Role of central nervous system glucagon-like Peptide-1 receptors in enteric glucose sensing

Abstract

Objective: Ingested glucose is detected by specialized sensors in the enteric/hepatoportal vein, which send neural signals to the brain, which in turn regulates key peripheral tissues. Hence, impairment in the control of enteric-neural glucose sensing could contribute to disordered glucose homeostasis. The aim of this study was to determine the cells in the brain targeted by the activation of the enteric glucose-sensing system.

Research design and methods: We selectively activated the axis in mice using a low-rate intragastric glucose infusion in wild-type and glucagon-like peptide-1 (GLP-1) receptor knockout mice, neuropeptide Y-and proopiomelanocortin-green fluorescent protein-expressing mice, and high-fat diet diabetic mice. We quantified the whole-body glucose utilization rate and the pattern of c-Fos positive in the brain.

Results: Enteric glucose increased muscle glycogen synthesis by 30% and regulates c-Fos expression in the brainstem and the hypothalamus. Moreover, the synthesis of muscle glycogen was diminished after central infusion of the GLP-1 receptor (GLP-1Rc) antagonist Exendin 9-39 and abolished in GLP-1Rc knockout mice. Gut-glucose-sensitive c-Fos-positive cells of the arcuate nucleus colocalized with neuropeptide Y-positive neurons but not with proopiomelanocortin-positive neurons. Furthermore, high-fat feeding prevented the enteric activation of c-Fos expression.

Conclusions: We conclude that the gut-glucose sensor modulates peripheral glucose metabolism through a nutrient-sensitive mechanism, which requires brain GLP-1Rc signaling and is impaired during diabetes.

FIG. 1.

A: Experimental design to analyze the effect of an intragastric glucose infusion. Mice bearing intragastric, femoral, and intracerebroventricular catheters underwent infusions after the surgery recovery period. On days 1, 2, 4, and 5 of the training period, mice were trained to the intragastric water infusion. On day 3, an intragastric glucose infusion (30 mg · kg⁻¹ · min⁻¹) was performed, and those mice developing immediate hyperglycemia demonstrated a correct intragastric catheter implantation and were studied on day 6 when they underwent an intragastric glucose or water infusion after 6 h of fasting. Two protocols were set up. In protocol 1, glucose turnover was assessed and the mice underwent a continuous intragastric glucose infusion for 2 h. In protocol 2, the mice underwent a 10-min intragastric glucose or water infusion, and 2 h later, the brain was fixed in vivo for histological studies. B: Schematic representation in gray of brain areas analyzed to quantify the number of c-Fos–positive cells (37). C: Glycemic profiles (mmol/l) in mice infused with glucose (▪) or saline (□). The inset represents the corresponding AUC (arbitrary units). No differences were noticed.

FIG. 2.

Glucose turnover and glycogen synthesis rates. A: Whole-body glucose turnover, glycolysis, and glycogen synthesis rates (mg · kg⁻¹ · min⁻¹). Glycogen synthesis rates (pg · mg⁻¹ · min⁻¹) in liver (B) and muscle (C) were assessed after a 2-h water or glucose intragastric infusion (protocol 1) in wild-type and GLP-1Rc knockout (GLP-1Rc KO) mice (see protocol 1 in Fig. 1). A subset of wild-type mice was infused simultaneously with Ex9 into the brain (Ex9). Five to nine mice per group were studied. *Statistically different from water-infused control mice when P < 0.05.

FIG. 3.

Pattern of c-Fos–positive cells after an intragastric glucose infusion. The number of c-Fos–positive cells was quantified in the NTS (A) and ARC, VMH, and DMN nuclei (B) (as described in Fig. 1B) of C57BL/6 mice infused with water (□) or glucose (▪) for 10 min (see protocol 2 in Fig. 1). C: Representative staining of c-Fos–expressing cells in each nuclei and condition. Eight mice per group were studied. *Statistically different from water-infused control mice when P < 0.05. **Statistically different from water-infused control mice when P < 0.01.

FIG. 4.

Pattern of c-Fos–positive cells after an intragastric glucose infusion in GLP-1Rc knockout mice. The number of c-Fos–expressing cells was quantified in the NTS (A) and ARC, VMH, and DMN nuclei (B) of GLP-1Rc knockout mice (as described in Fig. 1B) infused with water (□) or glucose (▪). Five mice per group were studied. No differences were noted between groups.

FIG. 5.

Pattern of c-Fos/POMC–and c-Fos/NPY–positive cells after an intragastric water or glucose infusion. c-Fos/POMC–positive cells were quantified in the ARC. Representative figures of c-Fos–positive cells (A) and POMC-positive cells (B) in the ARC. ARC was magnified. The POMC-positive cells showing clear staining in cytoplasm are indicated by white arrows. C: Representative figure of c-Fos/POMC–positive cells showing that POMC-positive cells were not colocalized with c-Fos in the magnified field. c-Fos/NPY–positive cells was quantified in the ARC. D and E: Immunohistochemical representation of cFos and NPY and NPY/cFos-positive cells, as indicated by arrows, in mice infused with water or glucose. F and G: Quantification of NPY and NPY/c-Fos-positive cells in mice infused with water (□) or glucose (▪). Five to six mice per group were studied. (Please see http://dx.doi.org/10.2337/db07-1788 for a high-quality digital representation of this figure.)

FIG. 6.

Pattern of c-Fos–positive cells and glucose utilization rates after an intragastric glucose infusion in high-fat diet–fed diabetic mice. The number of c-Fos–expressing cells was quantified in the NTS (A) and ARC, VMH, and DMN nuclei (B) of mice fed a high-fat, carbohydrate-free diet infused with water (□) or glucose (▪). Five mice per group were studied. Glucose flux (mg · kg⁻¹ · min⁻¹) was studied after 1 week (C) or 1 month (D) of high-fat, carbohydrate-free diet. TO, turnover; Glycol, glycolysis; and Gln Synth, glycogen synthesis. For all parameters, no difference was noted between groups.

FIG. 7.

Schematic representation of the role of GLP-1 in the brain as a master switch for the control of glucose fate. 1) Enteric sensors are the first site for glucose detection after an oral glucose load. They send a neural signal to the NTS. 2) Subsequently, the NTS sends a signal toward the hypothalamus, including GLP-1. The enteric signal triggers NPY-positive cells. 3) GLP-1–sensitive cells send a new signal, of unknown origin, toward peripheral tissues, i.e., muscles, to prepare cells to use glucose. 4) The brain would detect glucose. 5) Subsequently, a signal opposite to the one sent by the enteric glucose detector would be sent by the brain to the muscle, as previously described (23). Importantly, the enteric glucose signal is impaired during diabetes.