This ex-vivo/in-vitro study indicates that high dairy feeding in humans over 4 weeks results in significant systemic changes which may induce activation of SIRT1 and downstream targets of mitochondrial biogenesis in key target tissues as demonstrated here in muscle and adipose cells. These effects may contribute to observed changes in energy metabolism and oxidative and inflammatory stress. We previously reported that the high dairy intake in this clinical study resulted in significant suppression of oxidative and inflammatory biomarkers, including reductions in plasma malondialdehyde, 8-isoprostane-F2, TNF-α, IL-6 and MCP-1, and increased levels of the anti-inflammatory cytokine adiponectin . We have also shown that dairy components, especially calcium and leucine, up-regulated signaling pathways for fat oxidation, attenuated inflammatory pathways such as NF-κB signaling and stimulated insulin signaling in skeletal muscle and adipose tissue, suggesting a SIRT1 dependent regulation . Similarly, supplemental leucine intake by addition to drinking water in mice rescued metabolic changes to a high fat diet and was associated with improvement of glucose tolerance and insulin signaling as well as decrease in adipose tissue inflammation . Although the authors of this study did not assess SIRT1 activation, SIRT1 dependence is consistent with their observations. Yoshizaki et al  reported beneficial effects of SIRT1 activation on glucose uptake and insulin signaling, and improvement of inflammatory markers in 3T3-L1 adipocytes while SIRT1 depletion exerted the opposite effect. Moreover, modest global overexpression of SIRT1 in mice resulted in protection against metabolic damage from a high-fat diet by up-regulation of antioxidant proteins such as MnSOD and NRF1, and lower lipid-induced activation of pro-inflammatory cytokines such as TNFα and IL-6 via reduction of NF-κB . These effects were manifested in lower inflammation, improved glucose tolerance and nearly complete protection from hepatic steatosis. In contrast, heterozygous SIRT1 knockout (SIRT1+/-) mice developed severe hepatic steatosis on high-fat diets, accompanied by lower energy expenditure and increased inflammation . Accordingly, our observation of increased SIRT1 activity and expression in muscle and adipose tissue following incubation with serum from subjects fed a high dairy diet likely represents the mechanism of the observed reduction of oxidative and inflammatory stress in these subjects. However, the present data are limited by the opportunistic use of archival samples from our previous clinical trial ; accordingly, no direct comparison of the effects of increasing dairy food intake in lean vs. obese subjects is possible.
Mitochondrial loss and/or dysfunction play a key role in metabolic disorders such as insulin resistance, type II diabetes and cardiovascular disease [21–23], and stimulation of mitochondrial biogenesis with resveratrol increased insulin sensitivity and prevented obesity and insulin-resistance in mice fed a high-fat diet . The majority of mitochondrial proteins, including most of those involved in oxidative phosphorylation, are nuclear encoded and transported into the mitochondria from the cytoplasm while only 13 protein subunits involved in electron transport are encoded in the mitochondrial genome (mtDNA) . For mitochondrial biogenesis, both the nuclear and mitochondrial protein subunits have to form complexes and thus require a coordinated crosstalk and regulation for proper function. PGC-1α, a downstream-target of SIRT1, is a key regulator of mitochondrial biogenesis in response to external stimuli. Up-regulation of PGC-1α activates the expression of nuclear-encoded OxPhos components genes as well as of nuclear respiratory factor (NRF1), which also regulates the transcription of mitochondrial genes [25, 26].
Although it is possible that the previously observed systemic changes in oxidative and inflammatory biomarkers  may contribute to increased SIRT1 expression and/or activity, we have previously demonstrated that leucine administration stimulated mitochondrial biogenesis and metabolism in skeletal muscle and adipose cells and that these effects were mediated, in part, by SIRT1 , while calcitriol treatment exerted the opposite effects. Similarly, we demonstrate in this study that a leucine rich diet in form of dairy results in up-regulation of PGC-1α as well as in up-regulation of downstream target genes such as NRF-1, UCP2 and 3, NADH dehydrogenase and cytochrome c oxidase indicative of stimulated mitochondrial biogenesis in muscle and adipose tissue. Although we cannot directly attribute the observed ex vivo effects to the leucine content of the high dairy diet, the concentrations of leucine used in our in vitro studies are comparable to the plasma levels typically achieved in response to leucine-rich milk or whey-based diets , while soy protein isolate produced only ~30% the leucine level when studied at the same protein load (10 g/dose).
Data from this study indicate that not only leucine but also its metabolites HMB and KIC are direct activators of SIRT1 enzyme. It has been suggested that some of the leucine effects may be attributed to its metabolites, which have also variable effects on protein metabolism and immune function . The majority of the first step of leucine metabolism, the transamination to KIC, occurs in muscle. Orally administered alpha-KIC to food-deprived rats exerted stimulatory effects on protein-synthesis in skeletal muscle, similar to L-leucine administration, but not in liver; however, it was not clear whether these effects were direct effects of KIC or caused by the reversible conversion of KIC to leucine . Earlier results demonstrated that incubation with KIC could decrease protein degradation in rat diaphragms but did not stimulate protein synthesis while leucine incubation did both .
Although effects of HMB supplementation on muscle strength and gain are also conflicting, HMB has been used as a therapeutical supplement for years to attenuate muscle loss and damage under various conditions . Some of this anti-catabolic activity seems to be mediated by reduction of ROS formation . In addition, HMB may play a role as a potentially dietary immunomodulator since it has been shown to decrease proliferation and TNF-α production in stimulated human peripheral blood monocytes by 35%  and suppressed NF-κB expression in tumor-bearing Wistar rats, thereby reducing tumor growth and tumor cell proliferation . Since SIRT1 is a negative regulator of NF-κB [35, 36] and attenuation of NF-kB activity by SIRT1 results in suppression of TNF-α [37, 38], it is possible that the above mentioned effects of HMB are mediated, at least in part, by HMB activation of SIRT1. It is not yet clear how leucine or its metabolites KIC and HMB directly stimulate SIRT1 in a cell free system. We speculate that they may act as allosteric activators producing a conformational change in SIRT1, which increases binding to its substrate.
The stimulation of mitochondrial biogenesis in muscle cells was not associated with underlying changes in SIRT1 activity, although SIRT1 gene expression was up-regulated. Therefore, it is likely that SIRT1-independent pathways also modulate some of the effects of dairy components. AMP-activated protein kinase (AMPK), a key regulator of energy metabolism, is a likely target, as it also serves as an energy sensor and regulates cellular metabolism. In addition, there is a bidirectional interaction between AMPK and SIRT1; AMPK activates SIRT1 by increasing cellular NAD+ levels and, conversely, SIRT1 stimulates AMPK by activation of LKB1 . Since adiponectin activates AMPK , and we previously demonstrated the high dairy diet to increase adiponectin (16), adiponectin stimulation of AMPK may play a significant role in the observed effects.