Mitochondrial gene expression and increased oxidative metabolism: role in increased lifespan of fat-specific insulin receptor knock-out mice

Abstract

Caloric restriction, leanness and decreased activity of insulin/insulin-like growth factor 1 (IGF-1) receptor signaling are associated with increased longevity in a wide range of organisms from Caenorhabditis elegans to humans. Fat-specific insulin receptor knock-out (FIRKO) mice represent an interesting dichotomy, with leanness and increased lifespan, despite normal or increased food intake. To determine the mechanisms by which a lack of insulin signaling in adipose tissue might exert this effect, we performed physiological and gene expression studies in FIRKO and control mice as they aged. At the whole body level, FIRKO mice demonstrated an increase in basal metabolic rate and respiratory exchange ratio. Analysis of gene expression in white adipose tissue (WAT) of FIRKO mice from 6 to 36 months of age revealed persistently high expression of the nuclear-encoded mitochondrial genes involved in glycolysis, tricarboxylic acid cycle, beta-oxidation and oxidative phosphorylation as compared to expression of the same genes in WAT from controls that showed a tendency to decline in expression with age. These changes in gene expression were correlated with increased cytochrome c and cytochrome c oxidase subunit IV at the protein level, increased citrate synthase activity, increased expression of peroxisome proliferator-activated receptor gamma coactivator 1alpha (PGC-1alpha) and PGC-1beta, and an increase in mitochondrial DNA in WAT of FIRKO mice. Together, these data suggest that maintenance of mitochondrial activity and metabolic rates in adipose tissue may be important contributors to the increased lifespan of the FIRKO mouse.

Fig. 1

Body weight and body composition by dual energy X-ray absorptiometry (DEXA) scan of control and fat-specific insulin receptor knock-out (FIRKO) mice. Male and female FIRKO mice had consistently and significantly lower body weights than control mice (P < 0.05). DEXA scan analysis was performed at 10 months of age and decreased fat mass and percentage of body fat in both genders (P < 0.01), as well as decreased lean mass in female FIRKO mice (P < 0.01). Statistical analysis was done using a Student’s t-test (*P < 0.05), and results are expressed as average ± SEM.

Fig. 2

Metabolic parameters in fat-specific insulin receptor knock-out (FIRKO) mice assessed using a CLAMS apparatus. Ten- to 11-month-old male and female mice were acclimated to the apparatus for 2 days, then studied for two additional days. (a) Cumulative food intake was increased (10.9% on average) in FIRKO mice compared to controls. (b) Physical activity measured by light beam breaks was 46% lower (P < 0.005) in FIRKO mice during a light period, but did not differ during the dark period. (c) Oxygen consumption of FIRKO mice expressed per kg body weight was increased 14.5% ± 11.7 and 20.6% ± 10.0 during the light and a dark cycle, respectively (P < 0.001). (d) Heat production was slightly, but significantly higher in FIRKO compared to control mice during the dark period (6.8%, P < 0.005). (e) Respiratory exchange ratio (RER), a measure of total body metabolism, was significantly higher (3.9%, P < 0.001) during the dark period in FIRKO compared to control mice. (f) Basal body temperature was measured in both of male and female mice in the fed and fasted state as described in the Experimental procedures. In (a–e), analysis was done by comparing an average of all light vs. an average of all dark periods for all metabolic parameters. Statistical analysis was done using a Student’s t-test to compare light vs. dark period in each group of animals as well as light vs. light and dark vs. dark periods in different groups of animals, and results are expressed as average ± SEM.

Fig. 3

Analysis of microarray data obtained from RNA isolated from white adipose tissue from young, middle-aged and old mice. Tissues were isolated from mice at 6 months, 1.5 years and 2.5–3 years of age, RNA was extracted and level of gene expression assessed using Affymetrix murine 450A arrays. The CEL files generated by the Affymetrix Microarray Suite version 5.0 (MAS 5.0; Affymetrix) were analyzed using dChip version 1.3 software (http://www.dchip.org). Subset of genes (probesets) that were shown to be differentially expressed in old animals were additionally analyzed by analysis of variance (ANOVA) test with P < 0.01. Comparison of gene expression in control and FIRKO animals during aging showed four different patterns of expression as described in Results.

Fig. 4

Expression of genes in fat-specific insulin receptor knock-out (FIRKO) and control mice as a function of age. The relative fold expression in FIRKO vs. control mice obtained by microarray analysis was calculated for those genes whose protein products are involved in glycolysis, the tricarboxylic acid (TCA) cycle, oxidative phosphorylation and β-oxidation.

Fig. 5

Quantitative reverse transcriptase–polymerase chain reaction (RT-PCR) analysis of genes encoding mitochondrial proteins involved in oxidative phosphorylation (electron transport) and oxidative stress response. (a,b) Two subunits [NADH dehydrogenase (ub) Fe-S protein 1 (Ndufs1) and NADH dehydrogenase (ubiquinone) 1 α subcomplex, 5 (Nudfa5)] of mitochondrial Complex I. (c) Succinate dehydrogenase (Sdhd) (Complex II). (d) ATP synthase, H+ transporting, mitochondrial F1F0 complex, subunit e (Atp5k), subunit of Complex V. (e,f) Two subunits [ubiquinol–cytochrome c reductase (6.4 kDa) subunit (Uqcr) and ubiquinol–cytochrome c reductase core protein 2 (Uqcrc2)] of Complex III. (g,h) Two subunits [cytochrome c oxidase, subunit Vb (COX 5b) and cytochrome c oxidase, subunit VIIc (COX 7c)] of mitochondrial Complex IV. (i) Expression of cytochrome c1 (Cyc1). (j) Uncoupling protein 2 (Ucp2). Statistical analysis was performed using a Student’s t-test (*P < 0.05, **P < 0.01) for the comparison of different genotypes in each age group, and results are expressed as average ± SEM. All genes, except Nudfs1, Atp5k and Ucp2, had also a significantly decreased expression in old compared to middle-aged control mice. In addition, all oxidative phosphorylation genes had significantly increased expression in old FIRKO compared to young FIRKO mice (P ≤ 0.01).

Fig. 6

Conformation of microarray analysis and biological assays. (a) Western blot analysis of white adipose tissue isolated from 1.5–2-year-old mice for cytochrome c (Cyt c) and cytochrome c oxidase, subunit IV (COX IV). Note that SIRT1 expression was not different in fat-specific insulin receptor knock-out (FIRKO) compared to control mice. (b) Citrate synthase assay was performed as described in Experimental procedures and showed an almost threefold increased activity in FIRKO compared to control mice (P = 0.055). (c) Ratio of mitochondrial (COX II) vs. nuclear (β-globin) DNA obtained by quantitative reverse transcriptase–polymerase chain reaction (qRT-PCR) showed significantly increased (P = 0.02) mDNA copy number per cell in FIRKO compared to control mice. (d) Electron micrographs of adipose tissue of 2-year-old control and FIRKO mice (original magnification ×99750). (e,f) RT-PCR analysis showed almost significantly increased expression of peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) (P = 0.07) and a significant increase of expression of PGC-1β in old FIRKO compared to control mice (P = 0.017). Statistical analysis was done using a Student’s t-test (*P < 0.05) for the comparison of different genotypes in each age group, and results are expressed as average ± SEM.