Roles of IL-6 in metabolism

Besides its role in inflammation, 6 plays important roles in physiology and metabolism (345). IL-6 was demonstrated to be important for regulating glucose homeostasis, as its single injection improved insulin-stimulated glucose uptake in young healthy human subjects (296), but not in T2D patients (298).

Additional data suggested that it is rather the acute versus chronical character of elevated IL-6 levels, which will define whether the effects of IL-6 are beneficial or deleterious. An acute increase of IL-6 ameliorates insulin sensitivity, and impacts positively on glucose (346, 347) and fat metabolism (348), whereas chronic exposure causes insulin resistance in mice (299). Although IL6 KO mice develop mature-onset obesity and glucose intolerance (293), recent evidences in muscle-specific IL6 KO mice suggest that muscle IL-6 reduces utilization of carbohydrates during prolonged exercise by regulating pyruvate dehydrogenase activity (290).

IL-6 plasma levels are higher in obese people compared to non-obese, and we confirmed this in vitro (10). A recent article has shown that in obese mice fed on HFD, IL-6 acts independently of its receptor (IL-6R) via the trans-signaling pathway involving gp130 (also known as IL6ST), which activates STAT3 signaling in paraventricular nucleus of the hypothalamus (PVH), resulting in potent suppression of feeding and improvement of glucose homeostasis. Moreover, elevated levels of the soluble form of IL-6R and an increase of gp130 expression were specifically found in obese mice upon HFD, most likely to amplify IL-6 sensitivity of PVH (349). Thus, the identified signaling pathway in central nervous system might be an interesting potential target for drug to treat obesity and obesity-associated insulin resistance, as it exists for other pathologies, such as arthritis (350). A recent publication has revealed that myokine secretion is altered in cultured human myotubes established from T2D subject biopsies (302), agreeing with previous reports (351-359). However, in this study, authors measured only total secretion after 24 h or 48 h of culture, whereas our approach allows for obtaining continuous temporal secretion profile (302). Assessment of this conjunction in hSKM established from the tissue biopsies taken from obese and T2D individuals, by the developed here methodology, will represent an important direction for the continuation of this work (see chapter 6.8.3.), which we have already started to pursue. We hypothesize that the daily oscillation pattern of myokines might represent an advantage for their temporal

Page 152

coordination with the rest/activity cycle, which will likely result in optimal myokine response to external cues (10).

6.5. RNA-seq analyses revealed candidate genes involved in vesicle trafficking, glucose and lipid metabolism in hSKM synchronized in vitro, and in human skeletal muscle biopsies collected in vivo

The here presented study is the first that is depicting gene expression by RNA-seq analysis from human skeletal muscle biopsies and primary human myotubes derived from healthy subjects in the presence or absence of a functional circadian clock (Figure S1, Perrin et al. submitted to eLife). This design allowed to dissect the effects of the cell-autonomous muscle clocks on the gene transcription from the central effects driven by external cues, including rest-activity and feeding-fasting cycles (303).

Rhythmic mRNA oscillations have been observed for numerous functional gene groups, including cell cycle and DNA metabolic process (Table S2, Perrin L. et al. submitted to eLife). Of note, three major groups related to exocytosis, glucose uptake and lipid metabolism might provide explanation to our previous observations regarding the basal circadian myokine secretion and lipid metabolites oscillations in hSKM synchronized in vitro (Figure 13) (10, 336).

Figure 13. (Adopted from Perrin L. et al. submitted to eLife). Schema summarizing the main conclusions of the RNA-seq analysis. Clock disruption leads to an impaired insulin response and a decrease in glucose uptake (1), dysregulation of gene involved in vesicle trafficking (2) and lipid metabolism (3).

Page 153

6.5.1. Muscle clock disruption impairs glucose uptake in response to insulin

We found that basal and insulin-stimulated glucose uptake were reduced by about 30% upon siCLOCK (Figure 3D, Perrin L. et al. submitted to eLife). GLUT4 mRNA followed daily oscillation in vivo but not in vitro, (Perrin L. et al. submitted to eLife). Moreover, 14-3-3θ (YWHAQ) was downregulated upon siCLOCK at the mRNA and protein level by 14.7% and 28%, respectively which might lead to a weaker inhibition of the two Rab-GTPase-activating proteins (Rab-GAPs) TBC1D1 and TBC1D4 (known as AS160) (Figure 3C, Table 1, Perrin L. et al. submitted to eLife), triggering the maintenance of Rabs in their inactive state, and thus blocking GLUT4 movement to the plasma membrane (360-364). Of note, TBC1D4 (AS160) mRNA accumulation was found rhythmic in human skeletal muscle in vivo (Perrin L.

et al. submitted to eLife). Our results are in good agreement with studies demonstrating in muscle-specific Bmal1 KO or in Clock^∆19 mutant mice, that dysregulation of the clock leads to an impaired insulin-stimulated glucose uptake and glucose metabolism (145, 154, 365).

Despite mice models with whole-body or organ specific core clock gene deletion/mutation induce typical phenotype of metabolic disorder, mostly obesity and/or T2D (157, 186, 188, 221), and that HFD itself disrupts core clock genes expression in mice (190), this direct relationship between clock ablation and the development of a metabolic syndrome has not been well established in human. Two recent studies have characterized the transcriptional program of hSKM isolated from T2D and/or obese subjects (265, 266). A third one has depicted the temporal expression of selected genes by conventional qRT-PCR in hSKM from T2D subjects (344). hSKM derived in vitro from muscle biopsies of untrained/trained lean and from T2D or healthy obese subjects, displayed comparable core clock genes expression, except for REV-ERBα which presents a reduced rhythmicity pattern, whereas genes involved in carbohydrates and lipid metabolism exhibit various expression profiles depending of the donor characteristics (344). Therefore, it might be relevant to perform deeper large-scale circadian transcriptomic analyses on hSKM taken from T2D and/or obese subjects in order to identify the effects of the pathology on the circadian clockwork.

6.5.2. A functional muscle clock is required for proper myokine secretion

We did not find myokine encoding genes rhythmically expressed in vitro, except SERPINE1, whereas FABP3, TIMP-1 and VEGFA were oscillating in vivo (Supplemental Datasets 1 and 5, Perrin L. et al. submitted to eLife). We identified vesicle associated membrane protein 3 (VAMP3) and syntaxin

Page 154

6 (STX6) as downregulated upon siCLOCK in vitro (Table 1, Supplemental Dataset 1, Perrin L. et al.

submitted to eLife) in an agreement with the data obtained by us in human islets using a similar approach, and in mouse islets (157, 158). Importantly, VAMP3 and STX6 are required for proper IL-6 secretion by mouse macrophages (366) and by the synovial sarcoma cell line (367). Moreover, VAMP3 exhibited a rhythmic expression in human skeletal muscle biopsies in vivo (Perrin L. et al. submitted to eLife). We found that phosphatidylinositol 4-kinase type 2 alpha (PI4K2A) was downregulated in siCLOCK-treated myotubes, suggesting that the VAMP3-mediated recycling from plasma membrane might be impaired (Figure 2, Table 1, Perrin L. et al. submitted to eLife). This corroborates a previous study reporting that PI4K2A knockdown inhibited VAMP3 trafficking to perinuclear membranes (368).

Collectively, our RNA-seq data pinpoint on the exocytosis as a potential regulation site for clock regulation of myokine secretion. To validate this hypothesis, it will be important quantifying exocytosis events by total internal reflection fluorescence (TIRF) microscopy in this system around-the-clock, or in siCLOCK-transfected hSKM. Moreover, siRNA-mediated knockdown experiments for one or several of those genes, followed by perifusion and myokine assessment, could further provide insights into the mechanism of this regulation. Finally, as post-translational modifications can be subjected to circadian regulation, it would be of interest to visualize myokine secretion at the single cell level in synchronized human myotubes using time lapse microscopy, employing a combination of fluorescent dyes labelling the myokine and core clock gene in parallel. Immunofluorescence, co-immunoprecipitation and electronic microscopy might also be useful to assess this mechanism.

6.5.3. Circadian clock regulated lipid homeostasis in human skeletal muscle

Thanks to the developed in vitro hSKM model, our group has recently published that 18.6% of lipid metabolites oscillate in human primary myotubes in vitro and 20.3% in vivo (Figure 4 from (336)).

Employing our siRNA-mediated CLOCK knockdown assay, we demonstrated that this rhythmic pattern was abolished under not fully functional clock (Figure 8 from (336)). Moreover, correlation between temporal expression of enzymes involved in lipid biosynthesis and lipid species, including UGCG, SPTLC2, ELOVL5 and ACSL4, has been made (Figures 6 and 7 from (336)).

Based on our RNA-seq data, we confirmed the in vivo rhythmic expression of enzymes, such as those mentioned above, and the dysregulation upon siCLOCK of numerous genes involved in lipid metabolism, including SPTLC3 and UCGC, in fatty acid transport, such as CD36 and FABP3, and in

Page 155

lipolysis with PNPLA3 (Figure 2, Table S5, Supplemental Dataset 1, Perrin L. et al. submitted to eLife).

Importantly, these modifications in gene expression resulted in significant alterations in absolute lipid metabolite levels as analyzed by mass spectrometry based lipidomics, with total phosphatidylcholine levels being slightly downregulated, and glycosylceramide levels being upregulated (Figure 2, Perrin L.

et al. submitted to eLife), agreeing with results obtained in Bmal1^-/- skeletal muscle (146, 369) and liver (137). Altogether, these results suggest a change in energy consumption from glucose utilization toward lipolysis in siCLOCK muscle (140, 145, 146, 154).

6.6. Insulin sensitivity (IS) around-the-clock might suggest different rhythmic phenotypes