Fig 2: Skeletal muscle mitochondrial function and expression of genes/proteins regulating lipid homeostasis in SMAKO mice. A: FA oxidation (♂, 28 weeks, fasted 12 h, red gastrocnemius; n = 6/group). 14C-labeled incorporation into CO2 and acid-soluble metabolites (ASMs) represent complete and incomplete oxidation, respectively. B: Mitochondrial respiration in permeabilized muscle fibers (♂, 28 weeks, fasted 12 h, soleus; n = 6/group). Oxygen consumption was measured following the sequential addition of the following substrates: palmitoylcarnitine (P), malate (M), ADP (D), glutamate (G), succinate (S), and cytochrome c, oligomycin, and carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP). The corresponding respiratory states are noted: ADP-driven respiration (state 3), respiration in the absence of ADP (state 4), and uncoupled respiration (state U). C: Expression of oxidative phosphorylation proteins in complexes I–V (NDUFB8 [complex I], SDHB [complex II], UQCRC2 [complex III], MTCO1 [complex IV], and ATP5A [complex V]) (♂, 28 weeks, fasted 12 h, tibialis anterior; n = 6/group). Data are normalized to protein expression of β-actin. D: TEM of skeletal muscle (♂, 28 weeks, fasted 12 h, red quadriceps, representative images). mRNA expression of PPARα/PGC1α and their target genes (E) and genes for lipid breakdown (lipolysis), uptake, and synthesis as well as lipid droplet–associated proteins (LDAPs) of the Plin family (F) relative to cyclophilin control with expression in WT-chow normalized to 1 (♂, 28 weeks, tibialis anterior; n = 9–13/group). G: Protein expression of total HSL normalized to Ran GTPase (RAN) control (left), phosphorylated HSL normalized to total HSL (middle), and representative immunoblots (right) (♂, 28 weeks, tibialis anterior; n = 4/group). For mRNA and protein expression, samples were confirmed to have low or no expression of Plin1, thereby confirming absence of significant fat contamination. For overall effects having P < 0.05: D, diet; G, genotype. Where an interaction was identified, specific comparisons having P < 0.05: *for effect of genotype.

Fig 3: Direct effect of epinephrine alone or in combination with AG or UnAG on the phosphorylation of activating (Ser660/563) or inhibitory (Ser565) residues of HSL in oxidative, soleus skeletal muscle. Individual data points are shown with the mean indicated, in arbitrary protein units (phospho/total) normalized to vinculin. n = 8–12 per group. Data sharing a letter are not statistically different from each other. A value of P < 0.05 was considered statistically significant. HSL, hormone sensitive lipase; AG, acylated ghrelin; UnAG, unacylated ghrelin.

Fig 4: Expression of lipid droplet and cholesterol uptake-related genes in adrenals of Plin2+/+ and Plin2−/− mice. Female Plin2+/+ and Plin2−/− mice at 15 weeks of age had ad libitum access to chow diet (Fed) or were fasted for 24 h (Fast). Mice were euthanized at 8–10 AM. Adrenals were dissected free from the surrounding fat capsule prior to analyses. A: Expression of Plin1-5 and Lipe mRNAs relative to the expression of Tbp (n = 8). B: Expression of Ppars mRNAs relative to the expression of Tbp (n = 8). C: Protein content of Plin1-3, HSL, and HSL phosphorylated at Ser565 (p-HSL). Gapdh is shown to illustrate protein loading per lane. D: Protein content relative to levels measured for fed Plin2+/+ adrenals by quantification of immunoblots in C (n = 4). E: Expression of Scarb1, Ldlr, Hmgcr, Soat1, and Pcyt1a mRNAs relative to the expression of Tbp. Gene expression data and quantified immunoblots are presented as means ± SD (∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 indicate difference between Plin2+/+ and Plin2−/− mice; #P < 0.05, ##P < 0.01, ###P < 0.001 indicate difference between fed and fasted mice of the same genotype). Gapdh, glyceraldehyde 3-phosphate dehydrogenase; Hmgcr, 3-hydroxy-3-methylglutaryl-CoA reductase; HSL, hormone sensitive lipase; Ldlr, low density lipoprotein receptor; Lipe, lipase E (HSL); Pcyt1a, phosphate cytidylyltransferase 1, choline, alpha isoform; p-HSL, phosphorylated HSL; Ppara, peroxisome proliferator activated receptor alpha (PPARα); Ppard, peroxisome proliferator activated receptor delta (PPARδ); Pparg, peroxisome proliferator activated receptor gamma (PPARγ); Scarb1, scavenger receptor class B, member 1; Soat1, sterol O-acyltransferase 1; Srebf1c, sterol regulatory element binding transcription factor 1 isoform c (SREBP-1C); Tbp, TATA-box binding protein.

Fig 5: Ex vivo effects of CL, CL+AG and CL+UnAG compared to CON on glycerol release (Figure 2a), fatty acid release (Figure 2B) and reesterification (Figure 2 C) in both iWAT and RP after 5D of diet. Repeated measures one-way ANOVA showed significant differences in phosphorylation of HSL in treatments compared to CON. Superscript letters denote significant differences between treatment groups (p < 0.05). All data are presented as mean±S.E.M (n = 6–10 rats/group)