Mechanisms underlying the efficacy of a rodent model of vertical sleeve gastrectomy - A focus on energy expenditure

Abstract

Objective: Bariatric surgery remains the only effective and durable treatment option for morbid obesity. Vertical Sleeve Gastrectomy (VSG) is currently the most widely performed of these surgeries primarily because of its proven efficacy in generating rapid onset weight loss, improved glucose regulation and reduced mortality compared with other invasive procedures. VSG is associated with reduced appetite, however, the relative importance of energy expenditure to VSG-induced weight loss and changes in glucose regulation, particularly that in brown adipose tissue (BAT), remains unclear. The aim of this study was to investigate the role of BAT thermogenesis in the efficacy of VSG in a rodent model.

Methods: Diet-induced obese male Sprague-Dawley rats were either sham-operated, underwent VSG surgery or were pair-fed to the food consumed by the VSG group. Rats were also implanted with biotelemetry devices between the interscapular lobes of BAT to assess local changes in BAT temperature as a surrogate measure of thermogenic activity. Metabolic parameters including food intake, body weight and changes in body composition were assessed. To further elucidate the contribution of energy expenditure via BAT thermogenesis to VSG-induced weight loss, a separate cohort of chow-fed rats underwent complete excision of the interscapular BAT (iBAT lipectomy) or chemical denervation using 6-hydroxydopamine (6-OHDA). To localize glucose uptake in specific tissues, an oral glucose tolerance test was combined with an intraperitoneal injection of 14C-2-deoxy-d-glucose (14C-2DG). Transneuronal viral tracing was used to identify 1) sensory neurons directed to the stomach or small intestine (H129-RFP) or 2) chains of polysynaptically linked neurons directed to BAT (PRV-GFP) in the same animals.

Results: Following VSG, there was a rapid reduction in body weight that was associated with reduced food intake, elevated BAT temperature and improved glucose regulation. Rats that underwent VSG had elevated glucose uptake into BAT compared to sham operated animals as well as elevated gene markers related to increased BAT activity (Ucp1, Dio2, Cpt1b, Cox8b, Ppargc) and markers of increased browning of white fat (Ucp1, Dio2, Cited1, Tbx1, Tnfrs9). Both iBAT lipectomy and 6-OHDA treatment significantly attenuated the impact of VSG on changes in body weight and adiposity in chow-fed animals. In addition, surgical excision of iBAT following VSG significantly reversed VSG-mediated improvements in glucose tolerance, an effect that was independent of circulating insulin levels. Viral tracing studies highlighted a patent neural link between the gut and BAT that included groups of premotor BAT-directed neurons in the dorsal raphe and raphe pallidus.

Conclusions: Collectively, these data support a role for BAT in mediating the metabolic sequelae following VSG surgery, particularly the improvement in glucose regulation, and highlight the need to better understand the contribution from this tissue in human patients.

Keywords: Animal model; Bariatric surgery; Brown adipose tissue thermogenesis; Energy expenditure; Metabolic surgery; Vertical sleeve gastrectomy.

Graphical abstract

Figure 1

Changes in body weight, food intake, body composition and glucose tolerance following VSG in obese rats. (A) Study timeline, (B) comparison of the effects of sham operation (n = 5), VSG (n = 9) and pair-feeding (n = 5) on body weight gain (g), (C) daily food intake (g), (D) cumulative food intake (g) over 60-day measurement period in obese rats. Changes in body composition including (E) fat mass (g), (F) lean mass (g) and (G) bone mass (g) relative to baseline (pre-surgical) levels. (H) Blood glucose levels and AUC analysis following glucose (1.5 g/kg lean mass) in sham (n = 8) or VSG (n = 9) operated rats compared to Chow-fed (n = 6) controls. (I) Plasma insulin (ng/ml) at baseline and 15 min following glucose and (J) Change in insulin levels relative to baseline concentration. n = 4–9. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001, ∗ in H, comparing sham vs VSG.

Figure 2

Changes in energy expenditure following VSG in obese rats. (A) Mean hourly BAT temperature of obese rats during the (B) dark and (C) light period 10–14 days following sham operation (n = 5), VSG (n = 5) or pair-feeding (n = 5). (D), quantification of UCP1 protein expression in BAT (10 weeks post-op) following Western blot detection and a representative blot is shown (n = 6–9/group), (E) average energy expenditure (kcal/hr) every 30 min and regression analyses for energy expenditure during fasting and refeeding (7 weeks post-op) and (F) RER during a fast and following refeeding (0930h) (n = 8/group). n = 5–8. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.

Figure 3

Changes in glucose-stimulated glucose uptake following VSG and expression of brown and beige cell markers. Glucose uptake 30 min following the administration of glucose (1.5 g/kg lean mass) in (A) BAT, (B) eWAT, (C) iWAT, (D) rWAT, (E) gastrocnemius and (F) soleus skeletal muscle, (G) heart and (H) liver 6 weeks following sham (n = 8), or VSG (n = 9) surgery compared to Chow-fed (n = 6) controls. Gene expression of brown and beige cell markers in (I) BAT and (J) iWAT (n = 5–9). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.∗in I and J, compared to Sham., (K) Representative BAT and iWAT histology (H&E and UCP-1 Immunohistochemistry). Abbreviations: BAT, brown adipose tissue; eWAT, epidydimal white adipose tissue; iWAT, inguinal white adipose tissue; rWAT, retroperitoneal adipose tissue.

Figure 4

Changes in body weight (g) relative to the respective sham operations in low fat diet fed rats over 13 days post-surgery. Body weight was monitored following (A) sham operation (n = 5) or VSG (n = 5) with intact iBAT function, (B) complete excision of iBAT [sham + iBAT lipectomy (n = 5), VSG + iBAT lipectomy (n = 5)] and (C) chemical denervation of iBAT using 6-OHDA [sham+ 6-OHDA (n = 5), VSG+6-OHDA (n = 7)] and (D) comparison of VSG efficacy across all groups shown in A-C relative to individual control. Blood glucose (mmol/L) during oGTT with AUC in obese rats 6 weeks following sham (n = 6) or VSG (n = 4) surgery and Chow-fed controls (n = 6) with (E) intact iBAT and (G) one week following iBAT excision. Plasma insulin levels during oGTT at baseline (pre-glucose) and 15 min following oral glucose in rats with (F) intact iBAT and (H) following surgical excision of the iBAT. Comparison of (I) sham-operated, (J) VSG-operated and (K) Chow-fed rats prior to and one week following iBAT excision. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001. ∗ In H, Sham vs VSG; # in H, VSG vs Chow-fed.

Figure 5

Quantitative assessment of the extent of labelling of neurons containing Fos in chow-fed rats 2 weeks following Sham + Ccontrol (no infusion), Sham + Stretch (water), Sham + Nutrients (Ensure) or VSG + Control, VSG + Stretch, VSG + Nutrients. Counts were made through the (A) rostral, (B) mid and (C) caudal extent of the NTS and totalled across the entire nucleus (D). (E) Representative coronal sections of the NTS in sham and VSG operated rats. Scale bar in (D) = 200 μm. n = 3–4/group. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < .001, ∗∗∗∗P < 0.0001. Abbreviations: NTS, nucleus tractus solitarius.

Figure 6

Photomicrographs illustrating the distribution of H129-labelled cells in coronal sections through the caudal to rostral span of the brainstem and forebrain regions following H129-infection immediately following sham (column 1,3) or VSG surgery (column 2,4) (a) nodose ganglion (NG), (b) commissural NTS, (c) mid NTS, (d) parabrachial nucleus (PBN), (e) dorsal hypothalamic area (DHA), lateral hypothalamic area (LHA) and arcuate nucleus (ARC) and (f) mid paraventricular nucleus of the hypothalamus (mPVN). Scale bar = 200 μm. Abbreviations: 3V = 3rd ventricle; 4V = 4th Ventricle, DMV-X = dorsal motor nucleus of the vagus.

Figure 7

Photomicrographs of H129-RFP and PRV-GFP positive neurons within premotor regions in the brainstem and hypothalamus following injection of (A) H129-RFP into the stomach and PRV-GFP into iBAT and (B) H129-RFP into the small intestine and PRV-GFP into iBAT in the same rat. Co-localization of projections are seen in the rostroventromedial medulla (RVMM), dorsal raphe, raphe pallidus and paraventricular nucleus of the hypothalamus (PVN). Arrows indicate neurons receiving sensory information from the stomach or small intestine and projecting polysynaptically to both iBAT.