Loss of hypothalamic Furin affects POMC to proACTH cleavage and feeding behavior in high-fat diet-fed mice

Abstract

Objective

The hypothalamus regulates feeding and glucose homeostasis through the balanced action of different neuropeptides, which are cleaved and activated by the proprotein convertases PC1/3 and PC2. However, the recent association of polymorphisms in the proprotein convertase FURIN with type 2 diabetes, metabolic syndrome, and obesity, prompted us to investigate the role of FURIN in hypothalamic neurons controlling glucose and feeding.

Methods

POMC-Cre ^+/− mice were bred with Furin ^fl/fl mice to generate conditional knockout mice with Furin-deletion in neurons expressing proopiomelanocortin (POMC FurKO), and Furin ^fl/fl mice were used as controls. POMC FurKO and controls were periodically monitored on both normal chow diet and high fat diet (HFD) for body weight and glucose tolerance by established in-vivo procedures. Food intake was measured in HFD-fed FurKO and controls. Hypothalamic Pomc mRNA was measured by RT-qPCR. ELISAs quantified POMC protein and resulting peptides in the hypothalamic extracts of POMC FurKO mice and controls. The in-vitro processing of POMC was studied by biochemical techniques in HEK293T and CHO cell lines lacking FURIN.

Results

In control mice, Furin mRNA levels were significantly upregulated on HFD feeding, suggesting an increased demand for FURIN activity in obesogenic conditions. Under these conditions, the POMC FurKO mice were hyperphagic and had increased body weight compared to Furin ^fl/fl mice. Moreover, protein levels of POMC were elevated and ACTH concentrations markedly reduced. Also, the ratio of α-MSH/POMC was decreased in POMC FurKO mice compared to controls. This indicates that POMC processing was significantly reduced in the hypothalami of POMC FurKO mice, highlighting for the first time the involvement of FURIN in the cleavage of POMC. Importantly, we found that in vitro, the first stage in processing where POMC is cleaved into proACTH was achieved by FURIN but not by PC1/3 or the other proprotein convertases in cell lines lacking a regulated secretory pathway.

Conclusions

These results suggest that FURIN processes POMC into proACTH before sorting into the regulated secretory pathway, challenging the dogma that PC1/3 and PC2 are the only convertases responsible for POMC cleavage. Furthermore, its deletion affects feeding behaviors under obesogenic conditions.

Highlights

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  Hypothalamic Furin mRNA levels were significantly upregulated on high-fat feeding.

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  High-fat diet fed mice lacking Furin in POMC neurons were hyperphagic and had increased body weight.

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  POMC processing to proACTH was significantly reduced in the hypothalami of mice lacking Furin in POMC neurons.

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  FURIN is a POMC-processing enzyme.