Modified UCN2 peptide treatment improves skeletal muscle mass and function in mouse models of obesity‐induced insulin resistance

Figures

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  Figure 1. UCN2 treatment increases skeletal muscle mass in HFD‐fed mice. High‐fat diet (HFD)‐fed mice were either sedentary or had access to free wheel running (FWR) and treated with vehicle, Compound A, or Compound B for 14 days. Lean mass was assessed with EchoMRI and expressed as (A) absolute values and (B) a percentage of body weight. Absolute weight of the (C) extensor digitorum longus (EDL) and (D) presented as a percentage of body weight. Absolute weight of the (E) soleus and (F) presented as a percentage of body weight. Absolute weight of the (G) tibialis anterior (TA) and (H) presented as a percentage of body weight. Dotted line indicates mean of chow vehicle mice. n = 9–10 mice per group. αMain effect for Compound A treatment. βMain effect for Compound B treatment. ‡ P < 0.05 main effect for wheel running. *Compared with vehicle of same condition. ¤Compared with corresponding sedentary of same treatment as assessed via two‐way analysis of variance with Sidak's post hoc analysis.

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  Figure 2. UCN2 treatment affects genes that are mediators of skeletal muscle hypertrophy and degradation in HFD‐fed mice. Effects of Compound A or B treatment on relative mRNA expression of key mediators in pathways regulating muscle mass. Gene expression data were assessed via qPCR analysis of mRNA from the tibialis anterior (TA) after high‐fat feeding and 14 days of Compound A or B treatment. (A) Genes of thick filaments and structural proteins. (B) Genes and transcription factors involved in protein degradation. (C) Genes and transcription factors involved in hypertrophy. Dotted line indicates the mean of chow vehicle mice. Results are expressed relative to chow vehicle‐treated mice and normalized to the geometric mean of B2M and Tbp. n = 8–10 mice per group. § P < 0.05 effect as assessed via one‐way analysis of variance or Kruskal–Wallis test, *P < 0.05 versus vehicle as assessed via post hoc analysis.

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  Figure 3. UCN2 treatment affects proteins that are mediators of skeletal muscle hypertrophy and degradation in HFD‐fed mice. Effects of Compound A or B treatment on protein content and phosphorylation of key mediators in pathways regulating muscle mass assessed via western blot analysis of tibialis anterior (TA) after high‐fat feeding and 14 days of Compound A or B. (A) Upstream mediators in muscle mass regulation. (B) Proteins involved in hypertrophy and protein synthesis. (C) Proteins involved in ubiquitin‐proteasome system for protein breakdown. (D) Proteins involved in autophagy regulation and inflammation. Results calculated relative to ponceau stain and expressed relative to HFD vehicle‐treated mice. n = 8–10 mice per group. § P < 0.05 effect as assessed via one‐way analysis of variance or Kruskal–Wallis test, *P < 0.05 versus vehicle as assessed via post hoc analysis.

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  Figure 4. UCN2 acutely increases transcription factors mediating hypertrophy while rates of protein synthesis remain unchanged in skeletal muscle. (A) Schematic of acute injection experiments. Mice were fasted from 0 to 4 h. (B) In vivo muscle protein synthesis determined by puromycin‐labeled proteins in chow‐fed lean mice after acute Compound A or B injection assessed via the SUnSET method. Gene expression data were assessed via qPCR analysis of mRNA from the tibialis anterior (TA) after acute Compound A or B injection, (C) structural protein genes, (D) pro‐degradation genes, and (E) pro‐hypertrophy genes. Results are expressed relative to vehicle‐injected mice and normalized to the geometric mean of Hprt and Tbp. n = 6 mice per group. § P < 0.05 effect as assessed via one‐way analysis of variance or Kruskal–Wallis test, *P < 0.05 versus vehicle as assessed via post hoc analysis.

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  Figure 5. UCN2 treatment leads to improved skeletal muscle function during ex vivo contractions in HFD‐fed mice. At the end of 14 days of compound treatment in HFD‐fed mice, the EDL muscle was excised and subjected to electrical stimulation and contracted with a fatigue‐inducing protocol for 10 min for measurement of (A) absolute force and (B) relative force production during ex vivo contractions (electrical stimulation with 100 Hz, 0.2 ms pulse duration, 20 V at a rate of 0.2 s contraction every 2 s, while contralateral muscles were kept under resting passive tension conditions). βMain effect for Compound B treatment. †Main effect for time. #Interaction. *Compound B versus vehicle control as assessed via two‐way repeated measures analysis of variance with Sidak's post hoc analysis. (C) Time to 50% fatigue and (D) maximum force produced in EDL. (E) Glycogen content, (F) glycogen synthase phosphorylation at Ser641, and (G) total GLUT4 protein content in tibialis anterior (TA). Dotted line indicates the mean of chow vehicle mice. n = 9–10 HFD‐fed mice per group. § P < 0.05 effect as assessed via one‐way analysis of variance or Kruskal–Wallis test, *P < 0.05 versus vehicle as assessed via…

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  Figure 6. UCN2 treatment does not alter contraction‐induced glucose transport but increases TBC1D1 phosphorylation. Immediately following fatigue‐inducing contractions, glucose uptake and signaling was determined in EDL muscle. (A) Contraction‐induced glucose uptake assessed with radioactive labeled glucose. (B) Representative western blots. Quantification of (C) p‐AMPKα Thr172, (D) p‐ACC Ser79, (E) TBC1D1 phosphorylation at Ser237, and (F) TBC1D4 phosphorylation at Ser318, n = 5 chow‐fed and n = 9–10 HFD‐fed mice per group. α P < 0.05 main effect for Compound A treatment. βMain effect for Compound B treatment. † P < 0.05 main effect for contraction. # P < 0.05 interaction. *Compared with vehicle of same condition. ¤Compared with basal of the same treatment as assessed via two‐way repeated measures analysis of variance with Sidak's post hoc analysis.

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  Figure 7. UCN2 treatment increases lean mass and skeletal muscle protein synthesis in ob/ob mice. ob/ob mice were treated with vehicle, Compound A, or Compound B for 14 days. Wild‐type (WT) mice were treated with vehicle. (A) Change in body weight over the 14 day treatment period. Change in (B) fat mass and (C) lean mass over the 14 day treatment period expressed as a percentage of body weight, assessed with EchoMRI. Absolute weight of (D) extensor digitorum longus (EDL), (E) soleus, and (F) tibialis anterior (TA) muscles. Skeletal muscle weights expressed as percentage of body weight for (G) EDL, (H) soleus, and (I) TA muscles. In vivo muscle protein synthesis determined by puromycin‐labeled proteins in WT and ob/ob mice after the final compound injection, as assessed via the SUnSET method in 4 h fasted mice. (J) Representative western blots and (K) quantification of puromycin‐labelled proteins. n = 6 WT, n = 9 Veh, n = 9 Comp. A, n = 11 Comp. B. *P < 0.05 compared with vehicle as assessed via one‐way analysis of variance or Kruskal–Wallis test.