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Patients are encouraged to seek advice from ACT Pathology or their doctor before following any of the instructions below. To provide feedback or request an accessible version of a document please contact us or phone 13 22 81. Germline ablation of Sirt3 has major metabolic consequences, including increased susceptibility to metabolic damage and oxidative stress after high fat feeding. Similarly, there was no evidence for increased oxidative stress in muscle or liver when Sirt3 was ablated in a tissue-specific manner. These observations suggest that the mitochondrial hyperacetylation induced by Sirt3-deletion in a tissue specific manner is not necessarily linked to mitochondrial dysfunction and does not recapitulate the metabolic abnormalities observed in the germline Sirt3 knock-out mice.IntroductionSirt3 is one of the 7 members of the sirtuin family of NAD+-dependent deacetylases.
Sirt3 expression is highly enriched in metabolic tissues like liver, brown adipose tissue (BAT), heart, skeletal muscle and kidney3.
Sirt3 expression increases in muscle after exercise training, fasting and caloric restriction (CR), while it decreases upon long-term high fat feeding4,5. In contrast, Sirt3 overexpression in vitro enhances mitochondrial respiration13 and reduces ROS production3. Not surprisingly, whole body Sirt3 knock-out mice are sensitized to high fat diet (HFD)-induced obesity, insulin resistance, hyperlipidemia and steatohepatitis14. The etiology of such defects might be found in the ability of Sirt3 to enhance fat oxidation and improve anti-oxidant defences6,9,10,14,15,16.
However, while recent reports show that Sirt3 suppresses oxidative stress under CR9,10,12, it still remains to be elucidated whether Sirt3 might influence HFD-induced oxidative stress.All studies to date used germline Sirt3 deficient mice to study the role of Sirt3 on metabolism, making it impossible to distinguish the contribution of individual tissues to the phenotypic changes. Liver and muscle are two of the most important tissues determining whole body metabolism: skeletal muscle is the largest organ in mammals, contributing to ±40% of the body mass, and it plays a major role in whole body metabolism, as it is vital for insulin-mediated glucose disposal and lipid catabolism. Altered function of these tissues is, thus, likely to contribute to the systemic metabolic disturbances observed in the germline Sirt3 knock-out mice. Here, we report the generation of the first set of tissue-specific Sirt3 knockout mouse models in muscle and liver, and describe how Sirt3 deletion in these tissues, despite leading to mitochondrial protein hyperacetylation, has minor phenotypic consequences. Maps of the Sirt3 genomic locus showing the conditional allele (upper panel) and the KO allele (lower panel). The white arrowheads indicate the LoxP sites and the black vertical bars represent the respective exons.
1E), confirming that the Sirt3 deletion is efficient and restricted to the targeted tissue.The absence of Sirt3 had no impact on the expression of a vast set of metabolic genes in either muscle (Fig.


Of note, we did not detect any compensatory increase in the expression of the other mitochondrial sirtuins (Sirt4 and Sirt5) in the two Sirt3-deficient tissues.
We also determined Pgc1? and Scd1 expression, two genes whose levels were reported to be altered in germline Sirt3-KO tissues5,14; however, we could not detect any significant change in their expression in our mutant mice. These results suggest that muscle or liver-specific deletion of Sirt3 does not have a major impact on the expression of genes involved in metabolic control. During indirect calorimetry using the comprehensive lab animal monitoring system (CLAMS), no difference was observed in food intake (Fig.
At sacrifice, the weight of the liver, epididymal white fat or interscapular brown fat depots were not different between the genotypes (Fig.
To specifically test the contribution of Sirt3 deletion in the liver to glucose homeostasis we performed an intraperitoneal glucose tolerance test (ipGTT) and an insulin tolerance test (ipITT) both before (data not shown) and after HFD. Results are represented as mean values (n = 8?10 mice for each group) ± standard error of the mean (SEM).Full size imageGermline Sirt3-KO mice have also been reported to display altered aminoacid profiles in blood and liver15. 2K).Sirt3 has also been suggested to act as a key modulator of ketone body production, a glucose-sparing energy substrate in the fasting state mostly synthesized by hepatocytes19.
Consistent with the similarities in body weight, evaluation using CLAMS indicated that food intake (Fig. In addition, no significant differences were observed in the weight of different tissues at sacrifice (Fig. Also, the drop and recovery of blood glucose levels upon an intraperitoneal insulin tolerance test (ipITT) were, again, not distinct between the two genotypes (Fig. Mice from both genotypes similarly maintained their core body temperature for at least 6?hours after a 24?h fast (Fig.
1A), indicating that muscle Sirt3 is not essential for cold-induced adaptive thermogenesis. Exercise increases Sirt3 expression in muscle4,5, suggesting a role for Sirt3 in exercise physiology. However, this increased acetylation did not lead to any change in maximal complex I or complex IV activities (Fig. As an internal control, we measured the activity of citrate synthase, a mitochondrial enzyme from the Krebs cycle not belonging to the electron transport chain, whose activity was also unchanged in the absence of Sirt3 in both mouse models (Fig. Mitochondrial protein lysates were subjected to western blotting using an anti-acetylated lysine antibody (Ac-K) and porin as a loading control. Measurements in panels E–K were determined using samples from at least four mice per genotype. All the samples used for these studies come from mice subjected to the HFD treatment, sacrificed in fed state. Data are represented as mean ± SEM.Full size imageSirt3 deficiency has also been correlated with low levels of cellular ATP13.


2B).Sirt3 has been shown to reduce oxidative stress, mostly through deacetylating and activating the mitochondrial ROS scavenger Sod29,10,12,16. We therefore also determined Sod2 acetylation status in the absence of Sirt3; as reported, Sirt3-deficient tissues presented increased levels of acetylated Sod2 (Fig.
We next measured a few markers of oxidative stress, such as the level of 4-hydroxy-2-nonenal (HNE; a marker for lipid peroxidation) and glutathione, in liver and muscle from our mutant mouse models after HFD treatment.
No difference in these parameters was observed between Sirt3-deficient tissues and their respective controls (Fig. Consistent with the role of Sirt3 as the major mitochondrial deacetylase2, mitochondrial proteins were robustly hyperacetylated in Sirt3-deficient tissues.
In line with this observation, specific Sirt3 targets, such as mitochondrial complex I and the mitochondrial ROS scavenger, Sod2, were also markedly hyperacetylated in Sirt3-deficient tissues (Fig.
4H–I) were not changed in Sirt3-deficient tissues, which indicates that hyperacetylation of these proteins does not necessarily result in a change in their maximal activity.
This phenomenon might explain the lack of a metabolic phenotype in the tissue-specific Sirt3-deficient mice.Several in vivo and in vitro studies demonstrated a role for Sirt3 in the suppression of oxidative stress3,8,9,10,11,12,16. However, different groups have reported normal ROS levels in basal state in liver, brain or cochlea9,10.
In line with these last reports, we observed normal levels of surrogate markers for ROS in Sirt3-deficient tissues, as well as similar levels of activity of the mitochondrial ROS scavengers Sod2 and catalase (Fig.
Rather, our data indicate that either Sirt3 deficiency in another tissue or the coordinated defect of Sirt3 in multiple tissues accounts for these effects in the germline Sirt3 knock-out mice.
In this regard, our results do also not rule out the possibility that latent defects in muscle or liver function in our models can be fully compensated by another tissue or sirtuin function.
In this sense, further tissue-specific Sirt3 deficient mouse models exploring the brain, white or brown adipose, or macrophage function of Sirt3 will be key to fully evaluate the etiology of the metabolic alterations in the germline Sirt3 knock-out mice.
In this context it is interesting to note that no change in Sirt3 mRNA or protein expression in non-targeted tissues (Fig.
1F and 1G) was present, which suggest that there is no compensation by other mitochondrial sirtuins.
Since the two main tissues responsible for temperature homeostasis are muscle and brown fat, and Sirt3 is highly expressed in this last tissue, it is tempting to speculate that Sirt3 in brown fat, but not in skeletal muscle, plays an important role in temperature homeostasis, most probably via regulation of mitochondrial uncoupling. Brown fat is also an important tissue in fat and glucose clearance, and increased brown fat amounts or activity can significantly improve global metabolism20,21. Furthermore, differences in the developmental onset of the Sirt3 gene deletions need to be considered, as germline Sirt3-KO mouse model lacks Sirt3 from the beginning of embryonic development, while the tissue-specific promoters used in our work are activated only from a certain developmental stage.In summary, our work confirms the critical impact of Sirt3 on global mitochondrial acetylation.



Fasting blood glucose level 5.6 dangerous
Blood glucose reading 6.6


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