Hyperphagia and increased fat accumulation in two models of chronic CNS glucagon-like peptide-1 loss of function

Abstract

Central administration of glucagon-like peptide-1 (GLP-1) causes a dose-dependent reduction in food intake, but the role of endogenous CNS GLP-1 in the regulation of energy balance remains unclear. Here, we tested the hypothesis that CNS GLP-1 activity is required for normal energy balance by using two independent methods to achieve chronic CNS GLP-1 loss of function in rats. Specifically, lentiviral-mediated expression of RNA interference was used to knock down nucleus of the solitary tract (NTS) preproglucagon (PPG), and chronic intracerebroventricular (ICV) infusion of the GLP-1 receptor (GLP-1r) antagonist exendin (9-39) (Ex9) was used to block CNS GLP-1r. NTS PPG knockdown caused hyperphagia and exacerbated high-fat diet (HFD)-induced fat accumulation and glucose intolerance. Moreover, in control virus-treated rats fed the HFD, NTS PPG expression levels correlated positively with fat mass. Chronic ICV Ex9 also caused hyperphagia; however, increased fat accumulation and glucose intolerance occurred regardless of diet. Collectively, these data provide the strongest evidence to date that CNS GLP-1 plays a physiologic role in the long-term regulation of energy balance. Moreover, they suggest that this role is distinct from that of circulating GLP-1 as a short-term satiation signal. Therefore, it may be possible to tailor GLP-1-based therapies for the prevention and/or treatment of obesity.

Figure 1.

RNAi against PPG significantly decreases PPG expression in vitro and in vivo. A, Quantification of PPG mRNA and GLP-1 fiber density after infection with a lentivirus encoding a scrambled (control) or PPG-specific (PPG) shRNA. PPG mRNA levels in INS-1 cells (INS-1) were measured by quantitative reverse transcriptase-PCR 48 h after infection. PPG mRNA levels in rat NTS and ventrolateral medulla (VLM) were measured by in situ hybridization, and GLP-1 fiber density in rat PVN was measured by immunofluorescence 12 d after intra-NTS lentivirus infusion. B, Representative image of GFP immunoreactivity 12 d after intra-NTS lentivirus infusion. C, Schematic depicting the area shown in B (Paxinos and Watson, 1998). Gr, Gracile nucleus; gr, gracile fasciculus; SolC, commissural nucleus of the solitary tract; Sol, nucleus of the solitary tract; sol, solitary tract; A2, A2 noradrenergic cells; 10, dorsal motor nucleus of the vagus; CC, central canal; 12, hypoglossal nucleus. D–G, Representative images of NTS and VLM PPG mRNA (D, E) and PVN GLP-1 fiber density (F, G) 12 d after intra-NTS infusion of control (D, F) or PPG (E, G) lentivirus. Data are represented as mean ± SEM (n = 11 rats per group). *p < 0.05, **p < 0.01, ***p < 0.001 vs control.

Figure 2.

NTS PPG knockdown results in hyperphagia and increased body weight in chow-fed rats. A, B, Cumulative body weight change (A) and daily food intake (B) during the first 2 weeks of the study. C, D, Cumulative body weight change (C) and average daily food intake (D) for the 6-week duration of the study. E, F, Changes in fat mass (E) and lean mass (F) over time. Data are represented as mean ± SEM (n = 20 rats per group). *p < 0.05, **p < 0.01, ***p < 0.001 vs control.

Figure 3.

NTS PPG knockdown results in exacerbation of HFD-induced obesity and glucose intolerance. A, B, Cumulative body weight change (A) and average daily food intake (B). C, Cumulative food intake at various time points across a 24 h period, measured during week 3 of the study. D, Average light phase, dark phase and 24 h food intake during a 48 h measurement period while housed in the TSE continuous metabolic monitoring system during week 7 of the study. E, F, Changes in fat mass (E) and lean mass (F) over time. G, H, intraperitoneal glucose tolerance while maintained on chow (G) and HFD (H). Data are represented as mean ± SEM (n = 20 rats per group). *p < 0.05, **p < 0.01, ***p < 0.001 vs control.

Figure 4.

NTS PPG knockdown does not alter energy expenditure, respiratory quotient or locomotor activity. A–C, Average dark phase and light phase energy expenditure (A), respiratory quotient (B) and total locomotor activity (C) during a 48 h measurement period while housed in the TSE continuous metabolic monitoring system during week 7 of the study. Data are represented as mean ± SEM (n = 20 rats per group).

Figure 5.

Hindbrain PPG expression correlates positively with fat mass. A–C, PPG mRNA levels of HFD-fed control rats (n = 18) correlated significantly with body weight (A, r = 0.5384, p = 0.0174) and even more so with fat mass (B, r = 0.7207, p = 0.0005) but not with lean mass (C, r = −0.009578).

Figure 6.

Chronic ICV Ex9 results in hyperphagia, increased fat accumulation and glucose intolerance in chow- and HFD-fed rats. A, B, Cumulative body weight change (A) and food intake (B). C, D, Changes in fat mass (C) and lean mass (D). E, F, Intraperitoneal glucose tolerance. Data are represented as mean ± SEM (n = 10 rats per group). *p < 0.05, ***p < 0.001, Ex9 vs saline within chow or HFD.

Figure 7.

Chronic ICV Ex9-induced weight gain and glucose intolerance are secondary to hyperphagia and increased fat mass, respectively. A, B, Cumulative body weight change (A) and food intake (B). C, D, Changes in fat mass (C) and lean mass (D). E, F, Intraperitoneal glucose tolerance. Data are represented as mean ± SEM (n = 10 rats per group). **p < 0.01, ***p < 0.001 Ex9–AL vs saline. ^##p < 0.01, ^###p < 0.001 Ex9–AL vs Ex9–PF. ^$p < 0.05, ^$$p < 0.01, ^$$$p < 0.001 Ex9–AL vs Ex9–SC. ^@p < 0.05 Ex9–PF vs Ex9–SC.