Studies II-III

For Study II, we utilized the rat L6 skeletal myoblast cell lines to study the effects of strigolactone GR24 and pinosylvin. L6 cells initially described by Yaffe et al. propagate as mononucleated myoblasts but can differentiate into multinucleated primary myotubes with striated fibers (Yaffe et al. 1968). L6 myotubes are a well characterized and established cellular model of skeletal muscle origin to study GLUT4 translocation and glucose uptake. Upon insulin stimulation, L6 myotubes exhibit more glucose uptake compared to mouse C2C12 myotubes, the other cell line that is used to study muscle cell glucose metabolism (Sarabia et al. 1990).

Study III was performed using mouse 3T3-L1 preadipocyte fibroblasts. These cell lines were originally derived from Swiss mouse embryo tissue (Green and Meuth 1974) and have been a well-characterized model for the study of adipocyte differentiation, metabolism and inflammation. These cells exhibit a homogenous response to varied experimental conditions.

Hence, they have been widely used to evaluate the effects of compounds such as resveratrol (Chang et al. 2016) or nutrients on adipogenesis and to study their potential effects in the treatment of obesity-related disorders (Ruiz-Ojeda et al. 2016). Easier cell culture and cost

effectiveness compared to the usage of mature adipocytes are some other advantages offered by 3T3-L1 cells. We employed the adipocyte differentiation method developed by Zebisch et al., who modified the existing protocols (HPA (Health Protection Agency) cultures, Mehra et al.

2007) to effectively increase the efficiency and reproducibility of differentiation of adipocytes by including the PPARγ agonist rosiglitazone as an additional adipogenic agent in the adipogenic induction mixture (Zebisch et al. 2012).

Glucose uptake measurements were performed using incubations with tritiated 2-deoxyglucose (Smith et al. 2014). This method is based on the fact that glucose and 2-deoxyglucose are transported in a similar manner into cells and phosphorylated by hexokinase. 2-Deoxyglucose-6-phosphate, a stable and impermeable derivative formed from 2-deoxyglucose accumulates in the cells as it cannot be converted to an analog of fructose 6-phosphate or otherwise rapidly metabolized. Radiolabeled transporter substrates provide a better signal-to-noise ratio and higher specificity than fluorescent labels (Yamamoto and Ashida. 2012).

In-cell Western and near-infrared detection of the signal were used to assess the cell surface GLUT4 levels (Ganesan and Ito 2013). It is a quantitative and accurate immunocytochemical assay combining the specificity of western blotting with the throughput of ELISA. This method precisely detects target proteins in their cellular context thus eliminating variabilities caused by cell lysis and is highly sensitive in measuring minor changes in protein modification or amount (Olive 2004).

The cytotoxicity of the treatments in L6 myotubes were measured by sulforhodamine B (SRB) staining and colorimetric assay as previously described (Vichai and Kirtikara 2006). This assay screens the viability of adherent cells in a 96-well format, thus allowing the analyses of multiple samples simultaneously. Compared to the MTT assay, another popular cytotoxicity screening method that uses the tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), SRB assay has many advantages. The direct interference of some compounds with MTT reduction and the dependence on mitochondrial metabolic activity are some disadvantages of MTT assay, whereas SRB staining is rarely affected by compounds, is independent of the metabolic activity of the cell and needs fewer optimization steps than MTT assay (Keepers et al. 1991).

SIRT1 activity was estimated by the Fluor de Lys assay, which uses artificial fluorescently labeled substrate, which is based on the sequence of the tumour suppressor protein p53. As the labeled substrates can cause artefacts (Gertz et al. 2012), in SIRT1 activity assays, we also used the SIRTainty activity assay, which allows for the use of various unlabeled substrates (Hubbard et al. 2013).

The oil red O staining procedure is a well-established detection method to quantitatively and qualitatively detect lipid content in mature and differentiating adipocytes (Ramirez-Zacarias et al. 1992). It also allows for microscopic imaging of lipid droplets. Hence, we used this method to analyze lipid accumulation in 3T3-L1 adipocytes.

6.2 ASSOCIATION OF SIRT1 mRNA EXPRESSION WITH ENERGY EXPENDITURE AND INSULIN SENSITIVITY (STUDY I)

SIRT1 deacetylates several transcription factors playing a key role in energy metabolism and insulin sensitivity, and SIRT1 activation mimics the benefits of CR (Boily et al. 2008, Lagouge et al. 2006). Mitochondrial dysfunction is considered to contribute to IR (Petersen et al. 2003).

Skeletal muscle IR is influenced by downregulation of genes involved in mitochondrial oxidative metabolism including PGC-1α (Mootha et al. 2003, Patti et al. 2003), indicating a close association between energy metabolism and insulin sensitivity. SIRT1 interacts with PGC-1α (Nemoto et al. 2005) and increases mitochondrial biogenesis (Rasbach and Schnellmann 2008).

Our study showed that high EE during hyperinsulinemia, and particularly high ΔEE, were strongly associated with insulin sensitivity, in accordance with previous studies (Ravussin et al.

1983, Segal et al. 1992). Insulin infusion in humans for 8 hours has been shown to increase mitochondrial mRNA transcript levels, mitochondrial protein synthesis, and ATP production (Stump et al. 2003), a response that was, however, blunted in T2D patients. Lower ATP production in response to a high-dose insulin infusion was observed in diabetic patients than in nondiabetic subjects (Asmann et al. 2006), indicating that impaired mitochondrial fitness could be a consequence of impaired insulin action. On the other hand, primary mitochondrial dysfunction could lead to IR, which can be supported by the hypothesis that impaired OXPHOS capacity leads to intramyocellular lipid accumulation (Petersen et al. 2004), eventually resulting in impaired insulin signaling and IR (Petersen et al. 2005). Higher lipid levels in the blood impair insulin-stimulated ATP production in humans (Brehm et al. 2006). In our study, subjects with low insulin-stimulated EE also showed higher levels of FFAs, higher lipid oxidation, and a lower respiratory quotient during the hyperinsulinemic clamp, indicating changes in fuel selection that often leads to IR.

We demonstrated a significant correlation of adipose tissue SIRT1 mRNA expression with several genes regulating oxidative phosphorylation function, such as PGC-1β, ERRα, NRF1 and with genes regulating the respiratory chain. Furthermore, SIRT1 mRNA expression also correlated with the expression of superoxide dismutase 1 and catalase, two key regulator enzymes protecting from oxidative stress. These results indicate the role of SIRT1 in governing mitochondrial function and protection from reactive oxygen species to improve insulin sensitivity. Other studies in mice have also reported similar findings (Lagouge et al. 2006, Baur et al. 2006). SIRT1 overexpression mouse models (Bordone et al. 2007) or administration of SIRT1 activators including resveratrol (Lagouge et al. 2006, Baur et al. 2006 Milne et al. 2007) evoked many beneficial effects, such as enhancement of insulin sensitivity in mice and improvement of glycemic control in humans (Elliott et al. 2008). These reports suggest that SIRT1 activators can be potential therapies for treating T2D and obesity.

SIRT1 mRNA expression in adipose tissue negatively correlated with hs-CRP level, which supported an earlier report on the regulation of inflammation by SIRT1 (Yeung et al. 2004). A high correlation of SIRT1 mRNA expression in adipose tissue with skeletal muscle SIRT1 mRNA and correlation of skeletal muscle mitochondrial DNA with SIRT1 mRNA expression in skeletal muscle and adipose tissue indicates that the results from adipose tissue reflect metabolic changes in skeletal muscle. In addition, we observed a strong correlation between SIRT1 mRNA and protein levels.

We demonstrated that insulin-stimulated increase in EE strongly associates with insulin sensitivity in offspring of patients with T2D and showed for the first time the correlation of adipose tissue SIRT1 mRNA expression with EE and insulin sensitivity during hyperinsulinemia. Our study also showed that SIRT1 expression correlates with the expression of several genes regulating mitochondrial function. As our study was cross-sectional, we could not determine the direction of the relationship between impaired EE and insulin sensitivity.

Thus, SIRT1 activation may have therapeutic potential in the treatment of IR and T2D.

6.3 EFFECTS OF STRIGOLACTONE GR24, PINOSYLVIN AND RESVERATROL IN L6 MYOTUBES AND 3T3-L1 ADIPOCYTES