Heme oxygenase-1 drives metaflammation and insulin resistance in mouse and man

Abstract

Obesity and diabetes affect more than half a billion individuals worldwide. Interestingly, the two conditions do not always coincide and the molecular determinants of "healthy" versus "unhealthy" obesity remain ill-defined. Chronic metabolic inflammation (metaflammation) is believed to be pivotal. Here, we tested a hypothesized anti-inflammatory role for heme oxygenase-1 (HO-1) in the development of metabolic disease. Surprisingly, in matched biopsies from "healthy" versus insulin-resistant obese subjects we find HO-1 to be among the strongest positive predictors of metabolic disease in humans. We find that hepatocyte and macrophage conditional HO-1 deletion in mice evokes resistance to diet-induced insulin resistance and inflammation, dramatically reducing secondary disease such as steatosis and liver toxicity. Intriguingly, cellular assays show that HO-1 defines prestimulation thresholds for inflammatory skewing and NF-κB amplification in macrophages and for insulin signaling in hepatocytes. These findings identify HO-1 inhibition as a potential therapeutic strategy for metabolic disease.

Figure 1. HO-1 Levels Predict Insulin Resistance in Mouse and Man

(A and B) qPCR analysis of HO-1 mRNA in (A) liver and (B) visceral adipose biopsies obtained from obese insulin-sensitive (obIS) and obese insulin-resistant (obIR) humans. (C and D) Immunohistochemical staining for HO-1 in (C) liver and (D) obIR visceral fat biopsies. Scale bar, 50 µm. (E) Percentage of HO-1 positive hepatocytes in obIS and obIR liver biopsies. (F–I) HO-1 mRNA levels obtained from public microarray data sets; (F and G) liver (H and I) adipose tissue. Numbers in bars represent study participants. (J) Rank order of 968 visceral adipose tissue genes correlating with HOMA-IR and predicting metabolically healthy (251 protective genes) and unhealthy (717 risk genes) obesity. p values for Spearman correlation coefficients (HOMA-IR versus expression values) obtained from public data set. Dotted line represents the significance cut-off at p < 0.01, corrected for multiple testing. (K and L) qPCR analysis of HO-1 expression in (K) liver, and (L) epididymal adipose obtained from obese IS and obese IR C57BL/6J mice after 16 weeks on HFD. (M and N) Immunoblot analysis for HO-1 in (M) liver, and (N) epididymal adipose obtained from obese IR and obese IS C57BL/6J mice after 16 weeks of HFD. Results are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. See also Figure S1 and Tables S1 and S2.

Figure 2. Hepatocyte HO-1 Knockout Mice are Insulin Hypersensitive

(A) HO-1 immunoblot of liver obtained from control and Lhoko mice. (B) HO activity in liver homogenates, n ≥ 6 per group. (C) Immunohistochemical staining for HO-1 in livers of vehicle and hemin injected control and Lhoko mice. Scale bar, 100 µm. (D) Oral glucose and insulin tolerance tests on HFD. (E) HOMA-IR of control and Lhoko mice after 16 weeks on HFD. (F) Serum levels of liver enzymes after 16 weeks on 60% HFD, n ≥ 4 per group. (G) In vivo activation of liver insulin signaling. HFD-fed mice were injected with insulin, tissue biopsies taken at the indicated times, and analyzed by immunoblotting. A representative result from three independent experiments is shown. (H) Liver triglyceride content, n = 3 per group. (I) H&E, oil red O and HO-1 staining of representative liver sections obtained from HFD-fed control and Lhoko mice. Scale bar, 200 µm. (J) Insulin tolerance tests in AdLacZ and AdHO-1 injected C57BL/6J animals, n = 7 per group, p value 1 sided. (K) HOMA-IR of AdLacZ and AdHO-1 injected C57BL/6J animals, n = 7 per group. Results are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. See also Figure S2.

Figure 3. Macrophage HO-1 Knockout Mice Resist Metabolic Disease

(A) qPCR analysis of HO-1 mRNA levels in BMDMs generated from control and Macho mice, naive and polarized with LPS/IFN_γ (M1-like) or IL-4/IL-13 (M2-like) for 24 hr. (B) Immunoblot for HO-1 in naive BMDMs derived from indicated genotypes. (C) HO activity in naive BMDMs generated from LFD-fed control and Macho mice, n = 3 per group. (D) Body weight gain of control and Macho mice on 60% HFD. (E) Oral glucose and insulin tolerance tests, corresponding blood glucose and insulin levels, n = 10–15 per genotype. (F) Oral glucose and insulin tolerance tests, corresponding blood glucose and insulin levels in body-weight-matched animals, n = 7 per genotype. (G) Liver aspects and stainings of representative liver sections obtained from HFD-fed control and Macho mice for H&E, oil red O, HO-1 (red), and MAC-2 (brown). Scale bar, 50 µm. (H) Relative mRNA levels of inflammatory, lipogenic, and gluconeogenic genes in liver samples, n = 4 per group. (I) Liver triglyceride and cholesterol content, n ≥ 5 per group. (J and K) Energy expenditure and activity of body-weight-matched control and Macho mice, n = 7 per genotype. Results are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. See also Figure S3.

Figure 4. Macrophage HO-1 Knockout Mice Resist Metaflammation

(A) Serum-free fatty acid levels measured in fed and fasted states on HFD, n ≥ 7 per group. (B) H&E staining of epididymal adipose of HFD fed control and Macho mice. Scale bar, 200 µm. (C) Adipocyte size distribution in epididymal fat of HFD fed control and Macho mice as determined by quantitative morphometry of H&E stained tissue sections, n = 6 per group. n = number of adipocytes. (D) Immunofluorescence staining for MAC-2 and confocal imaging of whole-mount epididymal fat pads. A representative result from three independent experiments is shown. Scale bar, 200 µm. (E) Serum levels of proinflammatory cytokines, n ≥ 3 per group. (F) qPCR analysis of adipokines in epididymal fat isolated from control and Macho mice, n = 4 per group. (G) Serum adipokine levels in control and Macho mice, n = 4 per group. (H–J) Flow cytometric analysis of the SVF isolated from epididymal white adipose tissue (WAT) of HFD fed control and Macho mice; (H) Cells were gated for F4/80⁺ cells and examined for CD11b coexpression to obtain total macrophage numbers; (I) The number of CD11c⁺, CCR2⁺ and MGL⁺ adipose tissue macrophage subpopulations is presented; (J) distribution of CD11c⁺ MGL⁻ and CD11c⁻ MGL⁺ adipose macrophages, n = 3 mice per group. (K) Immunofluorescence staining for perilipin (red) and MAC-2 (green) of whole-mount epididymal fat. A representative result from three independent experiments is shown. Scale bar, 100 µm. Results are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. See also Figure S4, Tables S4 and S5.

Figure 5. Attenuated NF-κB Signaling and Anti-Inflammatory Metabolic Signature in Macrophage HO-1 KO Mice

(A) pIkBα and IkBα were measured by immunoblotting in BMDMs from control and Macho mice fed with LFD. Naive cells were stimulated with TNF-α (10 ng/ml) for the indicated times. β-Actin was used as internal loading control. (B) RelA/p65 nuclear translocation assay. Naive BMDMs were stimulated with TNF-α (10 ng/ml) for the indicated times and analyzed for RelA/p65 localization by immunofluorescent staining. Nuclei were stained with DAPI, and the percentage of nuclear RelA/p65 positive cells was counted. Arrows show nuclear RelA/p65. Scale bar, 50 µm. (C) EMSA. Nuclear extracts were prepared from control and Macho BMDMs stimulated with TNF-α (10 ng/ml) for the indicated times. Specific RelA/p65 and p50 binding was verified by adding a 100-fold excess of unlabeled double-stranded probe (cold oligo) harboring the NF-κB consensus site as well as supershift assays using anti-RelA/p65 and anti-p50 antibodies. (D) Relative mRNA expression of NF-κB target genes in BMDMs generated from LFD fed control and Macho mice after stimulation with vehicle or TNF-α (10 ng/ml) for 60 min, n = 3 per group. (E) qPCR analysis of NF-κB targets Tnf and Nos2 in chromatin immunoprecipitations prepared from control and Macho BMDMs stimulated with TNF-α (10 ng/ml). (F and G) Oxygen consumption rates (OCR) and mitochondrial function of naive BMDMs derived from control and Macho mice, n = 9 per group. (H) Cytokine secretion of LPS stimulated control and Macho BMDMs, n = 3 per group. Results are mean ± SEM n = 2–3 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 6. Lhoko and Macho Mice Display Skewed OxPhos, ROS Signaling, and NRF2/INSR Signaling Tone

(A) Oxygen consumption rates (OCR) and mitochondrial function of primary hepatocytes and naive BMDMs derived from Lhoko and Macho animals and their littermate controls. Note that for macrophages the same experiment as in Figure 5F is shown, n = 5 for hepatocytes, n = 9 for macrophages. (B) Relative mRNA levels of mitochondrial ROS detoxification genes in HO-1 knockout hepatocytes and macrophages as well as their corresponding controls, n = 3–4 per group. *, NRF2 target genes. (C) SOD activity measured in lysates obtained from primary hepatocytes and naive BMDMs, n = 4 per group. (D) Intracellular H₂O₂ levels in primary hepatocytes and naive BMDMs, n = 4 per group. (E) Oxygen consumption rates (OCR) and mitochondrial function of primary hepatocytes and naive BMDMs isolated from Lhoko and Macho animals as well as their littermate controls. N-acetyl-cysteine (NAC, 10 µM) was added 1 hr before measurements. Note that for untreated hepatocytes the same experiment as in (A) is shown, n = 4–6 per group. (F) Nuclear and cytosolic extracts were prepared from naive Macho and control BMDMs and analyzed for NRF2, Histone H3 (nuclear marker) and GAPDH (cytosolic marker) by immunoblotting. A representative result is shown. (G) NRF2 binding activity in nuclear extracts derived from naive Macho and control BMDMs, n = 4 per genotype. (H) Normalized PTP1B activity in protein extracts obtained from primary hepatocytes (Hep) as well as liver biopsies of LFD and HFD fed Lhoko and control animals, n = 3–4 per group. (I) In vivo activation of liver insulin signaling. LFD fed mice were injected with insulin, liver biopsies taken at the indicated times, and analyzed by immunoblotting. A representative result from three independent experiments is shown. Results are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. See also Figure S5.