Preproglucagon Neurons in the Brainstem Regulate Glucose Homeostasis and Insulin Sensitivity

Abstract

Background and ObjectiveThe gut‐brain axis plays an important role in energy balance and glucose homeostasis. As key, nutrient‐responsive brain‐gut peptides, glucagon‐like peptides (GLP‐1/2) are secreted from enteroendocrine L cells in the gut and from preproglucagon (PPG) neurons in in the nucleus of the solitary tract (NTS) of the brainstem. The physiological significance of endocrine GLP from L cells is highly appreciated, but the functional relevance of neural GLP from PPG neurons is unknown. We hypothesized thatPPG neurons' activities fine‐tune central autonomic outputs to improve peripheral glucose metabolism and insulin sensitivity. Our objective was to define if the acute control of PPG neurons' activities regulates peripheral glucose homeostasis and insulin sensitivity.Methods and Results1) Establishment of a pharmacogenetics mouse model for the remote control of PPG neurons' activities in vivo. First, we generated glucagon (Gcg) promoter‐driven Cre transgenic mouse in which Gcg‐Cre expression was co‐localized to GLP‐positive neurons in the brainstem. Second, we created a remote control of PPG neurons' activities in vivo mouse model using the DREADD approach. Cre‐dependent, excitatory AAV‐hM3Dq‐mCherry vectors or inhibitory AAV‐hM4Di‐mCherry vectors were injected into the brainstem NTS of 8‐wk old Gcg‐Cre mice. Third, we validated Gcg Cre‐dependent hM3Dq‐induced activation of PPG neurons using the whole‐cell patch clamp. hM3Dq (or hM4Di)‐mCherry was faithfully expressed in GFP‐positive PPG neurons. hM3Dq‐expressing neurons were excited upon clozapine‐N‐oxide (CNO) application as indicated by changes in membrane potential and firing rate of Gcg‐Cre::hM3Dq‐mCherryNTS neurons in brains slices, indicating that hM3Dq receptors rapidly activate infected PPG neurons in vivo. 2) Peripheral metabolic impact of PPG neuronal activation/ inhibition: With glucose tolerance test and hyperinsulinemic euglycemic clamp (coupled with dual stable isotopic tracers 6,6‐2H2‐d‐glucose and 2H2O), glucose homeostasis and insulin sensitivity were quantified in conscious Gcg‐Cre mice injected with Cre‐dependent hM3Dq (or hM4Di) vectors. In the Gcg‐Cre mice infected with hM3Dq virus after ip CNO, fasting glucose was decreased, while glucose tolerance was enhanced; and glucose infusion rate was increased as resulted from increased glucose utilization and decreased endogenous glucose production. In contrast, the Gcg‐Cre mice infected with hM4Di virus after ip CNO showed the opposite metabolic effects. 3) Central neural function of PPG neuronal activation/ inhibition: Activation of hM3Dq‐expressing neurons‐mediated neural circuitry was mapped by c‐Fos immunostaining in the brain after ip CNO. Increased c‐fos expression was mainly localized to central autonomic regions. Moreover, PPG neurons‐mediated neurotransmitter profiling was quantified by LC‐MS‐based metabolomics. As major neurotransmitters, GLP‐1/2 were released from activated PPG neurons.ConclusionsAcute control of PPG neurons' activities in vivo regulates glucose tolerance and insulin sensitivity, suggesting that PPG neurons play an important, physiological role in glycemic control and insulin sensitivity and targeting PPG neurons can be a new therapeutic approach for glycemic control.Support or Funding Information(Supported by the USDA CRIS grant 3092‐5001‐059 and the National NSFC grant 81570722)