Divergent Effect of Central Incretin Receptors Inhibition in a Rat Model of Sporadic Alzheimer's Disease

Abstract

The incretin system is an emerging new field that might provide valuable contributions to the research of both the pathophysiology and therapeutic strategies in the treatment of diabetes, obesity, and neurodegenerative disorders. This study aimed to explore the roles of central glucagon-like peptide-1 (GLP-1) and gastric inhibitory polypeptide (GIP) on cell metabolism and energy in the brain, as well as on the levels of these incretins, insulin, and glucose via inhibition of the central incretin receptors following intracerebroventricular administration of the respective antagonists in healthy rats and a streptozotocin-induced rat model of sporadic Alzheimer's disease (sAD). Chemical ablation of the central GIP receptor (GIPR) or GLP-1 receptor (GLP-1R) in healthy and diseased animals indicated a region-dependent role of incretins in brain cell energy and metabolism and central incretin-dependent modulation of peripheral hormone secretion, markedly after GIPR inhibition, as well as a dysregulation of the GLP-1 system in experimental sAD.

Keywords: Alzheimer’s disease; gastric inhibitory polypeptide; glucagon-like peptide-1; hippocampus; hypothalamus.

Figure 1

The underlying mechanism responsible for the beneficial effect of incretins in Alzheimer’s disease. Incretin receptor activation may include stabilization of the outer mitochondrial membrane, prevention of cytochrome c (CytC) efflux into the cytoplasm, and reduction of apoptosis and oxidative stress through activation of cAMP and other kinases. Together with ATP, the cAMP-dependent, protein kinase A (PKA) signaling pathway can regulate cytochrome c oxidase (COXIV) activity and mitochondrial function [22]. It can also activate AMP-activated protein kinase (AMPK), leading to increased glucose uptake by the cells in an insulin-independent manner. By increasing aerobic glycolysis, pyruvate is converted to acetyl-CoA by pyruvate dehydrogenase (PDH), which enters the citric acid cycle (TCA) to produce ATP. In this way, incretins shift the cell metabolism from oxidative phosphorylation and increased ROS production to aerobic glycolysis, leading to a neuroprotective effect. FOXO1—forkhead box protein O1; AKT—protein kinase B; Bcl—B-cell lymphoma. Figure created with BioRender.com (accessed on 27 December 2021).

Figure 2

Glucose, insulin, glucagon-like peptide-1, and gastric inhibitory polypeptide plasma concentrations. One month after intracerebroventricular (icv) streptozotocin (STZ) or vehicle (CTR) administration, rats were injected icv with 85 µg/kg of glucagon-like peptide-1 receptor antagonist (exendin fragment 9-39, GLP1Rinh; experiment 1) or 85 µg/kg of gastric inhibitory polypeptide receptor antagonist (Pro3-GIP, GIPRinh; experiment 2) dissolved in saline or with saline only (CTR and STZ). Animals were sacrificed 30 min after icv administration and blood was sampled for analysis of glucose (i), insulin (ii), total (iii) and active (iv) GLP1, and total (v) and active (vi) GIP concentrations in plasma (A,B). Values are presented as boxplots with marked outliers and data analyzed by a non-parametric Kruskal–Wallis one-way ANOVA test followed by a Mann–Whitney U test (* p < 0.05; ** p < 0.01; *** p < 0.001).

Figure 3

Impact of central glucagon-like peptide-1 and gastric inhibitory polypeptide inhibition on the levels of cytochrome C, cytochrome C oxidase IV, and pyruvate dehydrogenase in the hippocampus and hypothalamus. One month after intracerebroventricular (icv) streptozotocin (STZ) or vehicle (CTR) administration, rats were injected icv with 85 µg/kg of glucagon-like peptide-1 receptor antagonist (Exendin fragment 9–39, GLP1Rinh; experiment 1) or 85 µg/kg of gastric inhibitory polypeptide receptor antagonist (Pro3-GIP, GIPRinh; experiment 2) dissolved in saline or with saline only (CTR and STZ). Animals were sacrificed 30 min after icv administration and the hippocampus (HPC) and hypothalamus (HPT) were dissected, homogenized/sonicated, and protein concentration was measured. Cytochrome C (CytC; iii,iv), cytochrome C oxidase IV (COXIV; i,ii), and pyruvate dehydrogenase (PDH; v,vi) levels in the HPC and HPT were measured by Western blot analysis 30 min after central GLP1R (A) and GIPR (B) inhibition. Values are presented as boxplots with marked outliers and data analyzed by a non-parametric Kruskal–Wallis one-way ANOVA test followed by a Mann–Whitney U test (* p < 0.05; ** p < 0.01).

Figure 4

Levels and activity of AMP-activated protein kinase in the hippocampus and hypothalamus changed after central glucagon-like peptide-1 and gastric inhibitory polypeptide inhibition. One month after intracerebroventricular (icv) streptozotocin (STZ) or vehicle (CTR) administration, rats were injected icv with 85 µg/kg of glucagon-like peptide-1 receptor antagonist (Exendin fragment 9–39, GLP1Rinh; experiment 1) or 85 µg/kg of gastric inhibitory polypeptide receptor antagonist (Pro3-GIP, GIPRinh; experiment 2) dissolved in saline or with saline only (CTR and STZ). Animals were sacrificed 30 min after icv administration and the hippocampus (HPC) and hypothalamus (HPT) were dissected, homogenized/sonicated, and protein concentration was measured. Phosphorylated (Thr172) and total AMP-activated protein kinase (pAMPK (i,ii) and tAMPK (iii,iv)) levels in the HPC and HPT were measured by Western blot analysis 30 min after central GLP1R (A) and GIPR (B) inhibition. AMPK activity is expressed as a ratio between pAMPK and tAMPK (v,vi). Values are presented as boxplots with marked outliers and data analyzed by a non-parametric Kruskal–Wallis one-way ANOVA test followed by a Mann–Whitney U test (* p < 0.05; ** p < 0.01).

Figure 5

Influence of central glucagon-like peptide-1 and gastric inhibitory polypeptide inhibition on c-fos and cAMP levels and ATP concentration in the hippocampus and hypothalamus. One month after intracerebroventricular (icv) streptozotocin (STZ) or vehicle (CTR) administration, rats were injected icv with 85 µg/kg of glucagon-like peptide-1 receptor antagonist (exendin fragment 9–39, GLP1Rinh) or 85 µg/kg of gastric inhibitory polypeptide receptor antagonist (Pro3-GIP, GIPRinh) dissolved in saline or with saline only (CTR and STZ). Animals were sacrificed 30 min after icv administration and the hippocampus (HPC) and hypothalamus (HPT) were dissected, homogenized/sonicated, and protein concentration was measured. Neuronal activation, cAMP levels and ATP concentration were measured 30 min after central GLP1R (A) and GIPR (B) inhibition. Neuronal activation was measured indirectly through c-fos level by Western blot (i,ii) inhibition. cAMP levels were measured using a commercial cAMP Direct Immunoassay kit and Western blot analysis (iii,iv). ATP concentration was measured by bioluminescence assay (v,vi). Values are presented as boxplots with marked outliers and data analyzed by a non-parametric Kruskal–Wallis one-way ANOVA test followed by a Mann–Whitney U test (* p < 0.05; ** p < 0.01).

Figure 6

Experimental design. Two experiments with 4 groups each were conducted with the same procedure. Three-month-old male Wistar rats were intracerebroventricularly (icv) injected with streptozotocin (STZ/3 mg/kg) or vehicle only (controls/CTR) on days 1 and 3. In experiment 1 (Exp1) rats were randomly divided into four groups. Two groups (CTR + GLP1Rinh and STZ + GLP1Rinh) received icv 85 µg/kg of glucagon-like peptide-1 receptor antagonist (exendin fragment 9–39, GLP1Rinh) dissolved in saline. In experiment 2 (Exp2), two groups (CTR + GIPRinh and STZ + GIPRinh) received icv 85 µg/kg of gastric inhibitory polypeptide receptor antagonist (Pro3-GIP, GIPRinh) dissolved in saline. The other groups in both experiments (CTR and STZ) received saline only (bilaterally 2 µL/ventricle). All animals were sacrificed 30 min after icv. Blood was sampled and brain was removed and the hippocampus (HPC) and hypothalamus (HPT) were dissected out for further analysis. Figure created with BioRender.com (accessed on 21 December 2021).