Evidence for a neuromuscular circuit involving hypothalamic Interleukin-6 signaling in the control of skeletal muscle metabolism

Hypothalamic interleukin-6 (IL6) exerts a broad metabolic control, including energy expenditure 1 , food consumption 2 , glucose homeostasis 2 , etc. Here we demonstrated that Interleukin-6 (IL6) activates the ERK1/2 pathway in the ventromedial hypothalamus (VMH), stimulating AMPK/ACC signaling and fatty acid oxidation in mice skeletal muscle. Bioinformatics analysis revealed that the hypothalamic IL6/ERK1-2 axis is closely associated with ring-rate-related genes in the hypothalamus and with fatty acid oxidation- and mitochondrial-related genes in skeletal muscle of genetically diverse BXD mice strains and humans. Using surgical denervation, pharmacological approaches, and transgenic mice, we showed that the hypothalamic IL6/ERK1/2 pathway requires the a2-adrenergic pathway to modify the fatty acid skeletal muscle metabolism. To address the physiological relevance of these ndings, we demonstrated that this neuromuscular circuitry is required to underpin AMPK/ACC signaling activation and fatty acid oxidation post-exercise. Once the selective downregulation of IL6 receptor in VMH abolished the effects of exercise to sustain AMPK and ACC phosphorylation and fatty acid oxidation in the muscle postexercise. Altogether, these data demonstrated that IL6/ERK axis in VMH controls fatty acid metabolism in mice skeletal muscle.


Main Text
Neuronal activity in the ventromedial hypothalamus (VMH) controls energy expenditure 3 and skeletal muscle metabolism, including fatty acid oxidation and glucose uptake 4,5 . Hormones and neuropeptides, such as leptin and orexin, can trigger signals into VHM nucleus to control the muscle metabolism in murine models 4,6 . Thus, we sought to determine the existence of a neuromuscular circuit involving hypothalamic IL6 action in VMH nucleus toward the skeletal muscle metabolism, driving the lipid metabolism.
Interaction graph by using Genotype-Tissue Expression (GTEx) human database 7 from 81 individuals revealed a positive association (red lines) between hypothalamic IL6 gene expression and lipid metabolism-and mitochondrial-related genes in the skeletal muscle ( Figure 1A). No correlation was observed between the hypothalamic IL6 gene and glucose metabolism-and protein synthesis-related genes ( Figure 1A). Hypothalamic IL6 gene expression showed a positive correlation (red lines) with lipid metabolism-related genes ( Figure 1B -upper panels) and negative correlation (green lines) with lipid biosynthesis-related genes ( Figure 1B -lower panels). These data were con rmed by a two-factor analysis ( Figure 1C). We next employed analyses using a system data from the BXD mouse genetic reference population 8 , geared towards multiscalar integration of traits. This data set also showed positive interaction between hypothalamic Il6 gene expression and gene-sets involved in fatty acid oxidation in skeletal muscle (red lines). Conversely, negative interaction (green lines) was observed among hypothalamic Il6 gene expression and expression of some markers of lipid biosynthesis in skeletal muscle of BXD mice ( Figures  1D and E). No interaction was found among hypothalamic Il6 gene expression and glucose metabolism or protein synthesis-related genes ( Figure 1D). The principal component analysis (PCA) con rmed a strong positive correlation between hypothalamic Il6 levels and lipid oxidation-and mitochondrial-related genes in muscle ( Figure 1F). The heat map graph revealed that speci c strains of BXD mice with low levels of hypothalamic Il6 gene expression (BXD63, 90 and 65a) displayed high levels of transcripts related to skeletal muscle lipid biosynthesis ( Figure 1G). Conversely, BXD families with high levels of hypothalamic Il6 gene expression (BXD43, 99, and 84) present high lipid oxidation-and mitochondrialrelated genes in skeletal muscle ( Figure 1G). The two-factor loadings plot con rmed that the hypothalamic Il6 gene expression is positively correlated with fatty acid oxidation markers (Acadsb, Cpt1c, Mrpl41, Pcca, and Uqcrc) and showed a negative correlation with markers of lipid biosynthesis (Srebp1c and Scd1) in the skeletal muscle of strains of genetically diverse BXD mice ( Figure  1H).
Beyond the genetic variation and correlative analyses, we also tested our hypothesis experimentally by using recombinant IL6 icv microinjection in C57BL6/j mice ( Figure 1I). Recombinant IL6 (200ng) increased the oxygen consumption ( Figure 1J) and stimulated critical markers involved in fatty acid oxidation in the soleus muscle, including ACC Ser79/Ser212 and AMPK Thr172 phosphorylation ( Figure 1K) and Cpt1β and Pparα gene expression ( Figure 1L). Recombinant IL6 icv administration induced transient fatty acid oxidation in the soleus muscle, peaking 3 hours after the central injection ( Figure 1M). The mass spectrometry analysis was performed using the same experimental design to deeply assess the hypothalamic IL6 impact on the skeletal muscle lipid metabolism. The central action of IL6 markedly reduced the amount of fraction of saturated (C15:0, C16:0, C17:0, and C18:0), monosaturated (C18:1, C18:1t and C20:1), and polyunsaturated (18:2, 18:3 and 20:3) fractions of fatty acids in the skeletal muscle ( Figure 1N-P and S1A). Collectively, these initial data revealed that the hypothalamic IL6 action modulates the lipid metabolism in the skeletal muscle.
Several pieces of evidence are connecting the VMH nucleus and muscle metabolism 5,9 . In addition, it has been demonstrated that neurons expressing steroidogenic factor-1 (SF-1) 10 or extracellular signalregulated kinase 1/2 (ERK1/2) 11 in VMH elicit signals from the central nervous system toward the skeletal muscle. Curiously, IL6 stimulates both SF-1 expression and ERK1/2 phosphorylation in human 12 and murine 13 cell lines. To explore the possibility of IL6 to modulates targets such as SF-1 and/or ERK1/2 expressing neurons in VMH, we rst evaluated the presence of IL6 receptor (IL6R) in neurons of hypothalamic nuclei of mice. Double-staining analysis revealed that IL6R (red) is spread in neurons (green) of different hypothalamic nuclei of mice (Figure 2A, S1B, and S1C), including in the VMH ( Figure   2B). We also found IL6R in non-neuronal cells (yellow arrows) in VMH ( Figure 2B). Nevertheless, the presence of IL6R in SF-1 neurons of VMH was not clearly observed ( Figure S1D). We also found the presence of ERK1/2 phosphorylation (green) in IL6R-expressing cells (red) in VMH of mice ( Figure S2A).
Based on these ndings and previous studies that reported that hypothalamic ERK signaling plays a critical role in controlling sympathetic ow 14,15 , we hypothesized that the hypothalamic IL6/ERK axis in VMH could control the skeletal muscle lipid metabolism.
After that, we evaluated the capacity of exogenous IL6 to activate ERK1/2 in the hypothalamus. First, we observed that hypothalamic IL6 gene expression was strongly associated with ring-rate-related transcripts in mice and humans ( Figure S2B and C). Indeed, the recombinant IL6 icv microinjection stimulated ERK1/2 phosphorylation in the whole hypothalamus ( Figure 2C) and speci cally in VMH of mice ( Figure 2D). We observed that IL6 also stimulated ERK1/2 phosphorylation in Neuro 2A cells ( Figure   2E). These data revealed the potential of IL6 in stimulating neuronal activity and ERK1/2 phosphorylation, as previously reported 16,17 .
To test if hypothalamic IL6/ERK1/2 axis is involved in the skeletal muscle lipid metabolism, PD98059, a MEK/ERK signaling pharmacological inhibitor or saline, were icv administrated 30 minutes before the icv microinjection of IL6 recombinant, as detailed in the experimental design ( Figure 2F). Notably, PD98059 abolished hypothalamic IL6-induced ACC and AMPK phosphorylation ( Figure 2G) and fatty acid oxidation ( Figure 2H) in soleus muscle. We also evaluated the interaction between hypothalamic ERK1/2 signaling and lipid metabolism in skeletal muscle of humans. The interactome analysis and heat map graph con rmed a consistent association between hypothalamic ERK1 and ERK2 gene expression and several genes involved in fatty acid metabolism in human skeletal muscle (Figures 2I-K), but not glucose metabolism or protein synthesis ( Figure 2I). Altogether, bioinformatics and experimental data demonstrated that the hypothalamic IL6/ERK axis controls AMPK/ACC signaling and fatty acid oxidation in the soleus muscle. In the same line, leptin modulates muscle metabolism through ERK1/2 activation in VMH of rats 11 .
Next, we investigated the involvement of the sympathetic nervous system in this neuromuscular circuitry. The left hindlimb of mice was denervated, and the contralateral paw was used as control. Two days after the surgeries (cannula implant and denervation), the animals received recombinant IL6 (200ng) icv microinjection, and the soleus muscles were removed, as demonstrated in the experimental design ( Figure 3A). Interestingly, the surgical denervation reduced the ability of the central action of IL6 to phosphorylates ACC and AMPK in the soleus muscle of mice ( Figure 3B), demonstrating that the hypothalamic IL6 requires the nervous projections to alter the muscle metabolism.
Notably, a-adrenergic signals mediate the ACC phosphorylation and fatty acid oxidation in the skeletal muscle in response to intrahypothalamic injections of leptin 6 and the synthetic inhibitor of fatty acid synthase, C75 18 . Based on this information, we sought to determine the involvement of a-adrenergic signaling in this neuromuscular circuitry. Interactome analysis revealed that in humans, the hypothalamic Il6 gene is positively correlated with expression of gene-set linked to alpha-adrenergic signaling in the skeletal muscle, including ADRA2C, CREM, ADCY3, PDE4B, PDE4D, and AK4 genes ( Figure  3C). These data were also con rmed by Pearson's correlations ( Figure 3D) and two-component analyses ( Figure 3E). In the BXD cohort, we found that α2 adrenoceptors subunits (Adra2c, 2a and 2b), but not α1 subunits, were highly expressed in skeletal muscle of strains with high expression of hypothalamic Il6 ( Figure 3SA-C). These transcriptomic analyses led us to investigate the involvement of α2 adrenoceptors subunits as critical components to connect the central IL6 action with the skeletal muscle metabolism. For this, mice lacking both α2A and α2C adrenoceptors (α2AC KO) were generated. Wild-type and α2AC KO mice received an icv microinjection of IL6 recombinant and the soleus muscle were removed for analyses 3 hours later ( Figure 3F). Interestingly, the central action of IL6 increased ACC and AMPK phosphorylation in the muscle of wild-type, but not, in double-knockout mice ( Figure 3G). Furthermore, icv IL6 administration failed to stimulate fatty acid oxidation in the skeletal muscle of α2AC KO mice ( Figure  3H). No difference was observed in the IL6 serum levels between wild-type and α2AC KO mice, or after icv IL6 microinjection ( Figure 3I). Together, these data demonstrate that α2A and α2C receptors subunits are involved in the terminal sympathetic ow in muscle bers, integrating the hypothalamic IL6 effects.
Thereafter, we sought to evaluate this neuromuscular circuitry under some physiological conditions. It has been demonstrated that a single bout of exercise can underpin subsequent resting lipid oxidation for many hours in humans [19][20][21] . However, the mechanism by which our organism maintains the fatty acid oxidation in muscle ber post-exercise remains largely unknown. Intriguingly, high levels of IL6 were observed in the hypothalamus of sh 22 , mice 23 , and in the central nervous system of humans 24 postexercise. Thus, we hypothesized that hypothalamic IL6 could be involved in the fatty acid oxidation in the skeletal muscle post-exercise session. To address this issue, we initially evaluated the hypothalamic tissue immediately and skeletal muscle and blood samples 3 hours after an acute exercise session, as illustrated ( Figure 4A). High levels of Il6 gene expression were found immediately after the acute aerobic exercise ( Figure 4B). In addition, ERK1/2 phosphorylation was increased in whole hypothalamic tissue ( Figure S4B), particularly in VMH ( Figure 4C). Samples obtained 3 hours after the physical exercise revealed high levels of serum IL6 ( Figure 4D). In parallel, AMPK and ACC phosphorylation were still increased in the soleus muscle ( Figure S4C). Similar ndings were observed in AMPK and ACC phosphorylation levels post-exercise period in the skeletal muscle of rodents [25][26][27] and humans 28-30 .
RNA-sequencing analysis was performed to con rm the presence of the oxidative pro le in the skeletal muscle 3 hours after the acute exercise. Several genes involved in the mitochondrial function, lipid metabolism, and adrenergic signaling were upregulated 3 hours after the acute exercise ( Figure 4E), whereas, among 99 genes that were upregulated in response to exercise, almost half of these genes are related to the mitochondrial activity (24 genes), lipid metabolism (15 genes) and adrenergic signaling (7 genes) ( Figure 4E and F). Remarkably, in contrast to wild-type mice, the same exercise protocol failed to stimulate ERK1/2 phosphorylation in the hypothalamus and opped to sustain ACC and AMPK phosphorylation 3h post-exercise in the muscle of IL6-null mice (IL6KO) (Figure S4D-F).
To explore the relevance of IL6 action speci cally in the hypothalamus, exercised mice received anti-IL6 antibody (IL6 AB) icv microinjections 30 min before and immediately after the exercise session, and the muscle samples were analyzed 3 hours later, as illustrated in the experimental design ( Figure S5A). This experiment revealed that anti-IL6 AB icv microinjections abolished the fatty acid oxidation post-exercise in the skeletal muscle ( Figure S5B). We con rmed these preliminary ndings by employing a versatile tool for genome editing, targeting IL6 receptor (IL6R) speci cally in VMH of mice using lentiviral vector (shRNA) microinjection. Five distinct lentiviruses for inhibiting hypothalamic IL6R were tested. The TRCN94 lentivirus was the most e cient, reducing hypothalamic IL6R protein content by approximately 40% compared to scramble vector ( Figure S6A). Next, bilateral microinjections of TRCN94 were then performed to deliver IL6R lentivirus speci cally in VMH (IL6R KD VMH ). Three days after the lentivirus transduction, food consumption was normalized, and no difference was found in terms of food intake between scramble and IL6R KD VMH mice ( Figure S6B). Importantly, we con rmed that this lentiviral transduction was restricted to VMH, reducing the IL6R content in VMH, without affecting the presence of this receptor in adjacent hypothalamic nuclei, including in the arcuate nucleus ( Figure 4G-J). Seven days after lentivirus microinjection in VMH, the animals were submitted to an acute exercise session. The double-staining assay demonstrated that exercise increased the phospho-ERK1/2 positive cells in VMH in the scramble group ( Figure 4K and L). However, exercise did not promote the same effect in IL6R KD VMH mice ( Figure 4K and L). Interestingly, high levels of AMPK and ACC phosphorylation were observed 3 hours after exercise in the soleus muscle of control (scramble) but not in IL6R KD VMH mice ( Figure  4M). Finally, mass spectrometry analysis performed 3 hours after the exercise protocol con rmed that exercise markedly reduced the amount of fraction of saturated, monosaturated, and polyunsaturated fatty acids fractions in the skeletal muscle of control (scramble), but not in IL6R KD VMH mice ( Figure 4N and 6SC).
Altogether, our ndings demonstrate that the hypothalamic IL6/ERK axis in VMH drives the fatty acid metabolism in the skeletal muscle ( Figure S6D), and this neuromuscular circuitry seems to be critical to underpin the fatty acid oxidation post-exercise.
Declarations acid oxidation assays. R.S.G. performed the RNAseq analysis. L.J.EG. performed the bioinformatics analysis in gure S2C and B. P.C.B. provided a2AC AR knockout mice. P.O.P. and R.R.B. conducted the VMH dissection experiment. R.C., A.S.R.S., L.P.M., J.R.P., D.E.C, and L.A.V. provided the laboratory support and contributed to the discussion. E.R.R. wrote the manuscript.