Cristal M. Hill

Growth hormone-releasing hormone disruption extends lifespan and regulates response to caloric restriction in mice

We examine the impact of targeted disruption of growth hormone-releasing hormone (GHRH) in mice on longevity and the putative mechanisms of delayed aging. GHRH knockout mice are remarkably long-lived, exhibiting major shifts in the expression of genes related to xenobiotic detoxification, stress resistance, and insulin signaling. These mutant mice also have increased adiponectin levels and alterations in glucose homeostasis consistent with the removal of the counter-insulin effects of growth hormone. While these effects overlap with those of caloric restriction, we show that the effects of caloric restriction (CR) and the GHRH mutation are additive, with lifespan of GHRH-KO mutants further increased by CR. We conclude that GHRH-KO mice feature perturbations in a network of signaling pathways related to stress resistance, metabolic control and inflammation, and therefore provide a new model that can be used to explore links between GHRH repression, downregulation of the somatotropic axis, and extended longevity. DOI: http://dx.doi.org/10.7554/eLife.01098.001

Long-lived hypopituitary Ames dwarf mice are resistant to the detrimental effects of high-fat diet on metabolic function and energy expenditure.

Growth hormone (GH) signaling stimulates the production of IGF‐1; however, increased GH signaling may induce insulin resistance and can reduce life expectancy in both mice and humans. Interestingly, disruption of GH signaling by reducing plasma GH levels significantly improves health span and extends lifespan in mice, as observed in Ames dwarf mice. In addition, these mice have increased adiposity, yet are more insulin sensitive compared to control mice. Metabolic stressors such as high‐fat diet (HFD) promote obesity and may alter longevity through the GH signaling pathway. Therefore, our objective was to investigate the effects of a HFD (metabolic stressor) on genetic mechanisms that regulate metabolism during aging. We show that Ames dwarf mice fed HFD for 12 weeks had an increase in subcutaneous and visceral adiposity as a result of diet‐induced obesity, yet are more insulin sensitive and have higher levels of adiponectin compared to control mice fed HFD. Furthermore, energy expenditure was higher in Ames dwarf mice fed HFD than in control mice fed HFD. Additionally, we show that transplant of epididymal white adipose tissue (eWAT) from Ames dwarf mice fed HFD into control mice fed HFD improves their insulin sensitivity. We conclude that Ames dwarf mice are resistant to the detrimental metabolic effects of HFD and that visceral adipose tissue of Ames dwarf mice improves insulin sensitivity in control mice fed HFD.

Low protein-induced increases in FGF21 drive UCP1-dependent metabolic but not thermoregulatory endpoints

Dietary protein restriction increases adipose tissue uncoupling protein 1 (UCP1), energy expenditure and food intake, and these effects require the metabolic hormone fibroblast growth factor 21 (FGF21). Here we test whether the induction of energy expenditure during protein restriction requires UCP1, promotes a resistance to cold stress, and is dependent on the concomitant hyperphagia. Wildtype, Ucp1-KO and Fgf21-KO mice were placed on control and low protein (LP) diets to assess changes in energy expenditure, food intake and other metabolic endpoints. Deletion of Ucp1 blocked LP-induced increases in energy expenditure and food intake, and exacerbated LP-induced weight loss. While LP diet increased energy expenditure and Ucp1 expression in an FGF21-dependent manner, neither LP diet nor the deletion of Fgf21 influenced sensitivity to acute cold stress. Finally, LP-induced energy expenditure occurred even in the absence of hyperphagia. Increased energy expenditure is a primary metabolic effect of dietary protein restriction, and requires both UCP1 and FGF21 but is independent of changes in food intake. However, the FGF21-dependent increase in UCP1 and energy expenditure by LP has no effect on the ability to acutely respond to cold stress, suggesting that LP-induced increases in FGF21 impact metabolic but not thermogenic endpoints.

Longevity is impacted by growth hormone action during early postnatal period

Life-long lack of growth hormone (GH) action can produce remarkable extension of longevity in mice. Here we report that GH treatment limited to a few weeks during development influences the lifespan of long-lived Ames dwarf and normal littermate control mice in a genotype and sex-specific manner. Studies in a separate cohort of Ames dwarf mice show that this short period of the GH exposure during early development produces persistent phenotypic, metabolic and molecular changes that are evident in late adult life. These effects may represent mechanisms responsible for reduced longevity of dwarf mice exposed to GH treatment early in life. Our data suggest that developmental programming of aging importantly contributes to (and perhaps explains) the well documented developmental origins of adult disease. DOI: http://dx.doi.org/10.7554/eLife.24059.001

Original Research: Metabolic alterations from early life thyroxine replacement therapy in male Ames dwarf mice are transient

Ames dwarf mice are exceptionally long-lived due to a Prop1 loss of function mutation resulting in deficiency of growth hormone, thyroid-stimulating hormone and prolactin. Deficiency in thyroid-stimulating hormone and growth hormone leads to greatly reduced levels of circulating thyroid hormones and insulin-like growth factor 1, as well as a reduction in insulin secretion. Early life growth hormone replacement therapy in Ames dwarf mice significantly shortens their longevity, while early life thyroxine (T4) replacement therapy does not. Possible mechanisms by which early life growth hormone replacement therapy shortens longevity include deleterious effects on glucose homeostasis and energy metabolism, which are long lasting. A mechanism explaining why early life T4 replacement therapy does not shorten longevity remains elusive. Here, we look for a possible explanation as to why early life T4 replacement therapy does not impact longevity of Ames dwarf mice. We found that early life T4 replacement therapy increased body weight and advanced the age of sexual maturation. We also find that early life T4 replacement therapy does not impact glucose tolerance or insulin sensitivity, and any deleterious effects on oxygen consumption, respiratory quotient and heat production are transient. Lastly, we find that early life T4 replacement therapy has long-lasting effects on bone mineral density and bone mineral content. We suggest that the transient effects on energy metabolism and lack of effects on glucose homeostasis are the reasons why there is no shortening of longevity after early life T4 replacement therapy in Ames dwarf mice.

Effects of rapamycin on growth hormone receptor knockout mice

Significance In various animal species, including mammals, longevity can be extended by rapamycin, an inhibitor of mTOR (mechanistic target of rapamycin). mTOR acts through two complexes: mTORC1 and mTORC2. Antiaging effects of rapamycin are mediated by suppression of mTORC1, while the role of mTORC2 in aging remains to be elucidated. Here, we report that mTORC2 plays a positive role in regulating longevity via maintenance, or enhancement, of whole-body homeostasis. When mTORC2-mediated homeostasis was disrupted by rapamycin in the remarkably long-lived GHR-KO mice (in which mTORC1 signaling is low, while mTORC2 signaling is elevated), their life span was shortened. Hence, a selective approach toward mTORC1 inhibition without impairing mTORC2 is important in devising a strategy for slowing aging. It is well documented that inhibition of mTORC1 (defined by Raptor), a complex of mechanistic target of rapamycin (mTOR), extends life span, but less is known about the mechanisms by which mTORC2 (defined by Rictor) impacts longevity. Here, rapamycin (an inhibitor of mTOR) was used in GHR-KO (growth hormone receptor knockout) mice, which have suppressed mTORC1 and up-regulated mTORC2 signaling, to determine the effect of concurrently decreased mTORC1 and mTORC2 signaling on life span. We found that rapamycin extended life span in control normal (N) mice, whereas it had the opposite effect in GHR-KO mice. In the rapamycin-treated GHR-KO mice, mTORC2 signaling was reduced without further inhibition of mTORC1 in the liver, muscle, and s.c. fat. Glucose and lipid homeostasis were impaired, and old GHR-KO mice treated with rapamycin lost functional immune cells and had increased inflammation. In GHR-KO MEF cells, knockdown of Rictor, but not Raptor, decreased mTORC2 signaling. We conclude that drastic reduction of mTORC2 plays important roles in impaired longevity in GHR-KO mice via disruption of whole-body homeostasis.

Attenuation of epidermal growth factor (EGF) signaling by growth hormone (GH)

Transgenic mice overexpressing growth hormone (GH) show increased hepatic protein content of the epidermal growth factor receptor (EGFR), which is broadly associated with cell proliferation and oncogenesis. However, chronically elevated levels of GH result in desensitization of STAT-mediated EGF signal and similar response of ERK1/2 and AKT signaling to EGF compared to normal mice. To ascertain the mechanisms involved in GH attenuation of EGF signaling and the consequences on cell cycle promotion, phosphorylation of signaling mediators was studied at different time points after EGF stimulation, and induction of proteins involved in cell cycle progression was assessed in normal and GH-overexpressing transgenic mice. Results from kinetic studies confirmed the absence of STAT3 and 5 activation and comparable levels of ERK1/2 phosphorylation upon EGF stimulation, which was associated with diminished or similar induction of c-MYC, c-FOS, c-JUN, CYCLIN D1 and CYCLIN E in transgenic compared to normal mice. Accordingly, kinetics of EGF-induced c-SRC and EGFR phosphorylation at activating residues demonstrated that activation of these proteins was lower in the transgenic mice with respect to normal animals. In turn, EGFR phosphorylation at serine 1046/1047, which is implicated in the negative regulation of the receptor, was increased in the liver of GH-overexpressing transgenic mice both in basal conditions and upon EGF stimulus. Increased basal phosphorylation and activation of the p38-mitogen-activated protein kinase might account for increased Ser 1046/1047 EGFR. Hyperphosphorylation of EGFR at serine residues would represent a compensatory mechanism triggered by chronically elevated levels of GH to mitigate the proliferative response induced by EGF.

Dietary branched chain amino acids and metabolic health: when less is more

Within obesity research it is almost axiomatic that the consumption of adequate protein is healthy. A large body of work suggests that high protein diets reduce food intake, while maintaining protein intake but reducing caloric intake promotes fat loss while sustaining lean mass. Yet evidence is also accumulating to suggest that excess protein intake negatively impacts health, and that the restriction of dietary protein triggers beneficial metabolic effects. This body of work derives in part from efforts in the ageing field to define the mechanisms through which dietary restriction extends lifespan. In this issue of The Journal of Physiology, Cummings et al. (2018) provide compelling evidence that the restriction of branched chain amino acid (BCAA) intake is sufficient to restore metabolic health in obese mice. Although it has long been known that BCAAs are elevated in settings of obesity (Felig et al. 1974), work by Newgard and colleagues in 2009 rekindled interest in this field by demonstrating that elevated BCAAs contribute to a ‘metabolic signature’ predicting insulin resistance in obese humans (Newgard et al. 2009). Furthermore, supplementing high-fat-fed animals with excess BCAAs reduced food intake and body weight but failed to improve glucose tolerance. This negative effect of excess BCAAs is consistent with more recent work by several groups suggesting that restricting dietary protein intake improves glucose tolerance and other metabolic endpoints. Cummings et al. build on their prior work (Fontana et al. 2016) by testing the impact of BCAA restriction in mice with established obesity. The key question is whether reducing dietary BCAA intake alone is sufficient to improve glucose homeostasis in obese mice, and whether this effect rivals the beneficial effects produced by the restriction of dietary protein (all amino acids). This question is first tested by transitioning mice made obese with a high fat ‘Western diet’ (WD) to a low fat, low BCAA diet. Not surprisingly, swapping mice from WD to low fat diet reduced adiposity and improved glucose homeostasis. However, the low BCAA diet was even more effective, producing a larger weight loss and improvement in glucose tolerance than low fat alone. The BCAA restriction also largely replicated the effect of a diet that was low in all AAs. While this study suggested that BCAA restriction exerts beneficial effects, the authors recognized the weakness inherent in simultaneously manipulating energy density, macronutrient content and amino acid ratios. Therefore, a second study was designed in which mice were made obese by 12 weeks of WD exposure and then swapped to a WD specifically restricting BCAAs. In this context the restriction of dietary BCAAs significantly decreased body weight and adiposity, increased energy expenditure, and improved glucose tolerance and insulin sensitivity. BCAA restriction also largely recapitulated the metabolic effects induced by the restriction of all amino acids. These data provide compelling evidence that restricting BCAAs is sufficient to restore metabolic health in the context of continuous WD exposure. The observation that restriction of BCAAs produces metabolic benefits that are comparable to the restriction of all AAs (dietary protein restriction) leads to the logical question of whether the beneficial effect of low protein diets are mediated by BCAAs. While the current data are consistent with this conclusion, there are reasons to be cautious. Cummings et al. demonstrate that BCAA restriction is sufficient to trigger metabolic improvements, but do not test whether BCAA restriction is necessary for the effects of protein restriction. Answering this question requires restricting dietary protein but restoring BCAAs to control levels. Interestingly, a recent study by Maida et al. (2017) used this design to suggest that the normalizing BCAA intake only attenuates the metabolic effect of protein restriction in lean mice and has no effect in genetically obese NZO mice. Taken together, the work of Cummings et al. and Maida et al. suggest that although BCAA restriction reproduces aspects of the metabolic response to general protein restriction, BCAA restriction does not functionally account for all the effects of protein restriction. It should be noted that the metabolic effects of BCAA restriction and protein (all AA) restriction are similar, but not interchangeable (Fontana et al. 2016; Cummings et al. 2018). While Cummings et al. provide compelling evidence that BCAA restriction influences metabolic endpoints, the report does not identify the mechanistic pathways that drive these improvements. For instance, BCAAs and leucine in particular are known to stimulate mechanistic target of rapamycin (mTOR) signalling, and mTOR has been linked to the regulation of insulin sensitivity. Restricting or supplementing leucine alone would have further complemented their prior work (Fontana et al. 2016) by delineating whether leucine is the primary mediator of the BCAA effect. Finally, a growing body of work implicates the metabolic hormone fibroblast growth factor 21 (FGF21) as a key mediator of dietary protein restriction (Laeger et al. 2014), but its contribution to the effect of BCAA restriction remains unclear. Cummings et al. suggest that restriction of BCAAs has inconsistent effects on FGF21, which was increased after 12 days on diet but not after 12 weeks. This temporal variability in the FGF21 response is also associated with temporal variability in other endpoints, such as food intake and energy expenditure. Therefore, when and how BCAA restriction influences metabolism, particularly glucose homeostasis, remain unclear. The report by Cummings et al. provides strong evidence that reducing BCAA intake restores metabolic health in obese rodents, thereby adding to a growing body of work suggesting that restriction of dietary protein or select amino acids promotes beneficial metabolic adaptations. Future work is necessary to define how these various dietary interventions produce these effects, the extent to which the underlying mechanisms are similar or divergent, and finally whether these beneficial effects can be translated to humans.