Acute activation of GLP-1-expressing neurons promotes glucose homeostasis and insulin sensitivity

Abstract

Objective: Glucagon-like peptides are co-released from enteroendocrine L cells in the gut and preproglucagon (PPG) neurons in the brainstem. PPG-derived GLP-1/2 are probably key neuroendocrine signals for the control of energy balance and glucose homeostasis. The objective of this study was to determine whether activation of PPG neurons per se modulates glucose homeostasis and insulin sensitivity in vivo.

Methods: We generated glucagon (Gcg) promoter-driven Cre transgenic mice and injected excitatory hM3Dq-mCherry AAV into their brainstem NTS. We characterized the metabolic impact of PPG neuron activation on glucose homeostasis and insulin sensitivity using stable isotopic tracers coupled with hyperinsulinemic euglycemic clamp.

Results: We showed that after ip injection of clozapine N-oxide, Gcg-Cre lean mice transduced with hM3Dq in the brainstem NTS downregulated basal endogenous glucose production and enhanced glucose tolerance following ip glucose tolerance test. Moreover, acute activation of PPG neurons^NTS enhanced whole-body insulin sensitivity as indicated by increased glucose infusion rate as well as augmented insulin-suppression of endogenous glucose production and gluconeogenesis. In contrast, insulin-stimulation of glucose disposal was not altered significantly.

Conclusions: We conclude that acute activation of PPG neurons in the brainstem reduces basal glucose production, enhances intraperitoneal glucose tolerance, and augments hepatic insulin sensitivity, suggesting an important physiological role of PPG neurons-mediated circuitry in promoting glycemic control and insulin sensitivity.

Keywords: Endogenous glucose production; Glucagon-Cre mice; Glucagon-like peptides; Gluconeogenesis; Insulin sensitivity; Preproglucagon neurons.

Graphical abstract

Figure 1

Gcg-Cre transgenic mouse line was validated by Cre-dependent Rosa26-eGFP reporter mouse. Gcg-Cre-mediated eGFP reporter was expressed in distinct neurons in the brainstem the dorsal motor vagal nucleus (DMV, A), the nucleus tractus solitarius (NTS, B), and the intermediate reticular nucleus (IRT, C); and projected to the brainstem dorsal motor vagal nucleus (DMV, D) and hypothalamus [the paraventricular/dorsomedial hypothalamic nuclei (PVH/DMH) and arcuate nucleus (ARC), E–F]. Note that Gcg promoter-driven Cre expression is reported by eGFP (green) from the Gcg-Cre::Rosa26-eGFP mouse brain. CC, central canal; AP, area postrema.

Figure 2

Remote activation of hM3Dq-expressing Gcg neurons induced by CNO. A. Weak c-Fos expression in the basal level without CNO injection. B. Enlarged image of the squared region in A. c-Fos-positive cells (green) were largely segregated from hM3Dq-expressing cells (red). C. c-Fos expression was increased at 30 min after ip injection of CNO (0.3 mg/kg). D. Enlarged image of the squared region in C. c-Fos-positive cells (green) were largely localized to hM3Dq-expressing cells (red). Note that Gcg neurons are indicated by expression of mCherry (red) in the brainstem NTS-DMV from the Gcg-Cre mouse locally infected with AAV8-hM3Dq-mCherry viruses.

Figure 3

Acute excitation of hM3Dq-expressing Gcg neurons induced by CNO. A. Gcg Cre-mediated expression of eGFP (green) in the brainstem NTS–NTS from the Gcg-Cre::Rosa26-eGFP mouse line. B. PPG neurons (green) in the brainstem NTS did not respond to CNO (10 μM) application as control. C. Gcg neurons indicated by mCherry (red) in the brainstem NTS from the Gcg-Cre mouse locally infected with AAV8- hM3Dq-mCherry viruses. D. hM3Dq-expressing Gcg neurons was excited by CNO (10 μM) application as shown representative traces of the whole-cell current patch clamp.

Figure 4

A protocol for quantifying glucose kinetics and insulin sensitivity. A protocol for stable isotopic tracer method in conjunction with hyperinsulinemic euglycemic clamp is pictured . After a 7-d recovery, conscious mice after 12-h fast were primed-continuously infused with stable isotopic tracers (²H₂O and 6,6-²H₂-d-glucose) for 3 h to quantify glucose kinetics during the basal period; and then for 3 h to assess tissue-specific insulin sensitivity during hyperinsulinemic euglycemic clamp. Two weeks after viral injection, 10-wk-old mice were implanted with jugular vein catheters. Hyperinsulinemic euglycemic clamp was performed in stress-less, conscious mice at one week post cannulation. Note that CNO (0.3 mg/kg) was ip injected at 30 min prior to the primed-continuous infusion of 6,6-²H₂-d-glucose for glucose kinetics and then at the beginning of the insulin clamp. Blood glucose was clamped at ∼100 mg/dL (as shown in Fig. S2) with infusion of glucose during insulin infusion (2.5 mU/kg/h). Isotopic enrichments of blood glucose and water were measured by GC–MS and GC-IR-MS, respectively. Glucose kinetics (including GIR, glucose infusion rate; EGP, endogenous glucose production; GNG, gluconeogenesis; and R_d, the rate of glucose disappearance) were quantified at postabsorptive, steady state in conscious mice.

Figure 5

Remote activation of Gcg neurons reduces endogenous glucose production in mice fed regular chow. A. 12-h fasting glucose at 0 and 3.5 h post injection of CNO (ip 0.3 mg/kg). Excitatory AAV-hM3Dq-mCherry viruses (150 nL) were injected into the Gcg-Cre mouse brainstem NTS. B. Basal endogenous glucose production (EGP) in the conscious mice after an overnight fast (at 3 wks post viral injection). To quantify the basal EGP, a stable isotopic tracer (6,6-²H₂-d-glucose) was infused for 3 h to reach an equilibrium for glucose isotopologues. See the detailed stable isotopic tracer methodology in METHODS. Mice were ip injected with CNO (0.3 mg/kg) or saline prior to the tracer infusion. n = 6–8/group. *P < 0.05. C. Blood glucose concentrations (of mice at 3 wks post viral injection) measured in 6-h fast mice after ip glucose challenge test (ipGTT). CNO (0.3 mg/kg) was ip injected 30 min prior to ipGTT.

Figure 6

Remote activation of Gcg neurons enhances hepatic insulin sensitivity in mice fed regular chow. A. Glucose infusion rate (GIR, mmol/kg/h) during hyperinsulinemic euglycemic clamp. Higher GIR were needed to maintain the same level of blood glucose (clamped at ∼100 mg/dL, see Fig. S2) in conscious PPG^hM3Dq mice after ip injection of CNO. B. Tissue-specific insulin sensitivity of the conscious PPG^hM3Dq mice quantified by hyperinsulinemic euglycemic clamp coupled with dual stable isotopic tracers (²H₂O and 6,6-²H₂-d-glucose as shown in Figure 4). Glucose kinetics was determined at the steady status. Remote activation of Gcg neurons augments hepatic insulin sensitivity largely by further suppression of EGP and GNG. GIR, glucose infusion rate; Rd, rate of glucose disappearance; EGP, endogenous glucose production; GNG, gluconeogenesis. PPG^hM3Dq mice (at 3 wks post viral injection) were ip injected with CNO (0.3 mg/kg) or saline prior to the insulin clamp. n = 6–8/group. * or **P < 0.05 or 0.01.

Figure 7

A proposed model for Gcg neurons in glycemic control. Activation of Gcg neurons in the brainstem NTS promotes glycemic control and insulin sensitivity via fine-tuning autonomic outputs to peripheral tissues (liver), suppressing hepatic glucose production (HGP, mainly gluconeogenesis), which may involve Glp1r/2r-positive neurons in the hypothalamus (PVH and ARC) and brainstem (DMV). In addition, activation of Gcg neurons may release unidentified neurotransmitter(s) X that modulates postsynaptic neurons in the above nuclei.