Fibroblast Growth Factor 21 Is Not Required for the Reductions in Circulating Insulin-Like Growth Factor-1 or Global Cell Proliferation Rates in Response to Moderate Calorie Restriction in Adult Mice

<div><p>Calorie restriction (CR) delays aging and extends lifespan in numerous organisms, including mice. Down-regulation of the somatotropic axis, including a reduction in insulin-like growth factor-1 (IGF-1), likely plays an important role in CR-induced lifespan extension, possibly by reducing cell proliferation rates, thereby delaying replicative senescence and inhibiting tumor promotion. Accordingly, elucidating the mechanism(s) by which IGF-1 is reduced in response to CR holds therapeutic potential in the fight against age-related diseases. Up-regulation of fibroblast growth factor 21 (FGF21) is one possible mechanism given that FGF21 expression is induced in response to nutritional deprivation and has been implicated as a negative regulator of IGF-1 expression. Here we investigated alterations in hepatic growth hormone (GH)-mediated IGF-1 production and signaling as well as the role of FGF21 in the regulation of IGF-1 levels and cell proliferation rates in response to moderate CR in adult mice. We found that in response to moderate CR, circulating GH and hepatic janus kinase 2 (JAK2) phosphorylation levels are unchanged but that hepatic signal transducer and activator of transcription 5 (STAT5) phosphorylation levels are reduced, identifying STAT5 phosphorylation as a potential key site of CR action within the somatotropic axis. Circadian measurements revealed that the relative level of FGF21 expression is both higher and lower in CR vs. ad libitum (AL)-fed mice, depending on the time of measurement. Employing FGF21-knockout mice, we determined that FGF21 is not required for the reduction in IGF-1 levels or cell proliferation rates in response to moderate CR. However, compared to AL-fed WT mice, AL-fed FGF21-knockout mice exhibited higher basal rates of cell proliferation, suggesting anti-mitotic effects of FGF21. This work provides insights into both GH-mediated IGF-1 production in the context of CR and the complex network that regulates FGF21 and IGF-1 expression and cell proliferation rates in response to nutritional status.</p></div

Circulating and hepatic mRNA FGF21, IGF-1 and IGFBP-1 levels in WT/AL, WT/CR, FGF21-KO/AL and FGF21-KO/CR mice.

<p>Plasma levels of A) FGF21, C) IGF-1 and E) IGFBP-1 and relative hepatic mRNA expression levels of B) FGF21, D) IGF-1 and F) IGFBP-1 at 1500 h and 1900 h in WT and FGF21-KO mice fed AL or CR (n = 3–12 per genotype per diet per time point). Hepatic mRNA expression levels were normalized to 18S rRNA and then normalized to WT/AL mice at 1500 h. All between-group analyses were performed using a two-way ANOVA with a Bonferroni <i>post hoc</i> test at each time point (* p<0.05, ** p<0.01, *** p<0.001).</p

Circulating and hepatic mRNA levels of IGF-1 and FGF21 in CR mice.

<p>Plasma A) IGF-1 and B) FGF21 levels in AL and CR mice at 2100 h or 2200 h (n = 16–21 per diet). Relative hepatic mRNA expression levels of C) IGF-1 and D) FGF21 in AL and CR mice at 2100 h or 2200 h (n = 29–31 per diet). Hepatic mRNA expression levels were normalized to 18S rRNA and then normalized to the AL group. Student's unpaired two-tailed <i>t</i>-tests were used for all between-group analyses (* p<0.05, ** p<0.0043, *** p<0.0001).</p

GH secretory dynamics and hepatic GH signaling in CR mice.

<p>Plasma GH levels described as A) the integrated hourly concentration or B) the maximum pulse amplitude of GH over the 5.25 h sampling period in AL and CR mice (n = 8 per diet). Densitometric analysis and western blot images of hepatic protein levels of phosphorylated to total C) JAK2 and D) STAT5 in AL and CR mice at 2100 h (n = 5–7 per diet. Data normalized to AL). Student's unpaired two-tailed <i>t</i>-tests were used for all between-group analyses (** p<0.0071).</p

The effects of physiological adaptations to calorie restriction on global cell proliferation rates

Calorie restriction (CR) reduces the rate of cell proliferation in mitotic tissues. It has been suggested that this reduction in cell proliferation may mediate CR-induced increases in longevity. However, the mechanisms that lead to CR-induced reductions in cell proliferation rates remain unclear. To evaluate the CR-induced physiological adaptations that may mediate reductions in cell proliferation rates, we altered housing temperature and access to voluntary running wheels to determine the effects of food intake, energy expenditure, percent body fat, and body weight on proliferation rates of keratinocytes, liver cells, mammary epithelial cells, and splenic T-cells in C57BL/6 mice. We found that ∼20% CR led to a reduction in cell proliferation rates in all cell types. However, lower cell proliferation rates were not observed with reductions in 1) food intake and energy expenditure in female mice housed at 27°C, 2) percent body fat in female mice provided running wheels, or 3) body weight in male mice provided running wheels compared with ad libitum-fed controls. In contrast, reductions in insulin-like growth factor I were associated with decreased cell proliferation rates. Taken together, these data suggest that CR-induced reductions in food intake, energy expenditure, percent body fat, and body weight do not account for the reductions in global cell proliferation rates observed in CR. In addition, these data are consistent with the hypothesis that reduced cell proliferation rates could be useful as a biomarker of interventions that increase longevity

Reduced in vivo hepatic proteome replacement rates but not cell proliferation rates predict maximum lifespan extension in mice.

Combating the social and economic consequences of a growing elderly population will require the identification of interventions that slow the development of age-related diseases. Preserved cellular homeostasis and delayed aging have been previously linked to reduced cell proliferation and protein synthesis rates. To determine whether changes in these processes may contribute to or predict delayed aging in mammals, we measured cell proliferation rates and the synthesis and replacement rates (RRs) of over a hundred hepatic proteins in vivo in three different mouse models of extended maximum lifespan (maxLS): Snell Dwarf, calorie-restricted (CR), and rapamycin (Rapa)-treated mice. Cell proliferation rates were not consistently reduced across the models. In contrast, reduced hepatic protein RRs (longer half-lives) were observed in all three models compared to controls. Intriguingly, the degree of mean hepatic protein RR reduction was significantly correlated with the degree of maxLS extension across the models and across different Rapa doses. Absolute rates of hepatic protein synthesis were reduced in Snell Dwarf and CR, but not Rapa-treated mice. Hepatic chaperone levels were unchanged or reduced and glutathione S-transferase synthesis was preserved or increased in all three models, suggesting a reduced demand for protein renewal, possibly due to reduced levels of unfolded or damaged proteins. These data demonstrate that maxLS extension in mammals is associated with improved hepatic proteome homeostasis, as reflected by a reduced demand for protein renewal, and that reduced hepatic protein RRs hold promise as an early biomarker and potential target for interventions that delay aging in mammals. Aging Cell 2016; 15(1):118-27

RDH1 suppresses adiposity by promoting brown adipose adaptation to fasting and re-feeding.

RDH1 is one of the several enzymes that catalyze the first of the two reactions to convert retinol into all-trans-retinoic acid (atRA). Here, we show that Rdh1-null mice fed a low-fat diet gain more weight as adiposity (17% males, 13% females) than wild-type mice by 20&nbsp;weeks old, despite neither consuming more calories nor decreasing activity. Glucose intolerance and insulin resistance develop following increased adiposity. Despite the increase in white fat pads, epididymal white adipose does not express Rdh1, nor does muscle. Brown adipose tissue (BAT) and liver express Rdh1 at relatively high levels compared to other tissues. Rdh1 ablation lowered body temperatures during ambient conditions. Given the decreased body temperature, we focused on BAT. A lack of differences in BAT adipogenic gene expression between Rdh1-null mice and wild-type mice, including Pparg, Prdm16, Zfp516 and Zfp521, indicated that the phenotype was not driven by brown adipose hyperplasia. Rather, Rdh1 ablation eliminated the increase in BAT atRA that occurs after re-feeding. This disruption of atRA homeostasis increased fatty acid uptake, but attenuated lipolysis in primary brown adipocytes, resulting in increased lipid content and larger lipid droplets. Rdh1 ablation also decreased mitochondrial proteins, including CYCS and UCP1, the mitochondria oxygen consumption rate, and disrupted the mitochondria membrane potential, further reflecting impaired BAT function, resulting in both BAT and white adipose hypertrophy. RNAseq revealed dysregulation of 424 BAT genes in null mice, which segregated predominantly into differences after fasting vs after re-feeding. Exceptions were Rbp4 and Gbp2b, which increased during both dietary conditions. Rbp4 encodes the serum retinol-binding protein-an insulin desensitizer. Gbp2b encodes a GTPase. Because Gbp2b increased several hundred-fold, we overexpressed it in brown adipocytes. This caused a shift to larger lipid droplets, suggesting that GBP2b affects signaling downstream of the β-adrenergic receptor during basal thermogenesis. Thus, Rdh1-generated atRA in BAT regulates multiple genes that promote BAT adaptation to whole-body energy status, such as fasting and re-feeding. These gene expression changes promote optimum mitochondria function and thermogenesis, limiting adiposity. Attenuation of adiposity and insulin resistance suggests that RDH1 mitigates metabolic syndrome