Phosphoproteomic mapping reveals distinct signaling actions and 1 activation of protein synthesis and muscle hypertrophy by Isthmin-1 2

The secreted protein Isthmin-1 (Ism1) mitigates diabetes by increasing adipocyte and skeletal 25 muscle glucose uptake by activating the PI3K-Akt pathway. However, while both Ism1 and insulin 26 converge on these common targets, Ism1 has distinct cellular actions suggesting divergence in 27 downstream intracellular signaling pathways. To understand the biological complexity of Ism1 28 signaling, we performed phosphoproteomic analysis after acute exposure, revealing overlapping 29 and distinct pathways of Ism1 and insulin. We identify a 53 % overlap between Ism1 and insulin 30 signaling and Ism1-mediated phosphoproteome-wide alterations in ~ 450 proteins that are not 31 shared with insulin. Interestingly, we find several unknown phosphorylation sites on proteins 32 related to protein translation, mTOR pathway and, unexpectedly, muscle function in the Ism1 33 signaling network. Physiologically, Ism1 ablation in mice results in altered proteostasis, including 34 lower muscle protein levels under fed and fasted conditions, reduced amino acid incorporation 35 into proteins, and reduced phosphorylation of the key protein synthesis effectors Akt and 36 downstream mTORC1 targets. As metabolic disorders such as diabetes are associated with 37 accelerated loss of skeletal muscle protein content, these studies define a non-canonical 38 mechanism by which this anti-diabetic circulating protein controls muscle biology. 39 41 Taken together, these results demonstrate Ism1 is a circulating anabolic protein synthesis muscle function by maintaining high Akt activity and protein synthesis skeletal muscle. In conclusion, Ism1 is a circulating anabolic regulator of protein synthesis and muscle function.

3 Introduction upon acute stimulation, with BSA as control. First, there is a 53 % overlap between Ism1 and 125 insulin signaling ( Figure 1D). For example, insulin induces phosphorylation of 654 phosphosites, 126 out of which 347 phosphosites are also phosphorylated or dephosphorylated by Ism1 ( Figure 1D). or insulin (n = 2). Individual comparisons between conditions across phosphopeptides was performed using empirical Bayes statistics followed by adjustment for multiple testing using false discovery rate, *p < 0.05, 210 **p < 0.01, ***p < 0.001. The minimum normalized intensity across the dataset was subtracted from each 211 normalized data point, and phosphorylation was calculated as a fraction of the maximum value of all 212 samples for each phosphopeptide. Bars show mean ± SEM. C) Venn diagram of shared and unique 213 phosphosites between treatments for the GO pathways Insulin mTOR. D) Venn diagram of shared and 214 unique phosphosites between treatments for the GO pathways and Muscle. (E-N) Abundance of proteins 215 with indicated phosphosite in cells treated with BSA, Ism1, or insulin (n = 2). Individual comparisons 216 between conditions across phosphopeptides was performed using empirical Bayes statistics followed by 217 adjustment for multiple testing using false discovery rate, *p < 0.05, **p < 0.01, ***p < 0.001. The minimum 218 normalized intensity across the dataset was subtracted from each normalized data point, and    Akt S473 and ribosomal S6 S235/S236 starting at 5 min and remaining up to 4h ( Figure 3A). The effect 245 of Ism1 on Akt signaling is robust but lower than that of the skeletal muscle hypertrophy hormone 246 insulin-like growth factor-1 (Igf1) (Figure 3A-B). Similarly, undifferentiated C2C12 myoblasts are 247 also responsive to Ism1 in a dose-dependent manner ( Figure 3C). Notably, Ism1 treatment

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KO mice were either fasted for 12 h or fasted followed by a 12 h re-feeding period (fed state) 286 ( Figure 3A). As expected, at dissection, all fasted mice had a reduction in body weight compared 287 with fed mice (Figure 3B). Blood glucose levels were 150 mg/dl in the fed state, and < 80 mg/dl 288 in the fasted state ( Figure 3C), but no differences were seen between genotypes. Under the same       To understand the underlying mechanism by which Ism1-KO mice develop smaller muscle fiber 358 size, we next asked whether Ism1 directly controls protein content in mice. Quadriceps muscles due to reduced Akt pathway activity. Indeed, we found that phosphorylation of Akt S473 , mTOR 2448 , 375 and ribosomal S6 S235/S236 are markedly decreased in Ism1-KO muscle compared with WT mice 376 under both fed and fast conditions, providing an explanation for the lower protein synthesis rate 377 ( Figure 6G). Taken together, these results demonstrate that Ism1 is a circulating anabolic 378 regulator of protein synthesis and muscle function by maintaining high Akt activity and protein 379 synthesis in skeletal muscle. In conclusion, Ism1 is a circulating anabolic regulator of protein 380 synthesis and muscle function.

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Therefore, Ism1 appears to be an important protein regulating metabolism during fed and fasted 426 conditions.

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The signaling mechanism behind Ism1-induced muscle hypertrophy is not simply an activation of

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In this work, we observe a 60 % decrease in pAkt S473 levels in the muscles of Ism1-KO mice, 446 which leads to a ~10 % reduction in muscle protein content. Our data is consistent with previous 447 reports using muscle-specific Akt-KO mice that demonstrate a 40% reduction in protein synthesis whether other yet unidentified pathways distinct from Akt and FoxO regulate proteostasis.

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The physiological function of Ism1 in regulating muscle growth is expected given that Ism1