Role of Arginase 2 in Systemic Metabolic Activity and Adipose Tissue Fatty Acid Metabolism in Diet-Induced Obese Mice

Abstract

Visceral adipose tissue (VAT) inflammation and metabolic dysregulation are key components of obesity-induced metabolic disease. Upregulated arginase, a ureahydrolase enzyme with two isoforms (A1-cytosolic and A2-mitochondrial), is implicated in pathologies associated with obesity and diabetes. This study examined A2 involvement in obesity-associated metabolic and vascular disorders. WT and globally deleted A2(^-/-) or A1(^+/-) mice were fed either a high fat/high sucrose (HFHS) diet or normal diet (ND) for 16 weeks. Increases in body and VAT weight of HFHS-fed WT mice were abrogated in A2^-/-, but not A1^+/-, mice. Additionally, A2^-/- HFHS-fed mice exhibited higher energy expenditure, lower blood glucose, and insulin levels compared to WT HFHS mice. VAT and adipocytes from WT HFHS fed mice showed greater A2 expression and adipocyte size and reduced expression of PGC-1α, PPAR-γ, and adiponectin. A2 deletion blunted these effects, increased levels of active AMPK-α, and upregulated genes involved in fatty acid metabolism. A2 deletion prevented HFHS-induced VAT collagen deposition and inflammation, which are involved in adipocyte metabolic dysfunction. Endothelium-dependent vasorelaxation, impaired by HFHS diet, was significantly preserved in A2^-/- mice, but more prominently maintained in A1^+/- mice. In summary, A2 is critically involved in HFHS-induced VAT inflammation and metabolic dysfunction.

Keywords: AMPK-α; arginase; endothelial dysfunction; fatty acid oxidation; inflammation; metabolism; obesity.

Figure 1

A2 deletion attenuates HFHS diet-induced increases in body weight, adiposity, VAT expansion, fasting blood glucose, postprandial serum insulin, and lipid accumulation in the liver without affecting adipocyte differentiation. Growth curves (A), n = 8–17 per group; percentage of body fat mass measured by nuclear magnetic resonance spectroscopy (B), n = 5–9 per group; percentage of VAT weight relative to body weight (C), n = 8–14 per group; fasting blood glucose (D) and fed serum insulin (E), n = 5–8 mice per group are shown for WT and A2^−/− mice. Values are represented as mean ± SEM. * P < 0.05 when compared to ND-fed mice within the same genotype, # P < 0.05 when compared to WT on the same diet. Representative photomicrographs of hemotoxylin and eosin-stained liver tissue sections from WT and A2^−/− after 16 weeks on HFHS or ND (F), scale bar = 50 μM. Representative images of cytoplasmic lipid droplets in preadipocytes from SVF of VAT of WT and A2^−/− mice stained with Oil Red O after eight days of in vitro differentiation (G) and the corresponding quantification (H), n = 3 per group).

Figure 2

A2 deletion improves whole body metabolism. Oxygen consumption (VO₂) (A–C) and carbon dioxide produced (VCO₂) (D–F), represented as mL/kg/h; respiratory exchange ratio calculated as VCO₂/VO₂ (G–I) and energy expenditure (J–L) calculated as kcal of heat produced/h/body weight, fatty acid oxidation rate calculated as kcal/h during light and dark cycles (M,N). Values are means ± SEM; n = 5–9 mice/group. * P < 0.05 when compared to ND-fed mice within the same genotype, # P < 0.05 when compared to WT on the same diet.

Figure 3

A2 deletion limits HFHS-induced VAT fibrosis and adipocyte hypertrophy. Representative photomicrographs of VAT show Masson’s trichrome staining of collagen/fibrosis depicted in blue color (A), Scale bar = 20 μM and quantified as % of trichrome positive area to total VAT area (B). Quantification of HFHS diet-induced increases in adipocyte area is shown for WT and A2^−/− mice (C). Data are presented as mean ± SEM, n = 3–5 mice/group. * P < 0.05 when compared to ND-fed mice within the same genotype, # P < 0.05 when compared to WT on the same diet.

Figure 4

A2 deletion limits HFHS-induced increase in pro-inflammatory macrophages. Flow cytometry dot plots show F4/80⁺ SVF cells gated using PE channel against side scatter (SSC), subsets of F4/80⁺ cells were identified based on surface expression of CD11c and CD206 from WT and A2^−/− using unstained cells as negative control (A). Quantification of the plots shows the effect of A2 deletion on the percentage of F4/80⁺ cells (B), (F4/80⁺CD11c⁺) (C), and (F4/80⁺CD206⁺) (D) of SVF, representing total macrophage, pro-inflammatory M1-like and anti-inflammatory M2-like macrophages, respectively. Values are means ± SEM; n = 5–6 mice/group. Adipocyte mRNA expression of TNF-α (E) and MCP-1 (F), n = 4 mice/group. * P < 0.05 when compared to ND-fed mice within the same genotype, # P < 0.05 when compared to WT on the same diet.

Figure 5

HFHS increases A2 expression in VAT and adipocytes. Representative western blot (A) and densitometry analysis (B) showing increased A2 protein levels in VAT of WT mice fed HFHS diet (n = 4 per group). qRT-PCR showing increased A2 (C) and HIF-1α (D) mRNA levels in mature adipocytes (AC) isolated from VAT of WT mice fed HFHS diet (n = 6–9 per group). Results are shown as a percentage of levels in WT mice fed ND. Representative western blot (E) and densitometry analysis (F) showing increased A2 protein levels in differentiated 3T3-L1 cell line treated with 250 μM of palmitate (PA) and high-glucose (HG) media (25 mM) for 7 days compared to normal glucose media (n = 4 per group). qRT-PCR showing increased A2 (G) and HIF-1α (H) mRNA levels in differentiated 3T3-L1 cells treated with PA/HG (n = 3–4 per group). Data are presented as mean ± SEM. * P < 0.05.

Figure 6

A2 deletion enhanced adipocyte expression of genes involved in fatty acid metabolism and preserved mitochondrial density. Representative western blot (A) and densitometry analysis normalized to the loading control (β-Actin) (B) showing adiponectin protein levels in VAT, n = 3–4/group. Adipocyte mRNA expression of adiponectin (C), n = 5–7/group. Adipocyte mRNA expression of PGC-1α (D) and PPAR-γ (E), n = 5–8 per group. Representative western blot (F) and densitometry analysis (G) showing ratio of p-AMPK-α to total-AMPK-α in VAT, n = 5–8/group and mRNA expression of genes involved in fatty acid uptake, β-oxidation and oxidative phosphorylation (OXPHOS), n = 4–6 (H). Immunofluorescence images of VAT sections stained with MitoID-Red as an estimate of mitochondrial mass (I) with quantitation (J); scale bar = 50 μM. Values are means ± SEM; * P < 0.05 when compared to ND-fed mice within the same genotype, # P < 0.05 when compared to WT on the same diet.

Figure 6

A2 deletion enhanced adipocyte expression of genes involved in fatty acid metabolism and preserved mitochondrial density. Representative western blot (A) and densitometry analysis normalized to the loading control (β-Actin) (B) showing adiponectin protein levels in VAT, n = 3–4/group. Adipocyte mRNA expression of adiponectin (C), n = 5–7/group. Adipocyte mRNA expression of PGC-1α (D) and PPAR-γ (E), n = 5–8 per group. Representative western blot (F) and densitometry analysis (G) showing ratio of p-AMPK-α to total-AMPK-α in VAT, n = 5–8/group and mRNA expression of genes involved in fatty acid uptake, β-oxidation and oxidative phosphorylation (OXPHOS), n = 4–6 (H). Immunofluorescence images of VAT sections stained with MitoID-Red as an estimate of mitochondrial mass (I) with quantitation (J); scale bar = 50 μM. Values are means ± SEM; * P < 0.05 when compared to ND-fed mice within the same genotype, # P < 0.05 when compared to WT on the same diet.

Figure 7

A2 deletion limited HFHS-induced oxidative stress, preserved NO levels and endothelium-dependent vasorelaxation to acetylcholine (ACh). Lipid peroxides are expressed as micromolar of malondialdehyde (MDA) (A), n = 5–9 mice/group, and plasma NO is expressed as picomoles of nitrite (B), n = 5–9 mice/group. Effects of A2 deletion on endothelium-dependent vasorelaxation to acetylcholine (ACh) (C), n = 7–10 mice per group, and sodium nitroprusside (SNP) (D), n = 4–5 mice per group. Values are mean ± SEM. * P < 0.05 when compared to ND-fed mice within the same genotype, # P < 0.05 when compared to WT on the same diet.

Figure 8

A1 deletion enhanced endothelium-dependent vasorelaxation with no effect on the gain of body and VAT weight. Effect of WT and A1 heterozygous deleted (A1^+/−) mice challenged with HFHS diet on gain in body weight (A), VAT weights (B), and endothelium-dependent vasorelaxation to acetylcholine (ACh) (C). EC₅₀: half maximal effective concentration for the ACh. Values are mean ± SEM, n = 4–6 mice/group. * P < 0.05 when compared to WT HFHS.

Figure 9

Vascular endothelial dysfunction induced by conditioned medium (CM) from VAT of WT-HFHS mice involves arginase upregulation. Conditioned media (CM) was prepared by incubating 50 mg VAT from either WT-ND or WT-HFHS mice for 24 h in M199 media. Presented are mRNA expression of A1 (A), A2 (B), and NO production (C) in MAEC. Additionally presented are endothelial-dependent vasorelaxant responses to acetylcholine in aortic rings from WT-ND mice treated for 24 h with CM from VAT of WT-ND and WT-HFHS mice in the presence/absence of ABH (100 μM) (D). Values are mean ± SEM. * P < 0.05 vs. WT-ND CM, # P < 0.05 vs. WT-HFHS CM (n = 4–5).