Lan Ye

SIRT1 is required for AMPK activation and the beneficial effects of resveratrol on mitochondrial function

Resveratrol induces mitochondrial biogenesis and protects against metabolic decline, but whether SIRT1 mediates these benefits is the subject of debate. To circumvent the developmental defects of germline SIRT1 knockouts, we have developed an inducible system that permits whole-body deletion of SIRT1 in adult mice. Mice treated with a moderate dose of resveratrol showed increased mitochondrial biogenesis and function, AMPK activation, and increased NAD(+) levels in skeletal muscle, whereas SIRT1 knockouts displayed none of these benefits. A mouse overexpressing SIRT1 mimicked these effects. A high dose of resveratrol activated AMPK in a SIRT1-independent manner, demonstrating that resveratrol dosage is a critical factor. Importantly, at both doses of resveratrol no improvements in mitochondrial function were observed in animals lacking SIRT1. Together these data indicate that SIRT1 plays an essential role in the ability of moderate doses of resveratrol to stimulate AMPK and improve mitochondrial function both in vitro and in vivo.

Rapamycin-induced insulin resistance is mediated by mTORC2 loss and uncoupled from longevity

Dissecting Rapamycin Responses Long-term treatment of mice and other organisms with the drug rapamycin extends life span. But, at the same time, the drug disrupts metabolic regulation and the action of the hormone insulin. Lamming et al. (p. 1638; see the Perspective by Hughes and Kennedy) dissected the action of rapamycin in genetically modified mice and found, encouragingly, that these two actions of rapamycin can be separated. Rapamycin inhibits a protein kinase complex known as mTORC1, and this appears to provide most of the life-lengthening effects of the drug. However, rapamycin also acts on a related complex known as mTORC2, and it is the disruption of mTORC2 action that produces the diabetic-like symptoms of decreased glucose tolerance and insensitivity to insulin. The effect of the drug rapamycin on life span can be separated from its effects on metabolism. Rapamycin, an inhibitor of mechanistic target of rapamycin complex 1 (mTORC1), extends the life spans of yeast, flies, and mice. Calorie restriction, which increases life span and insulin sensitivity, is proposed to function by inhibition of mTORC1, yet paradoxically, chronic administration of rapamycin substantially impairs glucose tolerance and insulin action. We demonstrate that rapamycin disrupted a second mTOR complex, mTORC2, in vivo and that mTORC2 was required for the insulin-mediated suppression of hepatic gluconeogenesis. Further, decreased mTORC1 signaling was sufficient to extend life span independently from changes in glucose homeostasis, as female mice heterozygous for both mTOR and mLST8 exhibited decreased mTORC1 activity and extended life span but had normal glucose tolerance and insulin sensitivity. Thus, mTORC2 disruption is an important mediator of the effects of rapamycin in vivo.

Rapalogs and mTOR inhibitors as anti-aging therapeutics

Rapamycin, an inhibitor of mechanistic target of rapamycin (mTOR), has the strongest experimental support to date as a potential anti-aging therapeutic in mammals. Unlike many other compounds that have been claimed to influence longevity, rapamycin has been repeatedly tested in long-lived, genetically heterogeneous mice, in which it extends both mean and maximum life spans. However, the mechanism that accounts for these effects is far from clear, and a growing list of side effects make it doubtful that rapamycin would ultimately be beneficial in humans. This Review discusses the prospects for developing newer, safer anti-aging therapies based on analogs of rapamycin (termed rapalogs) or other approaches targeting mTOR signaling.

Young and old genetically heterogeneous HET3 mice on a rapamycin diet are glucose intolerant but insulin sensitive

Rapamycin, an inhibitor of the mechanistic target of rapamycin (mTOR) signaling pathway, extends the life span of yeast, worms, flies, and mice. Interventions that promote longevity are often correlated with increased insulin sensitivity, and it therefore is surprising that chronic rapamycin treatment of mice, rats, and humans is associated with insulin resistance (J Am Soc Nephrol., 19, 2008, 1411; Diabetes, 00, 2010, 00; Science, 335, 2012, 1638). We examined the effect of dietary rapamycin treatment on glucose homeostasis and insulin resistance in the genetically heterogeneous HET3 mouse strain, a strain in which dietary rapamycin robustly extends mean and maximum life span. We find that rapamycin treatment leads to glucose intolerance in both young and old HET3 mice, but in contrast to the previously reported effect of injected rapamycin in C57BL/6 mice, HET3 mice treated with dietary rapamycin responded normally in an insulin tolerance test. To gauge the overall consequences of rapamycin treatment on average blood glucose levels, we measured HBA1c. Dietary rapamycin increased HBA1c over the first 3 weeks of treatment in young animals, but the effect was lost by 3 months, and no effect was detected in older animals. Our results demonstrate that the extended life span of HET3 mice on a rapamycin diet occurs in the absence of major changes in insulin sensitivity and highlight the importance of strain background and delivery method in testing effects of longevity interventions.

Rapamycin has a biphasic effect on insulin sensitivity in C2C12 myotubes due to sequential disruption of mTORC1 and mTORC2

Rapamycin, an inhibitor of mTOR complex 1 (mTORC1), improves insulin sensitivity in acute studies in vitro and in vivo by disrupting a negative feedback loop mediated by S6 kinase. We find that rapamycin has a clear biphasic effect on insulin sensitivity in C2C12 myotubes, with enhanced responsiveness during the first hour that declines to almost complete insulin resistance by 24–48 h. We and others have recently observed that chronic rapamycin treatment induces insulin resistance in rodents, at least in part due to disruption of mTORC2, an mTOR-containing complex that is not acutely sensitive to the drug. Chronic rapamycin treatment may also impair insulin action via the inhibition of mTORC1-dependent mitochondrial biogenesis and activity, which could result in a buildup of lipid intermediates that are known to trigger insulin resistance. We confirmed that rapamycin inhibits expression of PGC-1α, a key mitochondrial transcription factor, and acutely reduces respiration rate in myotubes. However, rapamycin did not stimulate phosphorylation of PKCθ, a central mediator of lipid-induced insulin resistance. Instead, we found dramatic disruption of mTORC2, which coincided with the onset of insulin resistance. Selective inhibition of mTORC1 or mTORC2 by shRNA-mediated knockdown of specific components (Raptor and Rictor, respectively) confirmed that mitochondrial effects of rapamycin are mTORC1-dependent, whereas insulin resistance was recapitulated only by knockdown of mTORC2. Thus, mTORC2 disruption, rather than inhibition of mitochondria, causes insulin resistance in rapamycin-treated myotubes, and this system may serve as a useful model to understand the effects of rapamycin on mTOR signaling in vivo.

Rapamycin Blocks Induction of the Thermogenic Program in White Adipose Tissue

Rapamycin extends life span in mice, yet paradoxically causes lipid dysregulation and glucose intolerance through mechanisms that remain incompletely understood. Whole-body energy balance can be influenced by beige/brite adipocytes, which are inducible by cold and other stimuli via β-adrenergic signaling in white adipose depots. Induction of beige adipocytes is considered a promising strategy to combat obesity because of their ability to metabolize glucose and lipids, dissipating the resulting energy as heat through uncoupling protein 1. Here, we report that rapamycin blocks the ability of β-adrenergic signaling to induce beige adipocytes and expression of thermogenic genes in white adipose depots. Rapamycin enhanced transcriptional negative feedback on the β3-adrenergic receptor. However, thermogenic gene expression remained impaired even when the receptor was bypassed with a cell-permeable cAMP analog, revealing the existence of a second inhibitory mechanism. Accordingly, rapamycin-treated mice are cold intolerant, failing to maintain body temperature and weight when shifted to 4°C. Adipocyte-specific deletion of the mTORC1 subunit Raptor recapitulated the block in β-adrenergic signaling. Our findings demonstrate a positive role for mTORC1 in the recruitment of beige adipocytes and suggest that inhibition of β-adrenergic signaling by rapamycin may contribute to its physiological effects.

A germline-specific role for the mTORC2 component Rictor in maintaining spermatogonial differentiation and intercellular adhesion in mouse testis

STUDY QUESTION What is the physiological role of Rictor in spermatogenic cells? SUMMARY ANSWER Germline expression of Rictor regulates spermatogonial differentiation and has an essential role in coordinating germ cells and Sertoli cells in maintaining intact cell-cell adhesion dynamics and cytoskeleton-based architecture in the seminiferous epithelium. WHAT IS KNOWN ALREADY The mechanistic target of rapamycin (mTOR) resides in its functions as the catalytic subunits of the structurally and functionally distinct mTORC1 and mTORC2 complexes. In the mammalian testis, mTORC1 regulates spermatogonial stem cell self-renewal and differentiation, whereas mTORC2 is required for Sertoli cell function. In contrast to mTORC1, mTORC2 has been much less well studied. Rictor is a distinct component of the mTORC2 complex. STUDY DESIGN, SIZE, DURATION We investigated the effects of germ cell-specific ablation of Rictor on testicular development by using a mouse model of germline-specific ablation of Rictor. PARTICIPANTS/MATERIALS, SETTING, METHODS We analyzed the in-vivo functions of Rictor through different methods including histology, immunofluorescent staining, chromosome spreads, blood-testis barrier (BTB) integrity assays and RNA sequencing. MAIN RESULTS AND THE ROLE OF CHANCE Mutant mice did not show a defect in meiotic synapsis or recombination, but exhibited compromised spermatogonial differentiation potential, disorganized cell-cell junctions, impaired BTB dynamics and defective spermiogenesis. Concomitantly, RNA-seq profiling revealed that many genes involved in adhesion and migration were expressed inappropriately. LARGE SCALE DATA RNA-seq data are published in the SRA database (PRJNA419273). LIMITATIONS REASONS FOR CAUTION A detailed analysis of the mechanisms underlying the phenotype needs further investigations. WIDER IMPLICATIONS OF THE FINDINGS Our work provides previously unidentified in-vivo evidence that germline expression of Rictor plays a role in maintaining spermatogonial differentiation and cell-cell adhesion. These findings are important for understanding the regulation of spermatogenesis and have clinical implications for the effect of mTOR inhibitors on human fertility. STUDY FUNDING AND COMPETING INTEREST(S) This study was supported by National Key R&D Program of China (2016YFA0500902), National Natural Science Foundation of China (31471228 and 31771653), Jiangsu Science Foundation for Distinguished Young Scholars (BK20150047), and Natural Science Foundation of Jiangsu Province (BK20140897, 14KJA180005 and 14KJB310004) to K.Z. The authors declare no competing or financial interests.

Conditional ablation of Raptor in the male germline causes infertility due to meiotic arrest and impaired inactivation of sex chromosomes

Rapamycin is a clinically important drug that is used in transplantation and cancer therapy but which causes a number of side effects, including male infertility. Its canonical target, mammalian target of rapamycin complex 1 (mTORC1), plays a key role in metabolism and binds chromatin; however, its precise role in the male germline has not been elucidated. Here, we inactivate the core component, Raptor, to show that mTORC1 function is critical for male meiosis and the inactivation of sex chromosomes. Disruption of the Raptor gene impairs chromosomal synapsis and prevents the efficient spreading of silencing factors into the XY chromatin. Accordingly, mRNA for XY‐linked genes remains inappropriately expressed in Raptor‐deficient mice. Molecularly, the failure to suppress gene expression corresponded with deficiencies in 2 repressive chromatin markers, H3K9 dimethylation and H3K9 trimethylation, in the XY body. Together, these results demonstrate that mTORC1 has an essential role in the meiotic progression and silencing of sex chromosomes in the male germline, which may explain the infertility that has been associated with such inhibitors as rapamycin.—Xiong, M., Zhu, Z., Tian, S., Zhu, R., Bai, S., Fu, K., Davis, J. G., Sun, Z., Baur, J. A., Zheng, K., Ye, L. Conditional ablation of Raptor in the male germline causes infertility due to meiotic arrest and impaired inactivation of sex chromosomes. FASEB J. 31, 3934–3949 (2017). www.fasebj.org—Xiong, Mengneng, Zhu, Zhiping, Tian, Suwen, Zhu, Ruping, Bai, Shun, Fu, Kaiqiang, Davis, James G., Sun, Zheng, Baur, Joseph A., Zheng, Ke, Ye, Lan, Conditional ablation of Raptor in the male germline causes infertility due to meiotic arrest and impaired inactivation of sex chromosomes. FASEB J. 31, 3934–3949 (2017)

Sox30 initiates transcription of haploid genes during late meiosis and spermiogenesis in mouse testes

ABSTRACT Transcription factors of the Sox protein family contain a DNA-binding HMG box and are key regulators of progenitor cell fate. Here, we report that expression of Sox30 is restricted to meiotic spermatocytes and postmeiotic haploids. Sox30 mutant males are sterile owing to spermiogenic arrest at the early round spermatid stage. Specifically, in the absence of Sox30, proacrosomic vesicles fail to form a single acrosomal organelle, and spermatids arrest at step 2-3. Although most Sox30 mutant spermatocytes progress through meiosis, accumulation of diplotene spermatocytes indicates a delayed or impaired transition from meiotic to postmeiotic stages. Transcriptome analysis of isolated stage-specific spermatogenic cells reveals that Sox30 controls a core postmeiotic gene expression program that initiates as early as the late meiotic cell stage. ChIP-seq analysis shows that Sox30 binds to specific DNA sequences in mouse testes, and its genomic occupancy correlates positively with expression of many postmeiotic genes including Tnp1, Hils1, Ccdc54 and Tsks. These results define Sox30 as a crucial transcription factor that controls the transition from a late meiotic to a postmeiotic gene expression program and subsequent round spermatid development. Summary: Analysis of Sox30 mutants using ChIP-seq, meiotic analysis and RNA-seq identifies the direct targets of Sox30 and reveals that Sox30 is a transcription factor of postmeiotic genes in mouse testes.