differ between Hmgb1Δhep and Hmgb1f/f littermates, without signs of increased mitochondrial fragmentation in hepatocytes from Hmgb1Δhep mice. The induction of autophagy was analyzed in triple transgenic mice carrying GFP-tagged LC3 (Hmgb1f/f LC3-GFP-positive and Hmgb1Δhep LC3-GFP-positive, respectively), which allows for monitoring of autophagosome formation via confocal microscopy (Mizushima et al., 2004). Isolated primary hepatocytes from both groups of mice displayed low numbers of small GFP-positive puncta following isolation (Figure 3B), but readily upregulated GFP-positive cup- and ring-like structures representing isolation membranes and autophagosomes (Mizushima et al., 2004) after exposure to glucagon or rotenone, two well-established inducers of autophagy (Figure 3C). Co-localization of MitoTracker-stained mitochondria with GFP-positive lysosomes did not reveal signs of impaired mitophagy in the absence of HMGB1 (Figure 3D). Similar observations were made in vivo, where starvation, a well-established trigger of hepatic autophagy (Singh et al., 2009), induced a similar hepatic pattern of LC3-GFP puncta (Figure 3E), and comparable hepatic autophagic flux as measured by LC3-GFP cleavage (Figure 3F) in Hmgb1f/f and Hmgb1Δhep mice. There was also no difference in the hepatic LC3-II/actin ratio or p62 levels between groups after treatment with leupeptin as a measure of autophagic flux (Figure S2A–B). Importantly, the macroscopic appearance of livers as well as the respective liver-body weight ratios did not differ between Hmgb1f/f and Hmgb1Δhep mice, contrasting the profound hepatomegaly and disturbances of liver architecture that is typical for autophagy-deficient livers (Komatsu et al., 2005) and observed in age-matched Atg7Δhep mice following Cre-mediated recombination (Figures S2C–E). Similar to Hmgb1Δhep mice, a normal liver phenotype was also found after Mx1-Cre-mediated deletion of Hmgb1 (data not shown). Moreover, we also observed no difference in the LC3-II/actin ratio between chloroquine-treated Hmgb1f/f and Hmgb1-deleted macrophages (Figure S2F–G).

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

Figure 3

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(A) Electron microscopy of liver sections shows normal mitochondrial ultrastructure in Hmgb1Δhep livers. (B–D) Primary hepatocytes from Hmgb1f/f LC3-GFP-positive (n=3) and Hmgb1Δhep 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 Hmgb1f/f LC3-GFP-positive (n=3) and Hmgb1Δhep (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.

Having established that mitochondrial quality and function are unaltered by Hmgb1-deficiency in hepatocytes in vitro and in vivo, we next sought to rule out a liver-specific effect of our findings that might explain differences to the previously reported role of Hmgb1 in MEFs and transformed cell lines. As a first approach, we investigated effects of Hmgb1-deficiency on mitochondrial morphology and cellular function in the heart, another organ with abundant mitochondria and high energy demand. Cardiomyocyte-specific Hmgb1 deletion via MHC-Cre (Hmgb1ΔCM) efficiently reduced Hmgb1 mRNA and protein in the heart, but did not result in lethality or gross developmental abnormalities, and mice developed normally up to eight months of age (Figure 4A–C). Electron microscopy demonstrated similar mitochondrial morphology and size (1.68±0.48 μm in Hmgb1f/f vs. 1.68±0.47 μm in Hmgb1ΔCM mice) in the hearts in both groups (Figure 4D). Tissue ATP levels, respiratory chain enzyme activities, and COX and SDH histochemical activities were not significantly altered in the hearts of Hmgb1ΔCM mice (Figure 4E and 4F). Echocardiographic assessment of myocardial function in four month-old (Figure 4G) and eight month-old Hmgb1ΔCM mice (data not shown) did not reveal structural abnormalities or defects in myocardial function or contractility. Moreover, cardiac Hspb1 mRNA levels were comparable between Hmgb1f/f and Hmgb1ΔCM mice (Figure 4H). In contrast to the liver, HSPB1 protein was abundant in the heart, and we observed similar HSPB1 expression in both groups (Figure 4I), further confirming our finding that HMGB1 is not involved in the regulation of an HSPB1-dependent mitochondrial quality control pathway. As a second approach, we ablated Hmgb1 via adenoviral Cre in MEFs from Hmgb1-floxed mice in vitro. Hmgb1 deletion was highly efficient, but we again did not observe effects of Hmgb1 deletion on mitochondrial morphology, HSPB1 protein or mRNA expression, ATP levels, cellular respiration or autophagy (Figure S3A–I).

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

Figure 4

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(A) Growth curves of Hmgb1f/f and Hmgb1 CM mice do not diverge (n=5 mice per group and time point). (B–C) Efficient deletion of Hmgb1 from Hmgb1ΔCM hearts is demonstrated by qPCR and western blot (B), and immunohistochemistry (C) (n=4/group). (D) Electron microscopy demonstrates comparable mitochondrial morphology in Hmgb1f/f and Hmgb1ΔCM 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 Hmgb1f/f and Hmgb1ΔCM 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 Hmgb1ΔCM 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.

DISCUSSION

Our results conclusively show that HMGB1 is not required for ATP production, cellular respiration, mitochondrial architecture or autophagy in tissues such as the liver and the heart that have abundant mitochondria and require functional autophagy for metabolism and organ function (Jaber et al., 2012; Komatsu et al., 2005; Singh et al., 2009). Accordingly, Hmgb1 deficiency had no adverse effect on cardiac or hepatic function. These findings are consistent with the concept that the main function of HMGB1 in the adult organism is exerted in the extracellular space. Indeed, we observed that sterile inflammation after tissue injury is profoundly suppressed in Hmgb1Δhep mice (manuscript in preparation), supporting the hypothesis that extracellular HMGB1 predominantly functions endogenous danger signal that triggers inflammation in response to cell death (Harris et al., 2012; Sims et al., 2010). The key role of HMGB1 in danger responses and sterile inflammation may be a reason for the high degree of evolutionary conservation and robust expression of this protein in virtually all mammalian tissues, despite the apparent expendability of HMGB1 for cellular homeostasis in the adult organism. Our data, showing normal glucose levels and normal glucocorticoid-induced gene regulation in Hmgb1Δhep mice, also suggest that HMGB1 functions in the adult mouse differ substantially from HMGB1 functions in development, where altered glucocorticoid receptor function and glucose regulation contribute to perinatal mortality in the global knockout (Calogero et al., 1999).

We cannot fully explain differences to previous studies that described a key role of HMGB1 in mitochondrial quality control and autophagy (Tang et al., 2010; Tang et al., 2011; Yanai et al., 2013) In contrast to Tang et al, we even found no influence of HMGB1 on mitochondrial shape, respiration, HSPB1 expression or autophagy in vitro in MEFs. As we employed adenovirally-delivered Cre to compare MEFs from the same embryo, we can also exclude clonal differences. One major difference between the two studies was that Tang et al employed immortalized MEFs and cancer cell lines suggesting that genetic alterations, e.g. p53 status, may contribute to some of these differences. In summary, results from our in vitro and in vivo findings collectively suggest that HMGB1 does not exert a major role in mitochondrial quality control or autophagy in non-transformed cells, and that it is not essential for normal organ function. Further studies are required to unravel mechanisms through which HMGB1 contributes to development and perinatal survival.

EXPERIMENTAL PROCEDURES

Additional experimental procedures are provided in the Supplemental Information.

Animals

All animal procedures were approved by the Columbia University Institutional Animal Care and Use Committee, and are in accordance with the “Guide for the Care and Use of Laboratory Animals” by the National Institutes of Health. Mice were maintained on a 12h dark/light cycle with free access to food and water unless otherwise indicated. For all experiments, 8–10 week old mice were used unless otherwise indicated. Mx1-Cre, Albumin-Cre, Vav1-Cre and EIIa-Cre mice were obtained from Jackson. MHC-Cre mice have been described (Agah et al., 1997). GFP-LC3 mice (Mizushima et al., 2004) were obtained from RIKEN, Japan. Floxed Atg7 mice (Komatsu et al., 2005) and Alb-CreERT2 mice (Schuler et al., 2004) have been described. Alb-CreERT2 activity was induced by i.p. injection of 0.1 mg tamoxifen for five consecutive days.

Generation of Hmgb1-floxed mice

A loxP site (L83) and a FRT-Neo-FRT-LoxP (FNFL) cassette were engineered by recombineering to flank exons 2–4 of the Hmgb1 gene, thus generating the Hmgb1 conditional targeting construct on a bacterial artificial chromosome. The FNFL cassette conferred G418 resistance during gene targeting in PTL1 ES cells, and the DTA cassette provided autonomous negative selection to reduce random integration. After transfection of the targeting construct, five targeted ES cell clones were identified by PCR screening. Two clones were injected into C57BL/6 blastocysts to generate chimeric mice. Two chimeric males conferred germline transmission. Male chimeras were bred to mice expressing FLPe recombinase under the human ACTB promoter (ACTB:FLPe, obtained from Jackson labs) mice to remove the Neomycin cassette. Two founder lines, one from each ES clone, carrying the Hmgb1 conditional allele were selected to determine the effect of homozygosity and Cre-mediated deletion. As we observed no abnormalities in homozygous mice and efficient Hmgb1 deletion in both lines, we conducted all subsequent experiments in one of the two founder lines. For the current study, mice were backcrossed to C57Bl/6 mice for 5 generations.

Autophagy analysis

Hmgb1f/f or Hmgb1Δhep mice, crossed with LC3-GFP mice, were used to visualize GFP-labeled lysosomes by confocal microscopy. For in vitro experiments, cells were stimulated with rotenone (1 μM) for 6h, glucagon (2 μM) for 90 min or chloroquine (2 μM) for 24h to induce autophagy. 60 min before microscopy, mitochondria were labeled with MitoTracker Red (100 nM). Confocal microscopy was performed on a A1R MP confocal microscope (Nikon Instruments, Melville, NY), and LC3-GFP positive puncta were quantified in 20–50 cells from 5–10 random fields. Mitophagy was assessed by colocalization of LC3 with mitochondria (Kim and Lemasters, 2011). For in vivo assessment of autophagy, Hmgb1f/f LC3-GFP-positive and Hmgb1Δhep LC3-GFP-positive mice were starved for 24h, and GFP expression was assessed by confocal microscopy, and LC3-GFP cleavage by western blot as a measure of autophagic flux (Mizushima et al., 2010). LC3 western blots were performed as described (Singh et al., 2009).

Real-time assessment of oxidative phosphorylation

Oxidative phosphorylation was monitored with the Extracellular Flux Analyzer XF24 (Seahorse Bioscience Inc., North Billerica, MA) by monitoring the oxygen consumption rate (OCR) as an indicator of cellular respiration and a sensitive detection method for mitochondrial dysfunction in real time (Qian and Van Houten, 2010). 50,000 cells were seeded in 24-well plates and incubated overnight. Prior to measurements, cells were washed with unbuffered media twice, then immersed in 675 μl unbuffered media and incubated in the absence of CO2 for 60 min. After initial measurements of basal respiration, the OCR was measured in accordance to the manufacturer’s instructions following the injection of compounds that inhibit the respiratory mitochondrial electron transport chain, ATP synthesis, or glycolysis. Mitochondrial respiration was additionally assessed by incubating cells with 2 mM glutamine.

Statistical analysis

All data are expressed as mean ± standard deviation (SD). For comparison of two groups, two-sided unpaired t-test or Mann-Whitney test were used. For comparison of multiple groups, we performed one-way ANOVA followed by Tukey’s post-hoc multiple comparison test. A p value < 0.05 was considered significant.

Supplementary Material

01

HIGHLIGHTS.

  • HMGB1 is not required for normal mitochondrial architecture and function in vivo

  • HMGB1 is not required for organ function in the liver and heart

  • HMGB1 is dispensable for mitophagy, autophagy or gene expression in the liver

Acknowledgments

The study was supported by NIH grants 1U01AA021912, 5U54CA163111 and 5R01DK076920 (to RFS), 5R01DK061498 (to MJC), and HL45095 and HL73029 (to IJG). Peter Huebener was supported the German Research Foundation (Hu 1953/1-1) and Chad Trent by the American Heart Association (Founders Affiliate). Seahorse experiments were supported by NIH grant P60DK20541 (Diabetes Training and Research Center of Albert Einstein College of Medicine).

Footnotes

All authors declare no conflict of interest.

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