inal white adipose tissue.

DISCUSSION
Diminished fat browning and increased AT inflammation are key characteristics of obesity in animals and humans. Both are associated with glucose intolerance and insulin resistance. Intermittent fasting improves these phenotypes. Until now, the cellular and molecular links mediating IF and the associated phenotypic changes have remained poorly understood. We confirmed in this study that a 2:1 IF regimen protected mice from diet-induced obesity and metabolic disturbances without reducing food intake. These outcomes were accompanied by significant induction of Sirt6 protein expression in BAT, eWAT, and iWAT, implying a potential role of Sirt6 in metabolic adaptations to IF. Specifically, our study suggests that Sirt6 in adipocytes is a crucial player in IF-promoted metabolic adaptations. By mimicking the benefit of the IF diet regimen, activation of Sirt6 could serve as a defense against obesity and type 2 diabetes.
What was responsible for the observed improvement gaps between WT and aS6KO mice subjected to IF? The answer involves alterations in both WAT and BAT function, and several mechanisms are possible. First, enhanced adipose inflammation in eWAT could have led to the diminished IF response observed in aS6KO mice in terms of glucose and insulin responses. Obesity and type 2 diabetes are tightly linked to increased inflammation and M1 macrophage accumulation in WAT 41, whereas CR and IF have been found to suppress tissue inflammation in humans and animals 33,38,42,43, although the latter finding is controversial 44,45. CR/IF or acute fasting has been shown to either increase or decrease WAT macrophage numbers and inflammatory cytokine levels, and these differences could possibly be due to the degree of AT lipolysis, depending on the fasting period. Recently, a 2:1 IF regimen in HFD-fed mice was shown to reduce the number of M1 macrophages in eWAT by increasing AT vascular endothelial growth factor levels 33. Consistent with this report, our current findings showed (1) decreased numbers of total and M1 macrophages in the eWAT of HFD + IF mice ( Supplementary Fig. 1e), (2) high numbers of M1 macrophages and high levels of related genes in the eWAT of aS6KO mice, and (3) lower serum levels of the anti-inflammatory cytokine IL-10 in aS6KO mice than in WT mice. These results are consistent with our previous report showing that adipocyte Sirt6 decreases the M1 composition in eWAT 30. The question of how adipocyte Sirt6 regulates the AT macrophage subpopulation under IF conditions requires further study.
Second, insulin-stimulated phosphorylation of Akt in eWAT, muscle, and liver tissue was found to be lower in aS6KO mice than in WT mice, suggesting decreased insulin sensitivity in aS6KO mice under IF. A previous study showed that CR preferentially stimulated glucose uptake in white fat without affecting it in BAT or muscle 36, suggesting that white fat depots are major tissues of glucose disposal affected by CR. Therefore, the impairment of glucose homeostasis in aS6KO mice subjected to IF could be mainly attributable to eWAT with features of inflammation and insulin resistance, although decreased phosphorylation of Akt was also observed in the skeletal muscle and liver tissue of aS6KO mice under IF. Because eWAT inflammation was evident in the aS6KO mice we used in this study, adipose tissue-derived proinflammatory cytokines might act as a "second hit" for the development of insulin resistance in the skeletal muscle and liver tissues of aS6KO mice.
Third, decreases in beige fat development and BAT activation and the consequent repression of thermogenesis could have led to the increased body weight and insulin resistance observed in aS6KO mice. Previous studies have shown that increased beige and brown fat development enhances energy expenditure and boosts insulin sensitivity 46, and that IF-mediated metabolic improvement is attributable to adipose thermogenesis 33. We observed in the current study that (1) in normal mice, IF induced UCP1 protein expression to a similar degree in BAT and iWAT (Fig.  1d), and (2) in aS6KO mice, UCP1 protein expression in both BAT and iWAT was lower than that in WT mice, with a complete reduction in UCP1 expression in iWAT, supporting a selective role for Sirt6 in WAT browning (Fig. 5c). We found that Sirt6 protein expression correlated well with UCP1 expression only in iWAT (Fig.  1d) and that the impairment of the adaptive response to IF in aS6KO mice was severe only in iWAT, not in BAT (Fig. 5c), suggesting an adipose depot-specific response. While both BAT activation and WAT browning contribute to thermogenesis and energy expenditure, a few studies have suggested selective browning of WAT 10,47,48. It is possible that the underlying thermogenesis mechanism differs depending on the context. BAT is highly efficient and active in heat generation due to its constitutively high expression of UCP1 relative to that in WAT in mice. Yao et al. previously reported that fat Sirt6 expression is markedly induced by cold as well as β-3 adrenergic agonist treatment and is critical for thermogenesis of brown and beige fat 32. They showed that, mechanistically, Sirt6 interacts with phospho-ATF2 on the PGC-1α gene promoter to activate its expression and, in turn, increases UCP1 transcription. The requisite role of Sirt6 in the thermogenic response is similar between BAT and WAT, supporting the viability of the β-3 adrenergic receptor/ PGC1α/UCP1 axis. In this study, we observed that IF increased the phosphorylation of p38 MAPK, an upstream kinase for ATF2 49, and that this event was almost completely reduced in the iWAT of aS6KO mice. Concomitantly, the total expression of ATF2 was also suppressed in these mice. These results indicate that Sirt6 regulates UCP1 expression via p38 MAPK/ATF2 signaling in iWAT upon IF.
Interestingly, ATF2 phosphorylation in BAT did not differ between WT and aS6KO mice ( Supplementary Fig. 7a), despite the similar extent of UCP1 reduction in aS6KO mice in response to IF. Although we did not address the detailed mechanism of Sirt6 regulation of UCP1 induction in BAT, these results imply that there must be a difference in the intrinsic signaling pathway that leads to UCP1 expression in BAT.
Lipolysis in adipose tissue is tightly regulated by a series of complex mechanisms involving ATGL and HSL, as well as hormonal and biochemical signals that regulate the activity of these lipases. Deletion of adipose lipases results in excess storage of triglycerides, causing adipocyte hypertrophy and obesity 50,51, whereas overexpression of these enzymes promotes lipolysis 52. The impaired lipolysis pathway and adipocyte hypertrophy in aSKO mice under IF was attributable to reduced expression of phospho-ATGL, ATGL, phospho-HSL, and HSL. While it has been reported that Sirt6 in adipocytes upregulates ATGL transcription through FoxO1 activation 29, we did not observe reduced expression of ATGL in aS6KO-HFD mice (Fig. 3e) explain what caused this difference, but the longer periods of HFD feeding in the mice (eight weeks in the study by Kuang et al. vs 16 weeks in our study) might have contributed to the lack of difference in ATGL expression between WT and aS6KO mice. Instead, we noted a marked reduction in the phosphorylation of lipases upon IF, suggesting that the hormonal and biochemical environment was altered by Sirt6 deficiency. Future studies are needed to determine the mechanism by which Sirt6 ablation decreases the phosphorylation levels of ATGL and HSL.
Despite the marked elevations in the phosphorylation of ATGL and HSL in iWAT caused by IF, the serum levels of NEFAs and glycerol were consistently decreased by IF relative to HFD ad libitum feeding (Fig. 1b and Fig. 3d). The reason for this discrepancy is unclear, but there may be a dissociation between obesity and lipolysis. The association between lipolysis and adipose hypertrophy or obesity remains inconsistent across studies, with both defects 53,54 and increases in lipolysis 55 being reportedly linked with obesity.
In summary, adipose Sirt6 is necessary for metabolic adaptation to IF, enhances adipose tissue browning, ameliorates adipose tissue inflammation, and thereby improves insulin action on peripheral tissues, as depicted in Supplementary Fig. 8. Genetic or pharmacological activation of Sirt6 might be an alternative means of achieving the health advantages conferred by IF.This page documents how to add new provisioners to Vagrant, allowing Vagrant to automatically install software and configure software using a custom provisioner. Prior to reading this, you should be familiar with the plugin development basics.
Provisioners are defined with the provisioner method, which takes a single argument specifying the name of the provisioner. This is the name that used with config.vm.provision when configuring and enabling the provisioner. So in the case above, the provisioner would be enabled using config.vm.provision :custom.
The block argument then lazily loads and returns a class that implements the Vagrant.plugin(2, :provisioner) interface, which is covered next.
The provisioner class should subclass and implement Vagrant.plugin(2, :provisioner) which is an upgrade-safe way to let Vagrant return the proper parent class for provisioners.
This class and the methods that need to be implemented are very well documented. The documentation on the class in the comments should be enough to understand what needs to be done.
There are two main methods that need to be implemented: the configure method and