Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, PCB298

Poster Communications

Investigating the neuronal networks responsible for insulin sensing in the dorsal vagal complex

L. New1, B. Patel1, I. Letts1, B. Filippi1

1. University of Leeds, Leeds, United Kingdom.

Obesity is an epidemic that is spreading worldwide, by 2020 8/10 men and 7/10 women will be overweight or obese. Obesity leads to negative metabolic consequences including insulin resistance and diabetes. Obesity-related diseases e.g. diabetes will likely cost the NHS an extra £2.5 billion per year by 2035. New anti-obesity treatments are essential and recent research has begun to consider how targeting the CNS may be the answer. The dorsal vagal complex (DVC) of the brain can sense changes in insulin levels and regulate glucose metabolism and feeding behaviour. However, the neuronal circuitry in the DVC that senses insulin to regulate metabolic function are still unknown. Direct insulin infusion to the DVC lowers food intake and glucose levels in healthy rats but fails to do so following 3 days of high fat diet (HFD), indicating DVC insulin resistance. The exact mechanism for these changes following HFD is unknown, however changes in mitochondria, through alteration of mitochondrial dynamics, may be involved. HFD increases mitochondria fission in the DVC via activation of Dynamin Related Protein 1 (Drp1) which causes an increase in ER stress that leads to insulin resistance. It is still unclear how HFD-dependent activation of Drp1 causes ER stress and insulin resistance. A potential mediator of this process is iNOS as DVC iNOS levels increase following HFD. It is crucial to understand this event to prevent the loss of insulin sensitivity in the brain. We aim to characterise the neural cell types responsible for insulin sensing in the DVC and understand how changes in mitochondrial fission affect iNOS levels in these different cell types. Adenoviruses expressing a constitutively active form of Drp1 (S637A), a dominant negative form of Drp1 (K38A) and a GFP-expressing control were injected into the NTS of the DVC. IHC of DVC slices taken from GFP-expressing control rats showed that 24-26% of GFP-expressing cells were NeuN+, while 38-40% were GFAP+ glial cells. Close appositions between GFP+ cells and V-GAT+ or 5HT+ projections were also observed. On-going work will characterise Drp1-S637A+ and Drp1-K38A+ cells in the same way. The majority of GFP+ cells were glial cells, suggesting that glial-neuronal crosstalk may be a central aspect of NTS-dependent regulation of glucose levels and feeding behaviour. Current work aims to use Drp1 adenoviral constructs with a GFAP-promotor for glial-targeted expression of Drp1-S637A/K38A to further analyse this possibility. Fluorescence intensity of iNOS and the ER stress marker p-PERK are significantly greater in the NTS of HFD fed rats. iNOS and p-PERK IHC have also been carried out in NTS of Drp1- S637A- and Drp1- K38A-expressing DVC to determine whether these animals exhibit increased ER stress following changes in mitochondrial dynamics. Such results link HFD, ER stress, and insulin resistance, and experiments are currently underway to further examine this link by NTS-specific lentiviral-mediated KO of iNOS.

Where applicable, experiments conform with Society ethical requirements