1Universitätsklinikum Schleswig-Holstein Medizinische Klinik II / Kardiologie, Angiologie, Intensivmedizin Lübeck, Deutschland; 2Universitätsklinikum Mannheim I. Medizinische Klinik Mannheim, Deutschland; 3Institut für Experimentelle und Klinische Pharmakologie und Toxikologie Universität zu Lübeck Lübeck, Deutschland; 4Institute for Systemic Inflammation Research Universität zu Lübeck Lübeck, Deutschland
Background
Obesity represents a major risk factor for the development of diabetes, hypertension, hypercholesterolemia, and thus, atherosclerosis. Although both peripheral complement activation and lipid-mediated hypothalamic inflammation have been implicated in the development of obesity and subsequent atherosclerosis, the role of hypothalamic complement system in dysregulated energy homeostasis during obesity has not yet been investigated. As various complement inhibitors are currently being evaluated in clinical trials, and additional agents are in preclinical development, we here aim to elucidate the functional impact of hypothalamic complement activation in metabolic syndrome and subsequent cardiovascular disease, to pave the way for future translational approaches by using complement receptor antagonists in cardiovascular patients.
Methods
C57BL/6J (WT) mice were treated with complement 3a receptor (C3aR1) antagonist SB290157 or vehicle and fed with high fat diet (HFD) or chow for 6 or 12 weeks. C3 KO and novel generated hypothalamus specific C3aR1 KO mice (C3aR flox x LepR cre) were fed with HFD for 12 weeks. C3b expression as well as leptin receptor (LepR) and C3aR1 co-localization were evaluated by immunohistochemistry in the Nucleus arcuatus hypothalami (ARC). Hypothalamic leptin signaling was evaluated by STAT3 phosphorylation blots. Leptin resistance and glucose tolerance tests were performed in vivo. Hypothalamic mHypoE-46 neuronal-cell line was used to assess the impact of C3a on leptin-mediated release of orexigenic Agouti-related Protein (AgRP) secretion and SOCS3 expression in vitro.
Key Results
• Diet-induced obesity (DIO) leads to impaired insulin sensitivity and central leptin resistance, and increased hypothalamic C3a and C3b expression (A-D)
• C3aR1 antagonist SB290157 protects against DIO and maintains glucose tolerance and hypothalamic leptin sensitivity, shown by enhanced STAT3 phosphorylation after leptin injection (E-G)
• Confocal microscopy revealed that C3aR (green) is co-localized on hypothalamic appetite-regulating neurons that express the LepR (red, L). Thus, proving the crucial role of the hypothalamic C3aR axis in metabolic syndrome, both C3 KO and hypothalamus specific C3aR1 KO protect against DIO which is accompanied by a reduction of hypothalamic SOCS3 activation, preserving hypothalamic leptin sensibility (H-M)
• Accordingly, C3a diminishes leptin-mediated reduction of orexigenic AgRP secretion by mHypoE-46 cells (O)
• Mechanistically, we can confirm in vitro that C3aR1 activation enhances SOCS3 expression as a negative regulator of leptin receptor signaling (P)
Conclusion and perspective
For the first time we show that diet-induced obesity is accompanied by hypothalamic complement C3a/C3aR axis activation whereby complement receptor inhibition protects against the development of central leptin resistance and obesity. We finally verified these novel findings, by generating a hypothalamus-specific C3aR1 knock-out mice. Regarding the recently described importance of neuroimmune cardiovascular interfaces in the context of atherosclerosis, we will currently apply a PCSK9-AAV to render an atherosclerotic disposition in our novel conditional C3aR1 knock-out model, to elucidate the importance of hypothalamic complement system in atherosclerosis as a novel therapeutic approach.
Funding: ER is supported by the DZHK Clinician Scientist Programme and by a DGK Research Fellowship