Time-dependent gene set enrichment analysis revealed a coordinate increase of 13 OxPhos genes, belonging to respiration complexes I, III, IV, and V, in the livers of HFD-fed AJ but not B6 mice. Biochemical analysis confirmed higher mitochondrial respiration rates in the livers of AJ mice as well as marked uncoupling of mitochondrial respiration. These data demonstrate flexibility in the expression of the OxPhos genes in AJ mice in response to an acute increase in energy intake. This response may protect against the initial deleterious effect of HFD feeding by increasing energy dissipation and reducing reactive oxygen species production. It is part of a global protective response of the AJ mouse livers to HFD, which involves several lipid metabolic pathways, as previously described (8
GSEA results are usually difficult to confirm by direct quantitative analysis of gene expression because this statistical technique searches for groups of genes that belong to a given biological pathway and whose expression is coordinately regulated even though each individual gene may not be statistically differentially regulated between two conditions. Therefore, we did not attempt to confirm changes in mRNA expression by quantitative methods, but instead we measured mitochondrial respiration and ATP synthesis in mitochondria prepared from the livers of NC or HFD-fed AJ and B6 mice. Consistent with the GSEA analysis, we detected an increase in mitochondrial respiration in livers of AJ mice fed an HFD for 10 days.
A number of recent studies found a coordinately decreased expression of OxPhos genes in muscles (18
), livers, and fat (21
) of diabetic patients, leading to the suggestion that a reduced oxidative phosphorylation capacity can favor diabetes development. In contrast, in one study (24
), an increase in OxPhos gene expression was observed in the livers of obese patients with type 2 diabetes and this was correlated with increased measures of insulin resistance and diabetes. Thus, it is unclear whether changes in OxPhos gene expression cause disease progression or are a compensatory response to the diseased state. Studies in mice with muscle or liver-specific inactivation of the apoptosis-inducing factor (AIF
) gene, which reduces OxPhos activity as found in human patients, showed increased insulin sensitivity and resistance to diet-induced obesity (23
). The proposed explanation for this unanticipated observation is that reduced ATP production, as well as increased intracellular AMP levels, stimulates glucose utilization and fatty acid oxidation, thereby favoring nutrient absorption and catabolism. In both cases, however, it is difficult to assess the contribution of reduced OxPhos genes in the susceptibility to obesity or diabetes. Indeed, in the human studies, analysis has been performed at a single time point in the disease history of each patient and thus a possible causative role cannot be assessed. In the AIF knockout mice, the changes in OxPhos gene expression are induced early in the life of the animal and may induce unidentified compensatory mechanisms.
Our present study reveals a different aspect of OxPhos gene-regulated expression: its capacity to be rapidly and transiently increased in response to HFD feeding in mice that are protected against hepatic steatosis and obesity. This is also associated with increased mitochondrial uncoupling, which probably reduces the production of superoxide anions generated by electrons leaking from the OxPhos chain (25
) and which can induce oxidative damage in the liver of AJ mice. In this context, results from our recent study (8
) indicated that upon HFD feeding, expression of mitochondrial β-oxidation genes was increased in B6 but not AJ mouse livers. Thus, in response to fatty acid overload, more reduced nucleotides may be produced and channeled to the OxPhos chain in B6 mouse livers, but in the absence of an increased capacity to generate ATP or to uncouple mitochondria, this must further favor reactive oxygen species production (28
). Together, our observations suggest that upon initiation of HFD, the B6 mouse livers are prone to induction of ROS damage, whereas reduced efficiency of ATP generation in AJ mouse livers helps to utilize the excess of substrates while balancing ATP supply with cellular energy requirements (29
Adaptation of mice to HFD proceeds in different phases with considerable up- or downregulation of many genes in the first days of HFD, followed by a slow return of many genes to their basal levels of expression, with some gene expression levels remaining permanently modified (10
). In our previous study of AJ and B6 mouse liver adaptation to an HFD, we identified a rapid and coordinate increase in the expression of 10 peroxisomal genes, a microsomal elongase (Elovl5
), and two microsomal desaturases (Fads1
). This was associated with increased peroxisomal β-oxidation activity and increased production of the cannabinoid receptor agonist 2-AG, whose production from n-6 unsaturated fatty acids is favored by the action of the microsomal enzymes. Thus, in AJ mouse livers, there is a rapid adaptation of several metabolic pathways to HFD: 1
) increased peroxisomal β-oxidation; 2
) increased n-3 and n-6 fatty acid detoxification by desaturation and conversion to bioactive, protective lipids such as 2-AG; and, as shown in the current study, 3
) increased OxPhos and mitochondrial uncoupling. Collectively, these observations indicate that resistance to HFD-induced hepatic steatosis in AJ mouse livers and possible protection against obesity development is favored by the rapid adaptation of multiple metabolic pathways that protect the liver against a toxic lipid overload.
The participation of several pathways in resistance against diet-induced hepatosteatosis and obesity is consistent with analysis of mouse chromosome substitution lines that showed that AJ chromosomal loci able to protect B6 mice against diet-induced obesity were present in at least 17 different chromosomes (34
). Whether any of the pathways mentioned above has a dominant role in the development of hepatosteatosis and obesity is not clear. Studies with mice engineered to change the expression level or activity of any of these pathways could shed light on this question. Nevertheless, these studies suggest that the ability of an organ to rapidly adapt to changes in nutrient availability is an important aspect of the protective response to a metabolic stress and clearly show that this response is genetically encoded, although epigenetic programming may also be involved (10
). These studies also suggest that the ability to regulate gene expression may be as important as changes in protein structure and/or function in determining the susceptibility to metabolic diseases. This proposal is clearly in agreement with genome-wide association studies that usually find disease susceptibility variants in noncoding regions, which may impact gene transcription activity (35
). This is also reminiscent of the metabolic inflexibility hypothesis that proposes that a key defect in obesity and type 2 diabetes is a reduced capacity to switch from the use of glucose to lipid as oxidative substrates during the fed to postabsorptive phase (36
). Thus, long-term development of metabolic diseases may be associated with failure to acutely adapt metabolic pathways to a change in nutrient availability. Thus, we propose that the concept of metabolic flexibility be extended to include the capacity of organs, such as livers, to rapidly adapt their metabolic pathways to prevent transient generation of damaging metabolites that, over time, can accumulate to create pathological conditions.