Metabolic context regulates distinct hypothalamic transcriptional responses to antiaging interventions

Abstract

The hypothalamus is an essential relay in the neural circuitry underlying energy metabolism that needs to continually adapt to changes in the energetic environment. The neuroendocrine control of food intake and energy expenditure is associated with, and likely dependent upon, hypothalamic plasticity. Severe disturbances in energy metabolism, such as those that occur in obesity, are therefore likely to be associated with disruption of hypothalamic transcriptomic plasticity. In this paper, we investigated the effects of two well-characterized antiaging interventions, caloric restriction and voluntary wheel running, in two distinct physiological paradigms, that is, diabetic (db/db) and nondiabetic wild-type (C57/Bl/6) animals to investigate the contextual sensitivity of hypothalamic transcriptomic responses. We found that, both quantitatively and qualitatively, caloric restriction and physical exercise were associated with distinct transcriptional signatures that differed significantly between diabetic and non-diabetic mice. This suggests that challenges to metabolic homeostasis regulate distinct hypothalamic gene sets in diabetic and non-diabetic animals. A greater understanding of how genetic background contributes to hypothalamic response mechanisms could pave the way for the development of more nuanced therapeutics for the treatment of metabolic disorders that occur in diverse physiological backgrounds.

Figure 1

Caloric restriction and wheel running alter body weight gain and food intake in C57Bl/6 and leptin receptor-deficient mice. (a) C57Bl/6 mice maintained on 40% caloric restriction (CR) gain weight more slowly than wild-type mice on the ad libitum (AL) diet. db/db mice maintained on CR from one month of age did not significantly gain weight over the subsequent twelve weeks of the experiment, and voluntary wheel running decelerates body weight gain in db/db mice. (b) Running transiently suppresses food intake in db/db mice. Data were analyzed with 2 × 3 repeated measures ANOVA with Tukey's post hoc and significance set at P < 0.05. Error bars represent SEM.

Figure 2

Endocrine changes in C57Bl/6 and db/db mice following running or caloric restriction. For all graphs, asterisk (*) indicates significance at P < 0.05 relative to sedentary C57Bl/6 mice fed ad libitum. Black diamonds (♦) represent significance at P < 0.05 relative to sedentary db/db mice fed ad libitum. (a) Caloric restriction (CR) attenuates fasting hyperglycemia in db/db mice. (b) CR lowers circulating insulin concentrations in C57Bl/6 mice, and both running and CR reinstate normal insulin levels in db/db mice. (c) Although there is a trend towards reduced total cholesterol in runners, this did not reach statistical significance. (d) Serum triglycerides are elevated in db/db mice, but this elevation can be ameliorated following running or CR. (e) Serum leptin levels are increased in db/db mice across all conditions. (f) Running attenuates the elevated corticosterone levels observed in db/db mice. Abbreviations: wt = C57Bl/6; AL = ad libitum; CR = caloric restriction; RUN = wheel running; DB = db/db mice.

Figure 3

Differences in hypothalamic gene expression and pathway recruitment between C57Bl/6 and db/db mice under ad libitum, sedentary conditions. (a) Graph of Z-ratios showing that differences in gene expression between C57Bl/6 and db/db mice are primarily attributable to transcriptional upregulation, as opposed to down-regulation. Abbreviations: Eif3s1, eukaryotic translation initiation factor 3, subunit J; Ccdc94, coiled-coil domain containing 94; Dhx9, DEAH (Asp-Glu-Ala-His) box polypeptide 9; Enpp5, ectonucleotide pyrophosphatase/phosphodiesterase 5; Bat5, HLA-B associated transcript 5; Tomm22, translocase of outer mitochondrial membrane 22. (b) Pathways that differ between wild-type and db/db mice under ad libitum, sedentary conditions. Aplp2, amyloid beta (A4) precursor-like protein 2; App, amyloid beta precursor protein; Abcb4, ATP-binding cassette, subfamily B member 4; Mt3, metallothionein 3; Nr1h3, nuclear receptor subfamily 1, group H, member 3; Grm7, glutamate receptor, metabotropic 7; Cdkn1b, cyclin-dependent kinase inhibitor 1B. (c) Pias2 and Slc17a6 expression in C57Bl/6 and db/db mice under ad libitum, sedentary conditions. Arrowheads indicate the hypothalamic nuclei, which were traced over the rostrocaudal extent of the hypothalamus to generate an average index of gene expression that would be directly comparable to the microarray analysis conducted in whole-hypothalamus RNA extracts. Pias2, protein inhibitor of activated STAT 2; Slc17a6, solute carrier family 17 member 6. Associated graph indicating densitometric results of the expression analysis. Consistent with the microarray results, both Pias2 and Slc17a6 were significantly upregulated in the hypothalamus of db/db mice. Asterisk (*) indicates statistical significance at P < 0.05 following bidirectional student's t-tests. (d) Further validation through semiquantitative PCR reveals that, as shown by the microarray, Gpx7 mRNA is increased and Tomm22 mRNA is reduced in the hypothalamus of db/db mice.

Figure 4

Wheel running influences hypothalamic gene expression and pathway activation in C57Bl/6 mice. (a) Z-ratios for gene transcripts up- or downregulated following voluntary wheel running in C57Bl/6 mice. Fkbp5, FK506 binding protein 5; Vps72, vacuolar protein sorting 72; Sh3gl1, SH3-domain GRB2-like 1; Lcn2, lipocalin 2; Hcrt, hypocretin; Trim72, tripartite motif-containing 72. (b), Pathways responsive to running in the hypothalamus of C57Bl/6 mice. Abbreviations: Lep, leptin; Htr1b, 5-hydroxytryptamine receptor 1B; Tsn, translin; Sfrp1, secreted frizzled-related protein 1; Slc30a3, solute carrier family 30, member 3; Slit1, slit homolog 1; Pou3f3, POU domain, class 3, transcription factor 3; Chrd, chordin; Nkx2-1, NK2 homeobox 1; Pgr, progesterone receptor. (c), PCR validation of running-induced transcriptional alterations in the hypothalamus of wild-type mice. (d) Leptin mRNA was detected in the hypothalamus samples using microarray, PCR, and in situ hybridization techniques. White adipose tissue (WAT) expresses leptin mRNA at higher levels than hypothalamus (Hyp) by PCR; ob/ob mice lack leptin bands at 155 bp in the hypothalamus following restriction enzyme digest, while wild-type (wt) controls express leptin mRNA. As shown by the microarray, running reduced endogenous leptin mRNA expression in the hypothalamus. In situ hybridization techniques were also used to detect endogenous leptin mRNA expression in the hypothalamus of sedentary wild-type mice.

Figure 5

CR influences hypothalamic gene expression and pathway recruitment in wild-type mice. (a) Z-ratios for genes that were up- or down-regulated following CR in wild-type mice. Otof, otoferlin; Ylpm1, YLP motif containing 1; Wipf1, WAS/WASL interacting protein family, member 1; Igf1, insulin-like growth factor 1; Pltp, phospholipid transfer protein; Cln6, ceroid-lipofuscinosis, neuronal 6. (b) Pathway analysis of genes responsive to CR in wild-type mice. Abbreviations: Zic1, zinc finger protein of the cerebellum 1; E2f1, E2F transcription factor 1; Nde1, nuclear distribution gene E homolog 1; Adcyap1r1, adenylate cyclase activating polypeptide 1 receptor 1; Ntrk3, neurotrophic tyrosine kinase, receptor, type 3; Id2, inhibitor of DNA binding 2; Xrcc6, X-ray repair in Chinese hamster cells 6; Dicer1, Dicer1 homolog. (c) PCR validation of changes in gene expression following CR in wild-type mice.

Figure 6

db/db mice respond to running with a distinct transcriptional profile. (a) Leptin receptor-deficient db/db mice exhibit transcriptional upregulation, indicated by positive Z-ratios, following twelve weeks of voluntary wheel running. Rpe, ribulose-5-phosphate-3-epimerase; Glra1, glycine receptor, alpha 1 subunit; Snx1, sorting nexin 1; Naga, N-acetyl galactosaminidase, alpha; Hace1, HECT domain and ankyrin repeat containing, E3 ubiquitin protein ligase 1; Heatr5a, HEAT repeat containing 5A. (b) Pathway analysis of genes regulated by voluntary exercise in db/db mice. Abbreviations: Il6st, interleukin 6 signal transducer; Tbr1, T-box brain gene 1; Nrg1, neuregulin 1; Abcd2, ATP-binding cassette, sub-family D, member 2. (c) PCR validation of differences in gene expression following running in db/db mice.

Figure 7

CR activates a diverse set of transcripts and pathways in db/db mice. (a) Z-ratios for genes that were up- or downregulated following CR in db/db mice. Fpr2, formyl peptide receptor 2; Pmch, pro-melanin-concentrating hormone; Oxt, oxytocin; Kif2a, kinesin family member 2A; H2afj, H2A histone family, member J; Scn1a, sodium channel, voltage-gated, type I, alpha. (b) Pathway analysis of genes responsive to restricted feeding in leptin receptor-deficient mice. Abbreviations: Il6st, interleukin 6 signal transducer; Ccnd2, cyclin D2; Prkaa2, protein kinase, AMP activated, alpha 2 catalytic subunit; Abcd2, ATP-binding cassette, subfamily D member 2; Pmch, pro-melanin-concentrating hormone; Gad1, glutamic acid decarboxylase 1, Rac1, RAS-related C3 botulinum substrate 1; Wnt7a, wingless-related MMTV integration site 7A; Myo5a, myosin VA; Gal, galanin; Grin1, glutamate receptor, ionotropic, NMDA1; Slc6a4, solute carrier family 6 member 4; Cdkn1a, cyclin-dependent kinase inhibitor 1A; Fgfr3, fibroblast growth factor receptor 3; En2, engrailed 2; Gbx2, gastrulation brain homeobox 2. (c) PCR validation of differences in gene expression after CR in db/db mice.

Figure 8

db/db and C57Bl/6 mice respond differently to exercise and caloric restriction. (a) Heat map profiles showing that db/db and C57Bl/6 mice exhibit distinct transcriptional responses to energetic challenges. (b) Venn diagram showing the number of genes expressed following voluntary wheel running in C57Bl/6 and db/db mice. (c) Venn diagram showing the number of transcripts responsive to CR in C57Bl/6 and db/db mice. (d) Side-by-side comparison of the number of genes that differ between C57Bl/6 and db/db mice following running or CR.

Figure 9

Physiological background differentially modulates the effects of exercise and caloric restriction upon hypothalamic transcriptomes. (a) Venn diagram illustrates the number of significantly regulated genes (up- and downregulated) affected by exercise (Run) in WT (red) or db/db (blue) animals. The associated histogram indicates the significantly regulated genes common to the effects of exercise in both genetic backgrounds (WT and db/db). The yellow area indicates the genes whose direction of regulation was conserved in both genetic backgrounds while the grey area shows the genes that showed reversal of their exercise-mediated expression profile. (b) Venn diagram and associated histogram depict similar data to (a) but for the implementation of CR to the WT (blue) or db/db (red) mice. (c) Significantly regulated pathways associated with the conserved CR-induced transcriptional activity in both WT and db/db mice.