IL-6 Improves Energy and Glucose Homeostasis in Obesity via ...

Article

IL-6 Improves Energy and Glucose Homeostasis in Obesity via Enhanced Central IL-6 trans-Signaling Graphical Abstract

Authors Katharina Timper, Jesse Lee Denson, Sophie Marie Steculorum, ..., Stefan Rose-John, F. Thomas Wunderlich, € ning Jens Claus Bru

Correspondence [email protected] (F.T.W.), [email protected] (J.C.B.)

In Brief Timper et al. find that central IL-6 improves energy and glucose homeostasis via IL-6 trans-signaling. IL-6 trans-signaling is enhanced in the CNS of obese mice, allowing IL-6 to exert its beneficial metabolic effects even under conditions of leptin resistance.

Highlights d

Central IL-6 action suppresses feeding and improves glucose tolerance

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IL-6-mediated metabolic actions via the CNS are enhanced in obesity

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Beneficial central IL-6 effects on metabolism are exerted via IL-6 trans-signaling

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Blocking of IL-6 trans-signaling in the PVH attenuates its beneficial metabolic effects

Timper et al., 2017, Cell Reports 19, 267–280 April 11, 2017 ª 2017 The Authors. http://dx.doi.org/10.1016/j.celrep.2017.03.043

Cell Reports

Article IL-6 Improves Energy and Glucose Homeostasis in Obesity via Enhanced Central IL-6 trans-Signaling Katharina Timper,1,2,3,6 Jesse Lee Denson,1,2,3,6 Sophie Marie Steculorum,1,2,3 Christian Heilinger,1,2,3 Linda Engstro¨m-Ruud,1,2,3 Claudia Maria Wunderlich,1,2,3 Stefan Rose-John,4 F. Thomas Wunderlich,1,2,3,* € ning1,2,3,5,7,* and Jens Claus Bru 1Max

Planck Institute for Metabolism Research, Department of Neuronal Control of Metabolism, Gleueler Str. 50, 50931 Cologne, Germany for Endocrinology, Diabetes and Preventive Medicine (CEDP), University Hospital Cologne, Kerpener Str. 26, 50924 Cologne, Germany 3Excellence Cluster on Cellular Stress Responses in Aging Associated Diseases (CECAD) and Center of Molecular Medicine Cologne (CMMC), University of Cologne, Joseph-Stelzmann-Str. 26, 50931 Cologne, Germany 4Biochemical Institute, University of Kiel, 24098 Kiel, Germany 5National Center for Diabetes Research (DZD), Ingolsta ¨ dter Landstr. 1, 85764 Neuherberg, Germany 6Co-first author 7Lead Contact *Correspondence: [email protected] (F.T.W.), [email protected] (J.C.B.) http://dx.doi.org/10.1016/j.celrep.2017.03.043 2Center

SUMMARY

Interleukin (IL)-6 engages similar signaling mechanisms to leptin. Here, we find that central application of IL-6 in mice suppresses feeding and improves glucose tolerance. In contrast to leptin, whose action is attenuated in obesity, the ability of IL-6 to suppress feeding is enhanced in obese mice. IL-6 suppresses feeding in the absence of neuronal IL-6-receptor (IL-6R) expression in hypothalamic or all forebrain neurons of mice. Conversely, obese mice exhibit increased soluble IL-6R levels in the cerebrospinal fluid. Blocking IL-6 trans-signaling in the CNS abrogates the ability of IL-6 to suppress feeding. Furthermore, gp130 expression is enhanced in the paraventricular nucleus of the hypothalamus (PVH) of obese mice, and deletion of gp130 in the PVH attenuates the beneficial central IL-6 effects on metabolism. Collectively, these experiments indicate that IL-6 trans-signaling is enhanced in the CNS of obese mice, allowing IL-6 to exert its beneficial metabolic effects even under conditions of leptin resistance. INTRODUCTION Interleukin (IL)-6 is strongly associated with chronic inflammatory states, including the low-grade inflammation associated with obesity and type 2 diabetes mellitus (Wellen and Hotamisligil, 2005). In obesity, adipose tissue immune cells have emerged as the major source of elevated circulating IL-6 levels (Hotamisligil, 2008; Mohamed-Ali et al., 1997). Along this line, increased systemic IL-6 levels are associated with elevated fat mass, not only in rodent models, but also in obese human subjects

(Carey et al., 2004). As a result of these findings, IL-6 has long been recognized as an initiator of insulin resistance, particularly because acute peripheral IL-6 infusion can impair insulin action in mice (Kim et al., 2004, 2012), and because neutralization of IL-6 improves insulin resistance in distinct inflammatory mouse models (Cai et al., 2005). Despite its suggested detrimental role in glucose homeostasis, a growing body of evidence identifies IL-6 as a homeostatic regulator of energy and glucose metabolism. In support of this, mice lacking IL-6 expression develop systemic insulin resistance and late-onset obesity (Matthews et al., 2010; Wallenius et al., 2002b). Moreover, IL-6 promotes pancreatic alpha-cell expansion during obesity (Ellingsgaard et al., 2008), promotes insulin secretion via enhanced glucagon-like peptide (GLP)-1 production (Ellingsgaard et al., 2011), and is a crucial mediator for the regulatory action of glucose-dependent insulinotropic peptide (GIP) in glucose control (Timper et al., 2016). Finally, IL-6 receptor signaling, both in hepatocytes and macrophages, limits systemic inflammation to improve systemic glucose homeostasis in lean and obese mice (Mauer et al., 2014, 2015; Wunderlich et al., 2010). Taking these findings into consideration, elevation of IL-6 during obesity might serve as an adaptive mechanism in an attempt to increase insulin production and improve glucose tolerance to overcome obesity-associated insulin resistance. In contrast to the obese state, exercise induces secretion of muscle-derived IL-6, leading to an acute and robust increase in systemic IL-6 up to 100-fold higher than basal levels (Ostrowski et al., 1998; Pedersen and Febbraio, 2008; Steensberg et al., 2000), which is associated with improved peripheral fuel availability and increased insulin sensitivity in the periphery (Carey et al., 2006) as well as at the level of the hypothalamus (Ropelle et al., 2010). Furthermore, it is important to note that IL-6 can signal via two distinct pathways. Classical IL-6 signaling occurs upon binding

Cell Reports 19, 267–280, April 11, 2017 ª 2017 The Authors. 267 This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

of IL-6 to its membrane-bound IL-6 receptor alpha (IL-6Ra) on target cells, which in turn associates with transmembrane signaling receptor protein gp130, leading to initiation of downstream intracellular signaling pathways (Rothaug et al., 2016). However, IL-6 can also activate cells that do not express IL-6Ra through the alternative signaling pathway termed ‘‘trans-signaling’’ (Rose-John and Heinrich, 1994). Here, IL-6 binds to a soluble form of IL-6R (sIL-6R), which is abundant in extracellular fluid, including plasma and cerebrospinal fluid (CSF) (Ma¨rz et al., 1997; Michalopoulou et al., 2004). The IL-6/ sIL-6R-complex can then associate with the ubiquitously expressed transmembrane protein gp130, which activates IL-6dependent intracellular signaling cascades, such as the JAK/ STAT pathway, even in cells lacking the membrane-bound receptor IL-6Ra (Heinrich et al., 2003; Hibi et al., 1990). In addition to the modulatory effects of IL-6 on metabolism in multiple peripheral organs, several studies point to a role of IL-6 in the regulation of energy homeostasis at the level of the central nervous system (CNS). First, IL-6 mediates inflammationrelated cachexia via central mechanisms (Langhans, 2000) and activates the hypothalamic-pituitary-adrenal (HPA) axis at the level of the hypothalamus (Naitoh et al., 1988; Navarra et al., 1991). Furthermore, central administration of IL-6, as well as overexpression of IL-6 in the CNS, results in increased energy expenditure and decreased body weight gain (Scho¨bitz et al., 1995; Wallenius et al., 2002a, 2002b), whereas IL-6depletion in astrocytes leads to body weight gain (Quintana et al., 2013). Recently, several publications have suggested that the IL-6-dependent effects on body weight control are exerted at the level of the hypothalamus (Benrick et al., 2009; Sche´le et al., 2012, 2013). However, the exact molecular mechanisms involved and the specific brain region through which central IL-6 action exerts its beneficial metabolic effects remain enigmatic. RESULTS Central IL-6 Inhibits Feeding in Lean and Obese Mice Previous studies have indicated a role of central IL-6 action in the control of energy homeostasis (Langhans, 2000; Li et al., 2002; Scho¨bitz et al., 1995; Wallenius et al., 2002a, 2002b). However, the molecular mechanisms of central IL-6 action in lean and obese mice remain largely unknown. Thus, we first aimed to investigate the effect of centrally applied IL-6 on food intake in mice. To this end, C57BL/6 control mice were exposed to a normal-fat control diet (NCD) or a high-fat diet (HFD). Expectedly, HFD feeding led to a significant increase in body weight, glucose intolerance, and insulin resistance in these animals (Figures S1A–S1C). Following 16 weeks of NCD or HFD feeding, mice were administered intracerebroventricularly (i.c.v.) with 400 ng of IL-6 upon an overnight fasting period, and food intake was monitored over 24 hr. Central application of IL-6 in lean mice resulted in a 35% suppression of food intake within the first 2 hr of re-feeding (Figure 1A). Interestingly, i.c.v. injection of IL-6 in HFD-fed mice led to suppression of food intake by 50% within 2 hr of re-feeding, which continued up to 4 hr following re-feeding (Figure 1B). Given that leptin resistance represents a major obstacle for successful

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leptin-based weight reduction in obese mice and humans, we compared the effect of central IL-6 and leptin action in lean and obese mice. To this end, lean control and HFD-fed obese mice were injected with an equimolar dose of leptin i.c.v., and food intake was monitored upon re-feeding. Although leptin exhibited a similar food-intake-suppressive effect compared to IL-6 in lean mice (Figure 1C), it failed to reduce food intake in HFD-fed obese mice (Figure 1D). To investigate whether the observed effects are depended on central IL-6 action or might have resulted from leaky IL-6 effects in the periphery, we injected the same dose of IL-6 intraperitoneally (i.p.) in lean and HFD-fed obese mice. i.p. injection of 400 ng of IL-6 did not affect food intake (Figures S1D and S1E), clearly indicating that the observed effect of i.c.v. IL-6 on food intake is indeed mediated via the CNS. In order to address whether IL-6 originating from the periphery can reach the CNS to affect food intake similar to CNS-applied IL-6, lean and obese mice were injected with 50 ng/g body weight IL-6 i.p. (z4-fold higher than the i.c.v. injected dose). Peripheral injection of IL-6 at a higher dose significantly reduced food intake during re-feeding in both lean (Figure 1E) and HFD-fed obese mice (Figure 1F). Moreover, inhibiting central IL-6 action by injecting HFD-fed obese mice i.c.v. with a neutralizing IL-6 antibody before i.p. injection of high-dose IL-6 completely prevented the suppressive IL-6 effects on food intake (Figure 1G). In conclusion, peripherally derived IL-6, when present at high concentrations, is able to reach the CNS to reduce feeding in both lean and HFD-fed obese mice. Collectively, our experiments reveal that central IL-6 acts in the CNS of obese mice to inhibit feeding, even under conditions of central leptin resistance, and to a greater extent than in lean mice. Central IL-6 Improves Hepatic Insulin Action and Glucose Tolerance in Obese Mice Both central insulin and leptin, in addition to the regulation of energy homeostasis and feeding, can improve peripheral insulin sensitivity and glucose homeostasis (Belgardt et al., 2009; €ning, 2013). Thus, we aimed Ko¨nner et al., 2007; Vogt and Bru at studying whether central IL-6 also improves peripheral glucose metabolism in a similar fashion. To this end, lean and HFD-fed obese mice were injected i.c.v. with either vehicle or 400 ng of IL-6 prior to a glucose or insulin tolerance test. While only moderately improving glucose tolerance in lean mice (Figure 2A), central IL-6 significantly improved glucose tolerance in obese, HFD-fed mice (Figure 2B). Additionally, central IL-6 had no effect on insulin sensitivity in lean mice (Figure 2C), while significantly improving insulin sensitivity in obese mice (Figure 2D). Because leptin-dependent STAT3 activation of hypothalamic neurons impacts on insulin sensitivity primarily via the liver (Buettner et al., 2006), we determined the effect of centrally applied IL-6 to modulate hepatic insulin-induced Akt phosphorylation. Consistent with the inability of central IL-6 to significantly modify systemic insulin sensitivity in lean control mice, insulin-stimulated Akt phosphorylation did not significantly differ between lean mice that had been centrally injected with either vehicle or IL-6 (Figure 2E). In contrast, central IL-6 administration significantly increased the ability of insulin to promote

Figure 1. Central IL-6 Inhibits Feeding in Lean and Obese Mice (A–G) Relative food intake of lean NCD-fed control mice and HFD-fed obese mice upon (A and B) injection of 400 ng of IL-6 versus vehicle (saline 0.9%) i.c.v. (n = 7–9 per treatment group), (C and D) 500 ng of leptin versus vehicle (PBS) i.c.v. (n = 10–12 per treatment group), (E and F) injection of 50 ng/g body weight (bw) IL-6 versus vehicle (saline 0.9%) i.p. (n = 6–11 per treatment group), and (G) injection of 2 mg of neutralizing IL-6 antibody (AB) versus vehicle (saline 0.9%) i.c.v. 30 min before injection of 50 ng/g bw IL-6 versus vehicle (saline 0.9%) i.p. (n = 14–15 per treatment group). Data are represented as mean ± SEM. *p % 0.05, **p % 0.01 as determined by two-tailed, unpaired Student’s t test (A–F) or one-way ANOVA followed by Bonferroni post hoc test (G). See also Figure S1.

hepatic Akt phosphorylation by 2-fold in obese, HFD-fed mice (Figure 2F). Central IL-6 application failed to affect insulin-stimulated Akt phosphorylation in skeletal muscle (Figures S2A and S2B) or white adipose tissue (Figures S2C and S2D) in both lean control and HFD-fed obese mice. Of note, in the absence of insulin, central IL-6 administration did not affect hepatic Akt phos-

phorylation in HFD-fed mice (Figure S2E), while again robustly inducing hepatic Akt phosphorylation in the presence of insulin (Figure S2F). In conclusion, although mildly improving systemic glucose homeostasis in lean mice, centrally applied IL-6 leads to a significant enhancement of systemic glucose tolerance and insulin

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Figure 2. Central IL-6 Action Improves Hepatic Insulin Action and Glucose Tolerance in Obese Mice (A and B) Glucose tolerance test (GTT) in lean NCD-fed control mice (A) and HFD-fed obese mice (B) upon i.c.v. injection of 400 ng of IL-6 versus vehicle (saline 0.9%) (n = 10–11 per treatment group). (C and D) Insulin tolerance test (ITT) performed in NCD (C) and HFD (D) animals upon injection of 400 ng of IL-6 versus vehicle (saline 0.9%) i.c.v. (n = 12–14 per treatment group).

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sensitivity in leptin-resistant HFD-fed obese mice, predominantly via improved hepatic insulin action. Classical, Neuronal IL-6R Signaling in the Hypothalamus or the Forebrain Is Dispensable for the Beneficial Metabolic Effects of Central IL-6 Next, we aimed at investigating through which cellular compartment in the CNS IL-6 exerts its beneficial metabolic effects. Insulin and leptin primarily act through hypothalamic neurons to regulate feeding, energy homeostasis, and systemic insulin €ning, 2012; Vogt and Bru €ning, sensitivity (Ko¨nner and Bru 2013), and central IL-6Ra expression was shown to be densely localized in the hypothalamus (Sche´le et al., 2013). To determine if central IL-6 action on metabolism was mediated via expression of its receptor in hypothalamic neurons, we generated mice, which lack the transmembrane IL-6R and thus classical IL-6R signaling specifically in hypothalamic neurons. Therefore, we crossed mice carrying a loxP-flanked IL-6Ra allele with those expressing the Cre recombinase specifically in hypothalamic neurons under the control of the Nkx2.1 promoter (Xu et al., 2008). Further intercrossing of these animals yielded IL6Raflox/floxNkx2.1Cre-negative (IL-6Raflox/flox control mice) and IL-6Raflox/floxNkx2.1Cretg/wt mice, i.e., mice with IL-6R deficiency restricted to hypothalamic neurons (IL-6RaDNkx2.1-mice) (Figure S3A). Surprisingly, despite the lack of hypothalamic IL-6Ra expression, i.c.v. injection of IL-6 continued to suppress feeding in HFD-fed-IL-6RaDNkx2.1 mice (Figure 3A). Moreover, detailed phenotypical analysis of these animals revealed unaltered body weight, fat mass, systemic glucose tolerance, and insulin sensitivity as well as circulating insulin and leptin concentrations as compared to their littermate controls (Figures S4A–S4F). Taken together, these experiments clearly revealed that classical IL-6R membrane-bound signaling in hypothalamic neurons is dispensable not only for the acute effects of centrally applied IL-6 to suppress food intake but also for maintaining steadystate energy and glucose homeostasis in obesity. To determine whether classical membrane-bound IL-6Rmediated signaling in neuronal populations other than the hypothalamus is required for the beneficial metabolic effects of central IL-6 action, mice lacking neuronal IL-6Ra expression throughout the forebrain were generated. Therefore, we crossed mice carrying the loxP-flanked IL-6Ra gene with those expressing the Cre recombinase specifically in forebrain neurons under the control of the CamKIIa promoter. Further intercrossing of these animals yielded IL-6Raflox/floxCamKIIaCre-negative (IL6Raflox/flox control) mice and IL-6Raflox/floxCamKIIaCretg/wt mice, i.e., mice with IL-6R deficiency restricted to forebrain neurons (IL-6RaDCamKIIa mice) (Figure S3B). Similar to IL-6RaDNkx2.1 mice, i.c.v. injection of IL-6 continued to induce suppression of feeding in IL-6RaDCamKIIa HFD animals (Figure 3B). Moreover, body weight, fat mass, systemic glucose homeostasis, and circulating insulin and leptin concentrations were unaltered

in HFD-fed IL-6RaDCamKIIa mice compared to littermate controls (Figures S4G–S4L). In conclusion, neuronal IL-6Ra expression in the hypothalamus or the forebrain is not required for central IL-6mediated suppression of food intake or for the maintenance of energy and glucose homeostasis. IL-6R trans-Signaling Mediates the CNS-Dependent Metabolic Effects of IL-6 Both hypothalamic- and forebrain-neuron-restricted deficiency of IL-6Ra expression failed to abrogate the IL-6-dependent effects on feeding. Thus, we next aimed at investigating whether the alternative mechanism of IL-6 trans-signaling in the CNS might account for the IL-6-dependent regulation of feeding. It is well documented that cells, which do not express the membrane-bound IL-6R can still be rendered IL-6 responsive via the alternative mechanism of IL-6 trans-signaling (Jones et al., 2011). Here, IL-6 binds the sIL-6Ra, and the IL-6/sIL-6Ra complex can bind to the common cytokine signaling chain gp130 expressed on cells to elicit IL-6-dependent signaling, including the activation of STAT3, which is a critical component in the hypothalamic control of feeding and glucose homeostasis (Belgardt €ning, 2010; Heinrich et al., 2003; Xu et al., 2007). To and Bru test whether IL-6 trans-signaling mediates the acute regulatory effects of centrally acting IL-6 on food intake, we compared the food-intake-suppressing effect of central IL-6 to that of a designer fusion protein consisting of IL-6 bound to the soluble IL-6Ra (HyperIL-6), which specifically activates the IL-6 transsignaling pathway (Fischer et al., 1997). i.c.v. injection of HyperIL-6 at a dose 10 times lower than that of IL-6 suppressed feeding in control mice (Figure 3C). Although the ability of IL-6 to suppress feeding was abrogated in lean conventional IL6Ra-deficient mice (IL-6RaD/D-mice), which lack both classical and IL-6 trans-signaling, i.c.v. injection of HyperIL-6 continued to elicit its full food-intake-suppressive effect in the absence of any endogenous IL-6Ra expression to the same extent as centrally applied IL-6 in lean control mice (Figure 3C). In line with the ability of IL-6 and HyperIL-6 to suppress feeding, central application of IL-6 resulted in a marked STAT3 phosphorylation in the arcuate nucleus of the hypothalamus (ARH) of control mice, whereas the ability of IL-6 to induce STAT3 phosphorylation in the ARH was completely abrogated in IL-6Ra-deficient mice (Figure 3D). On the other hand, the low dose of HyperIL-6 stimulated STAT3 phosphorylation to a comparable extent in lean control and IL-6Ra-deficient mice (Figure 3D). Furthermore, cFos staining in the ARH revealed a comparable neuronal activation upon centrally applied IL-6 and HyperIL-6 in control mice as well as upon i.c.v. application of HyperIL-6 in IL-6Ra-deficient mice, whereas IL-6 failed to induce cFos expression in IL-6RaD/D mice (Figure S4M). These experiments suggest that the food-intake-suppressive action of IL-6 is mediated via trans-signaling.

(E and F) Western blot analysis depicting phosphorylated Akt (pAkt) and Akt in liver explants upon 5-min insulin stimulation in vivo (0.5 U of insulin were administered into the vena cava) from (E) NCD and (F) HFD animals that were injected with 400 ng of IL-6 versus vehicle (saline 0.9%) i.c.v. 30 min beforehand (n = 5 to 6 per treatment group). Data are represented as mean ± SEM. *p % 0.05 as determined by two-way ANOVA followed by Bonferroni post hoc test (A–D) and two-tailed, unpaired Student’s t test (E and F). See also Figure S2.


Figure 3. Classical, Neuronal IL-6R Signaling in the Hypothalamus or the Forebrain Is Dispensable for the Beneficial Metabolic Effects of Central IL-6 (A–C) Relative food intake of (A) HFD-fed obese IL-6Raflox/flox control and IL-6RaDNkx2.1 mice upon injection of 400 ng of IL-6 versus vehicle (saline 0.9%) i.c.v. (n = 7–8 per treatment group), (B) HFD-fed obese IL-6Raflox/flox control and IL-6RaDCamKIIa mice upon injection of 400 ng of IL-6 versus vehicle (saline 0.9%) i.c.v. (n = 5 per treatment group), and (C) IL-6Raflox/flox control and IL-6RaD/D mice upon injection of 400 ng of IL-6 versus 40 ng of HyperIL-6 versus vehicle (saline 0.9%) i.c.v. (n = 9–11 per treatment group).



IL-6R trans-Signaling Is Enhanced in the CNS of HFDFed Obese Mice To investigate if enhanced IL-6 trans-signaling could account for the amplified IL-6 effects on feeding and glucose control in HFDfed obese animals compared to lean control animals, we tested the ability of IL-6 to suppress feeding and improve glucose metabolism in the absence or presence of co-injected soluble gp130 receptor (sgp130Fc). sgp130Fc can bind IL-6/sIL-6Ra complexes, thus inhibiting their interaction on cell-surfacebound gp130 signaling chains and thereby selectively abrogating IL-6R trans-signaling without affecting classic IL-6 signaling via the membrane-bound IL-6Ra (Jostock et al., 2001; Narazaki et al., 1993). Co-injection of sgp130Fc together with IL-6 completely abrogated the food-intake-suppressing effect of centrally applied IL-6 in both lean (Figure 4A) and HFD-fed obese animals (Figure 4B). Similarly, the ability of centrally applied IL-6 to improve glucose tolerance and insulin sensitivity was partially attenuated in the presence of sgp130Fc in HFD-fed obese mice, but not in lean control animals (Figures S5A–S5D). Furthermore, pSTAT3 in the ARH upon i.c.v. IL-6 injection was enhanced in HFD-fed obese mice compared to lean control mice and was abolished in the presence of sgp130Fc (Figure 4C). Interestingly, centrally applied IL-6 also induced pSTAT3 in the paraventricular nucleus of the hypothalamus (PVH) in HFD-fed obese animals, but not in lean control animals, and, again, the induction of pSTAT3 upon IL-6 in HFD-fed obese mice was completely abrogated upon co-treatment with the IL-6 trans-signaling inhibitor sgp130Fc (Figure 4D). Collectively, these experiments reveal that enhanced IL-6 effects on feeding and partially on glucose regulation in HFD-fed obese mice are dependent on increased IL-6R trans-signaling. Moreover, alternative IL-6 trans-signaling activates STAT3 phosphorylation in the PVH only in HFD-fed mice. To elucidate the underlying mechanisms of enhanced IL-6R trans-signaling in HFD-fed obese mice, we first measured the concentration of sIL-6R in the CSF and serum of lean and HFD-fed obese mice. sIL-6R concentrations in the CSF of HFD-fed obese mice were increased compared to those of lean control mice, whereas serum concentrations of sIL-6R did not differ between the two dietary cohorts (Figures 5A–5C). Furthermore, mRNA expression of gp130 (IL-6st) was increased in the hypothalamus of HFD-fed obese compared to lean control mice (Figure 5D). Because centrally applied IL-6 strongly activated pSTAT3 in the PVH exclusively in HFD-fed obese mice, we compared gp130 mRNA expression in the PVH of lean and HFD-fed obese mice via quantitative in situ hybridization. This analysis revealed increased gp130 mRNA expression in the PVH of HFD-fed obese mice compared to lean control mice, whereas there was no difference in mRNA expression levels of the neuronal marker NeuN between the dietary groups (Figure 5E). Overall, these experiments revealed that IL-6 trans-

signaling is enhanced in the CNS, both as a consequence of increased concentrations of sIL-6R in the CSF and increased expression of gp130, predominantly in the PVH, under HFDfed obese conditions. Ablation of gp130 in the PVH in Obesity Attenuates IL-6’s Beneficial Metabolic Effects To elucidate whether enhanced IL-6 trans-signaling in the PVH might contribute to the enhanced beneficial effects of centrally applied IL-6 on feeding and glucose control in HFD-fed animals, we ablated IL-6 trans-signaling in the PVH by injecting a Creexpressing adeno-associated virus (AAV) or a control AAV, expressing GFP, in the PVH of gp130flox/flox mice before they were exposed to HFD feeding. Correct bilateral delivery of the virus to the PVH was confirmed by immunohistochemical detection of GFP expression in the PVH of injected mice (Figure 6A). After 5 weeks of HFD feeding, control-AAV- and Cre-AAVinjected mice were centrally applied with 400 ng of IL-6, and food intake was monitored over a period of 24 hr after an overnight fasting period. This analysis revealed that central application of IL-6 in control-AAV-injected mice resulted in a robust and sustained suppression of food intake within the first 2 hr, lasting until 10 hr of re-feeding (Figure 6B). Strikingly, the ability of IL-6 to suppress feeding was completely abolished in CreAAV-injected mice, i.e., HFD-fed obese mice that lacked gp130 expression in the PVH. Furthermore, centrally applied IL-6 improved glucose tolerance in both control-AAV- (Figure 6C) and Cre-AAV-injected mice (Figure 6D), whereas central IL-6 profoundly improved insulin sensitivity selectively in controlAAV-injected mice (Figure 6E), but not in Cre-AAV-injected mice (Figure 6F). Consistently, central IL-6 failed to induce cFos immunoreactivity in the PVH of Cre-AAV-injected mice, whereas a robust induction of cFos was observed in the PVH of control-AAV-injected mice (Figure S6). In summary, these experiments revealed a significant contribution of IL-6R transsignaling in the PVH to the beneficial effects of enhanced central IL-6R trans-signaling on feeding and insulin sensitivity in HFDfed obese mice. DISCUSSION IL-6 has been demonstrated to engage the same key molecular effector pathways as leptin to control energy and glucose homeostasis, most prominently through the activation of the transcription factor STAT3 (Mauer et al., 2015). A major obstacle in the treatment of obesity is the fact that effector neurons of leptin action, such as pro-opiomelanocortin (POMC) and agoutirelated peptide (AgRP) neurons, become leptin and insulin resistant during obesity development (Belgardt et al., 2010; Ernst €ning, et al., 2009; Plum et al., 2006a, 2006b; Vogt and Bru 2013). Obesity-induced activation of stress kinases, such as

(D) Representative confocal images and quantification comparison of pSTAT3 immunoreactive cells (pSTAT3, green) and corresponding nuclear counterstaining (DAPI, blue) in the arcuate nucleus of the hypothalamus (ARH) of IL-6Raflox/flox control and IL-6RaD/D mice upon injection of 400 ng of IL-6 versus 40 ng of HyperIL-6 versus vehicle (saline 0.9%) i.c.v. (n = 9–16 per treatment group). Scale bars, 100 mm; 3V, third ventricle. Data are represented as mean ± SEM. *p % 0.05 and **p % 0.01 (IL-6 versus vehicle or HyperIL-6 versus vehicle), as determined by two-tailed, unpaired Student’s t test (A, B, D) or one-way ANOVA followed by Bonferroni post hoc test (C). See also Figures S3 and S4.


Figure 4. IL-6 trans-Signaling Mediates the CNS-Dependent Metabolic Effects of IL-6 (A and B) Relative food intake in lean control-diet (NCD)-fed (A) or high-fat-diet (HFD)-fed (B) obese mice upon i.c.v. injection of 400 ng of IL-6 in the presence or not of 200 ng of soluble gp130 (sgp130Fc) (n = 8–9 per treatment group).



IkBa-Kinase (IKK) and c-Jun N-terminal Kinase (JNK), in neurons not only contributes to hyperphagia, but also deteriorates systemic insulin sensitivity and glucose homeostasis (Hill et al., 2010; Kleinridders et al., 2009; Ko¨nner et al., 2007; Lin et al., 2010; Tsaousidou et al., 2014). Thus, alternative activators of leptin-evoked downstream signaling components might represent an ideal pharmacological target to overcome obesity-associated central leptin and insulin resistance. Here, we demonstrate that IL-6-activated STAT3 signaling in hypothalamic neurons potently suppresses feeding and improves peripheral glucose homeostasis. We also report that central IL-6R activation predominantly in obese mice enhances hepatic insulin action and that central IL-6 action is even enhanced in obese mice. Through complementary pharmacological and genetic approaches, we reveal that IL-6 in the CNS acts independently of the classical membrane-resident IL-6R-dependent signaling, but mediates its effects via the alternative transsignaling pathway. So far, observations on IL-6 trans-signaling in the CNS have been controversial. On the one hand, transsignaling in the brain has been shown to transduce detrimental IL-6 effects in a distinct astrocyte-overexpressing IL-6 mouse model, which is associated with astrocytosis and neurodegeneration (Campbell et al., 1993, 2014), as well as in lipopolysaccharide-induced neuroinflammation models (Burton et al., 2011, 2013). On the other hand, and supportive to our findings, neurons and astrocytes are almost exclusively responsive to IL-6 transsignaling, but not to classical signaling via the membrane-bound IL-6R (Ma¨rz et al., 1997, 1998; Rothaug et al., 2016). Moreover, a recent report in which inhibition of IL-6 trans-signaling in the periphery of obese mice prevents adipose tissue inflammation, without effects on overall insulin sensitivity and glucose tolerance (Kraakman et al., 2015), is consistent with the role of CNS IL-6 trans-signaling in systemic control of glucose homeostasis in obesity, as described in the present report. Future experiments will have to define the cellular source of sIL-6R in the CNS, the concentration of which is increased in the CSF of obese animals. A likely candidate comprises microglia, which have previously been demonstrated to be active in the CNS of obese mice (Milanski et al., 2009; Thaler et al., 2012; Valdearcos et al., 2014) and that express IL-6R at both the mRNA and protein level (Hsu et al., 2015). However, in addition to elevated sIL-6R concentrations in the CNS of obese mice, expression of gp130 was specifically increased in the PVH upon HFD feeding, likely contributing to an amplified IL-6 sensitivity via enhanced IL-6R trans-signaling. In line with this evidence, PVH-specific activation of pSTAT3 upon central IL-6 application was observed exclusively in HFD-fed obese mice, but not in lean control mice. Consistently, ablation of IL-6R trans-signaling upon AAV-Cre-dependent deletion of gp130 expression in the PVH fully abrogated the ability of central IL-6 to inhibit feeding and partially to improve peripheral insulin

sensitivity in HFD-fed obese mice. Region-specific disruption of trans-signaling can only be achieved by ablation of gp130 because sIL-6Ra is freely distributed in the extracellular fluid compartment. Therefore, we chose the approach of region-specific delivery of a Cre-expressing AAV to the PVH of gp130-floxed mice to ablate IL-6 trans-signaling in this specific hypothalamic region. It remains to be determined which neuronal subpopulations within the PVH translate the IL-6 effect on feeding control. A likely candidate might depict parvocellular oxytocin neurons in the PVH, which have been reported to show pSTAT3 activation upon central leptin application and to transduce the leptin-dependent effect on body weight control in rats (Perello and Raingo, 2013). Taken together, our experiments uncovered the PVH as an essential mediator to transduce the beneficial effects of enhanced IL-6R trans-signaling on energy and glucose homeostasis under obese conditions. Here, future research is needed to unravel the neuronal subpopulations within the PVH that transduce the enhanced IL-6 effects in obesity and to decipher which other neuronal downstream circuits are involved. In summary, our experiments define IL-6 or modified IL-6 variants, which effectively activate IL-6 trans-signaling in the CNS, as potential drug targets for obesity and obesity-associated insulin resistance. EXPERIMENTAL PROCEDURES Animal Care All animal procedures were conducted in compliance with protocols approved by the local government authorities (Bezirksregierung Ko¨ln) and were in accordance with NIH guidelines. Mice were housed in groups of 3–5 at 22 C–24 C using a 12-hr light/12-hr dark cycle. Please see Supplemental Experimental Procedures for the diet compositions. Animals had ad libitum access to water and the prescribed diet at all times, and food was only withdrawn if required for an experiment. All experiments were performed in adult male mice at the indicated ages. C57BL/6N Mice C57BL/6N mice were obtained from Charles River Laboratories at 4 to 5 weeks of age and fed an NCD or HFD for 16–20 weeks unless otherwise indicated. Genetic Mouse Models Generation of Il6RaDNkx2.1 and Il6RaDCaMKIIa Mice To generate Il6RaDNkx2.1 and Il6RaDCaMKIIa mice, IL-6Raflox/flox mice (Wunderlich et al., 2010) were crossed to either Nkx2.1Cretg/wt mice (Xu et al., 2008) (purchased from The Jackson Laboratories) or CamKIIa Cretg/wt mice (Tsien et al., 1996), respectively. Transgenic Mice IL-6Raflox/flox and IL-6RaD/D mice were obtained from our own breeding facility, and their generation have been described elsewhere (Wunderlich et al., 2010, 2012). Intracerebroventricular Injections i.c.v. Cannulation i.c.v. cannulations were performed as described previously (Klo¨ckener et al., 2011) and in the Supplemental Experimental Procedures.

(C and D) Representative confocal images and quantification comparison of pSTAT3 immunostaining (pSTAT3, green) and corresponding nuclear counterstaining (DAPI, blue) in the (C) arcuate nucleus of the hypothalamus (ARH) and (D) paraventricular nucleus of the hypothalamus (PVH) of NCD or HFD animals upon i.c.v. injection of 400 ng of IL-6 in the presence or not of 200 ng of sgp130Fc (n = 7–10 per treatment group). Scale bars, 100 mm; 3V, third ventricle. Data are presented as mean ± SEM. *p % 0.05, ***p % 0.001 (IL-6 versus vehicle), tp % 0.05, tttp % 0.001 (IL-6 versus sgp130Fc+IL-6), as determined by one-way ANOVA followed by Bonferroni post hoc test (A–D). See also Figure S5.


Figure 5. IL-6 trans-Signaling Is Enhanced in the CNS of HFD-Fed Obese Mice (A–C) Soluble IL-6 receptor (sIL-6Ra) concentrations in (A) cerebrospinal fluid (CSF) and (B) serum as well as (C) CSF/serum ratio of normal control diet (NCD) and high-fat diet (HFD) animals (n = 13–15 per group, each data point consists of two pooled animals). (D) gp130 (IL-6st) mRNA expression in the hypothalamus of NCD and HFD animals (n = 9–10 per group). (E) Representative confocal images and quantification comparison of in situ hybridization of gp130 (IL-6st, red) and NeuN (green) and corresponding nuclear counterstaining (DAPI, blue) in the paraventricular nucleus of the hypothalamus (PVH) of NCD or HFD animals. Scale bars, 100 mm; 3V, third ventricle. Data are presented as mean ± SEM. *p % 0.05 as determined by unpaired two-tailed Student’s t test.

Compound Administration For i.c.v. injection, recombinant mouse IL-6 (400 ng/mouse; carrier free, R&D Systems), HyperIL-6 (human origin, 40 ng/mouse, [Fischer et al., 1997]), sgp130Fc (murine origin, 200 ng/mouse), leptin (mouse origin, 500 ng/mouse;


Sigma), or neutralizing mouse IL-6 antibody (goat IgG, 2 mg/mouse; R&D Systems) were used. Saline 0.9% or sterile 0.1 M PBS (pH 7.5) was used as the control solution (vehicle), as indicated. Mice were administered 2 mL of each compound.

Figure 6. Ablation of IL-6 trans-Signaling in the PVH of HFD-Fed Obese Mice Abrogates IL-6’s Beneficial Regulatory Effects on Feeding and Glucose Control (A) Representative confocal images of GFP immunoreactive cells (GFP, green) and corresponding nuclear staining (DAPI, blue) in the paraventricular nucleus of the hypothalamus (PVH) of gp130flox/flox mice that were injected with AAV5-GFP (control-AAV) or AAV5-GFP-iCre (Cre-AAV) in the PVH. Scale bars, 100 mm; 3V, third ventricle. (B) Relative food intake (n = 10–11 per treatment group).



Intraperitoneal Injections For intraperitoneal injection, recombinant mouse IL-6 (400 ng/mouse or 50 ng/g body weight; carrier-free, R&D Systems) was used. Saline 0.9% was used as the control solution (vehicle). Glucose Tolerance and Insulin Tolerance Tests Please see Supplemental Experimental Procedures. Food Intake Measurement For food-intake studies, mice were single caged and acclimated to custom-made food racks for a week prior to measurements. After 16-hr starvation overnight, mice were provided with fresh cages to avoid the leftover of food spilling in the bedding. Food intake was measured after i.c.v. injection of the respective compound or control vehicle, as indicated by weighing the food racks containing food pellets before (time 0) and 2, 4, 8, and 24 hr after compound or control vehicle administration, respectively. The food hopper was placed into the cage directly following i.c.v. injection. CSF and Serum for Analysis of sIL-6Ra Collection of CSF was performed as described previously (Liu and Duff, 2008) and in the Supplemental Experimental Procedures. Assessment of Insulin Signaling in Peripheral Organs To assess insulin action in peripheral organs, 16-hr fasted mice received an i.c.v. injection with recombinant mouse IL-6 (400 ng/mouse; carrier free, R&D Systems) or vehicle (saline 0.9%). 30 min thereafter, mice were deeply anesthetized, and the abdomen was opened under aseptic conditions. 0.5 U of insulin (100 mL of total volume; Actrapid [Novo Nordisk]) or vehicle (saline 0.9%) was injected in the vena cava just below the liver hilus. After 5 min, liver, skeletal muscle, and perigonadal white adipose tissue were dissected, snap frozen in liquid nitrogen, and stored at 80 C. AAV Injection Please see Supplemental Experimental Procedures. Western Blot Analysis Western blot analysis was conducted as previously described (Gruber et al., 2013). Antibodies were obtained from Cell Signaling Akt (#4686), p-Akt(S473) (#4060), and Calbiochem Calnexin (#208880). Please see Figure S2, which depicts the original membranes and blots from all western blot analyses in this study. Measurement of Plasma Insulin and Leptin Please see Supplemental Experimental Procedures. Analysis of Gene Expression Please see Supplemental Experimental Procedures. Body Composition Body composition was analyzed using the NMR Analyzer Minispec (Bruker Optik). Histology Please see Supplemental Experimental Procedures. Statistical Analyses All values were expressed as mean ± SEM. Statistical analyses were conducted using GraphPad PRISM (version 6.0a). Datasets with only two independent

groups were analyzed for statistical significance using unpaired, two-tailed Student’s t test, unless otherwise specified in the figure legends. Datasets with more than two groups were analyzed using one-way ANOVA, followed by Bonferroni post hoc test. Datasets subjected to two independent factors were analyzed using two-way ANOVA, followed by Bonferroni post hoc test. All p values below or equal to 0.05 were considered significant. *p % 0.05, **p % 0.01, and ***p % 0.001. SUPPLEMENTAL INFORMATION Supplemental Information includes Supplemental Experimental Procedures and six figures and can be found with this article online at http://dx.doi.org/ 10.1016/j.celrep.2017.03.043. AUTHOR CONTRIBUTIONS K.T. and J.L.D. contributed equally to this work. K.T., J.L.D., F.T.W. and J.C.B. conceived the project, designed the experiments, analyzed the data, and wrote the manuscript with input from the other authors. K.T. and J.L.D. performed all the experiments apart from western blot analysis (C.H.) and fluorescent in situ hybridization (L.E.-R.). S.R.-J. generated and provided the compounds (HyperIL-6, sgp130Fc). C.M.W. provided the IL-6Raflox/flox and IL-6RaD/D mice. S.M.S. was involved in experimental design and writing of the manuscript. All authors discussed the data, commented on the manuscript before submission, and agreed with the final submitted manuscript. ACKNOWLEDGMENTS We acknowledge Hella Bro¨nneke for outstanding support, Brigitte Hampel, Pia Scholl, and Nadine Spenrath for outstanding technical assistance with immu€ngst from CECAD nohistochemistry, as well as Astrid Schauss and Christian Ju Imaging Facility Cologne for excellent technical assistance with confocal imaging. This work was supported by a grant from the Deutsche Forschungsgemeinschaft (DFG) (BR 1492/7-1) to J.C.B. J.C.B. and F.T.W. also received funding by the DFG within the framework of the TRR134 and within the Excellence Initiative by German Federal and State Governments (CECAD). This work was funded (in part) by the Helmholtz Alliance ICEMED Imaging and Curing Environmental Metabolic Diseases, through the Initiative and Networking Fund of the Helmholtz Association. Moreover, the research leading to these results has received funding from the European Union Seventh Framework Program (FP7/2007-2013) under grant agreement number 266408. K.T. was funded by an Advanced Postdoc Grant from the Swiss National Science Foundation and received an Early Career Research Grant from the University of Basel, Switzerland. Received: October 7, 2016 Revised: February 3, 2017 Accepted: March 13, 2017 Published: April 11, 2017 REFERENCES €ning, J.C. (2010). CNS leptin and insulin action in the Belgardt, B.F., and Bru control of energy homeostasis. Ann. N Y Acad. Sci. 1212, 97–113. €ning, J.C. (2009). Hormone and glucose Belgardt, B.F., Okamura, T., and Bru signalling in POMC and AgRP neurons. J. Physiol. 587, 5305–5314. Belgardt, B.F., Mauer, J., Wunderlich, F.T., Ernst, M.B., Pal, M., Spohn, G., Bro¨nneke, H.S., Brodesser, S., Hampel, B., Schauss, A.C., et al. (2010).

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