Fig 1: Time-controlled fasting prevents high fat diet-induced metabolic inflexibility in skeletal muscle.(A) High fat diet causes a massive flux of fatty acids into skeletal muscle cells triggering an uncontrolled mitochondrial oxidative metabolism. A persistence of such dietary pattern induces a decrease of Atgl and metabolic inflexibility in skeletal muscle causing elevation of circulating levels of glucose, lipids and insulin. The occurrence of such systemic metabolic gridlock might be the consequence of a protective response (insulin resistance) of metabolically exhausted cells (aging phenotype). (B) Cycles of time-controlled fasting (>24h) boosts mitochondrial oxidative metabolism in skeletal muscle that confers an adaptive stress resistance to nutrient fat overload. Under fasting, the induction of Atgl represents the key molecular event that promotes a controlled release and gradual oxidation of fatty acids into mitochondria. These adaptive responses result in an effective metabolic flexibility preventing high fat diet-induced aging phenotype (geroprotection).
Fig 2: TCF limits mitochondrial damage and increases Atgl levels.(A) Western blot analysis of protein carbonyls in crude mitochondria from different tissues of mice fed with HFD/ND or HFD/TCF. vDAC1 was used as loading control. Immunoblots reported are representative of two mice per group out of five giving similar results. The same vDAC1 was used as loading control in Panel C in S1 Fig. (B) mtDNA damage measured by calculating the ratio of long fragment intensity (LFI) and short fragment intensity (SFI) in different tissues of mice fed with HFD/ND or HFD/TCF. BAT: brown adipose tissue, GSCN: gastrocnemius. (C) Western blot analysis of Atgl in GSCN homogenates of mice fed with HFD/ND or HFD/TCF. Immunoblot reported is representative of three mice per group out of five giving similar results. Ponceau staining was used as loading control. Student’s t-test was used to compare HFD/ND vs HFD/TCF.
Fig 3: Atgl controls mitochondrial functionality in muscle cells.(A) Western blot analysis of Atgl in total homogenates of GSCN of mice fed with normal diet (ND) or high fat diet (HFD). Immunoblots reported are representative of three mice per group out of five giving similar results. Below are reported the densitometric analyses of the immunoreactive bands normalized to Ponceau staining (entire line). (B) mRNA expression analysis in gastrocnemius (GSCN) upon treatment with HFD. (C) Western blot analysis of Atgl in C2C12 myotubes transfected with a siRNA against Atgl (Atgl-) or with a scramble siRNA (Scr) (left panel). Immunoblots reported are representative of one experiment out of three giving similar results. Tubulin was used as loading control. Measurement of mitochondrial membrane potential (??M) and citrate synthase activity in crude mitochondrial fractions of Atgl- C2C12 myotubes (right panel). (D) Lipid content measured by Oil Red O staining (right panel) in C2C12 myotubes transfected with the Atgl cDNA (Atgl+) or with empty vector (Empty) and treated with 300 µM palmitic acid (PA) for 72h. Atgl immunoblot (left panel) is representative of three independent experiments giving similar results. Tubulin was used as loading control. (E) Measurement of mitochondrial membrane potential (??M) in crude mitochondrial fractions of Atgl+ C2C12 myotubes. (F) Measurement of citrate synthase activity in crude mitochondrial fractions of Atgl+ C2C12 myotubes. All data are expressed as mean ±S.D. (n = 5 mice/group). In vitro data are representative of at least three independent experiments. Student’s t-test was used for two groups comparisons (A-C). One-way ANOVA analysis followed by Turkey’s test corrections was used for multiple comparisons (D-F).
Fig 4: TCF increases Atgl levels in SkM.(A) Heatmap obtained by analysing data from the publicly available Gene Expression Omnibus (GEO) data set (GDS3893) revealed an increase of lipid catabolic genes in SkM of mice subject to fasting versus mice fed with ND. (B) Schematic representation of alternate fasting (AF) and time-controlled fasting (TCF). (C) Western blot analysis of Atgl in total homogenates of GSCN and TA from mice fed with ND or subject to AF for 2 cycles. Immunoblots reported are representative of three mice per group out of five giving similar results. Tubulin was used as loading control. (D) Western blot analysis of Atgl in total homogenates of GSCN and TA from mice fed with ND or subject to TCF for 2 cycles. Immunoblots reported are representative of three mice per group out of five giving similar results. Below are reported the densitometric analyses of the immunoreactive bands normalized to Tubulin. Student’s t-test was used to compare ND vs TCF. (E) Western blot analysis of CV-ATP5A and CIII-UQCRC2 in crude mitochondria isolated from GSCN or TA of mice fed with ND or subject to 2 cycles of TCF. Immunoblots reported are representative of three mice per group out of five giving similar results. Grp75 was used as loading control.
Supplier Page from MilliporeSigma for Anti-ATGL/Desnutrin antibody produced in goat