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Vertical sleeve gastrectomy activates GPBAR‐1/TGR5 to sustain weight loss, improve fatty liver, and remit insulin resistance in mice

Vertical sleeve gastrectomy (VSG) is one of the most commonly performed clinical bariatric surgeries used for the remission of obesity and diabetes. However, the precise molecular mechanism by which VSG exerts its beneficial effects remains elusive. We report that the membrane‐bound G protein‐coupled bile acid receptor, GPBAR‐1 (also known as TGR5), is required to mediate the effects of anti‐obesity, anti‐hyperglycemia, and improvements of fatty liver of VSG in mice. In the absence of TGR5, the beneficial metabolic effects of VSG in mice are lost. Moreover, we found that the expression of TGR5 increased significantly after VSG, and VSG alters both BA levels and composition in mice, resulting in enhancement of TGR5 signaling in the ileum and brown adipose tissues, concomitant with improved glucose control and increased energy expenditure. Conclusion: Our study elucidates a novel underlying mechanism by which VSG achieves its postoperative therapeutic effects through enhanced TGR5 signaling. (Hepatology 2016;64:760‐773)


Glucose-stimulated GLP-1 and insulin secretion in vivo
For insulin secretion test, mice were fasted overnight, and then gavaged with D-glucose (1.5 g kg -1 BW). Blood was collected (40 μL) by retro-orbital puncture at the indicated times. Next, serum insulin levels were measured using an ELISA kit (Crystal Chem Inc.). For in vivo plasma GLP-1 measurements, mice were fasted and gavaged with the dipeptidyl-peptidase (DPP)-IV inhibitor sitagliptin (3 mg kg -1 BW) 60 min prior to oral administration of D-glucose (1 g kg -1 BW). Blood was collected (80 μL) by retro-orbital puncture at the indicated times, transferred to EDTA tubes, and kept on ice. Plasma was collected by centrifugation and total GLP-1 levels were assessed using an ELISA kit (Millipore). Mice that were 13 weeks post-surgery or sham surgery were used.

Euglycemic-hyperinsulinemic clamps
Hyperinsulinemic euglycemic clamp was performed on VSG and sham-operated mice at 14 weeks post-surgery at Baylor College of Medicine, as described previously (3). One week prior to the clamp procedure, indwelling catheters were placed into the right internal jugular vein extending to the right atrium for sampling and infusions, respectively. After an overnight fast, [3-3 H] glucose (HPLC purified; PerkinElmer, Boston, MA) was infused at a rate of 0.05 μCi per min for 2 h to assess basal glucose turnover. After this basal period, hyperinsulinemic-euglycemic clamping was performed for 120 min with a primed/continuous infusion of human insulin (126 pmol kg -1 , prime; 18 pmol kg -1 ·min −1 , infusion) (Novo Nordisk, Princeton, NJ) to raise plasma insulin within the physiological range. Blood samples (10 μL) were collected at 10-to 20-min intervals for immediate measurement of plasma glucose, and 20% dextrose was infused at various rates to maintain plasma glucose at basal concentrations (≈6.7 mM). To estimate insulin-stimulated whole-body glucose fluxes, [3-3 H] glucose was infused at a rate of 0.1 μCi per min throughout the clamps and 2-deoxy-D-[1-14 C] glucose (PerkinElmer) was injected as a bolus at minute 75 of the clamp to estimate the rate of insulin-stimulated tissue glucose uptake. Blood samples (10 μl) for the measurement of plasma 3 H and 14 C activities were taken at the end of the basal period and during the last 45 min of the clamp.
Data were calculated as previously described (4,5). Briefly, whole body glucose and basal glucose production (mg/kg/min) were calculated by dividing the [3-3 H] glucose infusion rate by the plasma glucose-specific activity corrected to the body weight. During the clamp, hepatic glucose production was calculated as the difference between the tracer-derived rate of glucose appearance and the infusion rate of glucose.

Body fat analysis
Body fat was measured at 14 weeks post-surgery by 1 H magnetic resonance spectroscopy (Bruker BioSpin, Billerica, MA).

Indirect calorimetry and locomotor activity assays
Mice from each group were put in a comprehensive animal metabolic monitoring system (Columbus Instruments, Columbus, OH) at 14 weeks post-surgery. The volume of O 2 consumption and CO 2 production were recorded over a 24 h period. Energy expenditure is calculated as previously described (6). Locomotor activity was evaluated as previously described (7). Activity in the open field was collected by a computer-operated optical animal activity system (Versamax by AccuScan Instruments, Inc.). Total distance (locomotor activity), vertical activity (rearing measured by number of photobeam interruptions), and center distance (i.e., the distance traveled in the center of the arena) were recorded. Results are presented for the last 12 h of the light cycle, dark cycle, and the total activity during both dark and light phases.

Biochemical analysis of sera, liver tissues, and feces
After fasting overnight, mice were sacrificed in the early light phase and blood and tissue samples

Histological analysis of liver and adipose tissues
Liver and adipose tissue were fixed in 4% formalin and then embedded in OCT or paraffin. Oil red O staining and hematoxylin and eosin (HE) staining were performed as described (9).

Bile acid profile analysis
Bile acids composition was analyzed by ultra performance liquid chromatography-mass spectrometry with modifications (10). BAs were extracted from serum as follows: 150 μL of methanol was added to 50 μL of serum, and mixed by vortexing for 2 min. Next, samples were centrifuged at 20,000 g (4°C) for 10 min. BAs in feces were extracted using 50% methanol. Specifically, 450 μL of 50% methanol was added to 20 mg dry feces powder which was collected using metabolic cages for 24 h, and mixed by vortexing for 5 min. Samples were then centrifuged at 20,000 g (4°C) for 10 min. The entire supernatant was separated and evaporated. Residues were stored at -20°C and reconstituted in 100 μL of methanol-water (55:45v/v; containing a mixture of 5 mM ammonium acetate and 0.1% formic acid) before analysis. Fecal total bile acids levels were measured using the total bile acids kit (Wako Chemicals, Japan). The sample solution was centrifuged at 20,000 g for 10 min at 4°C, and a 5 μL of aliquot was injected for UPLC-MS analysis.
BAs were determined using the Waters ACQUITY ultra-performance lipid chromatograph system A and 45% B, which was linearly changed to 62% A and 38% B in 2.6 min, and then the proportion of B was increased to 80% for the next 8.8 min. The sample injection volume was 5 μL. MS analysis was performed using ZQ 2000 quadrupole spectrometry equipped with an ESI probe operated with Selective Ion Monitoring (SIM) in the negative-ion mode (Waters, MA, USA). The capillary and cone voltages were set at 3.0 and 55 V, respectively. The source temperature was 120°C, and the desolvation temperature was 300°C. The desolvation gas flow was set at 700 L h -1 , and the cone gas flow rate was set at 50 L h -1 . Data were acquired and processed using MassLynx 4.1 software.

Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)
Total RNA was extracted from liver, ileum, and BAT tissues of mice at 14 weeks post-surgery using

Sampling of cecal contents and microbiome analysis
WT and Tgr5 KO mice on HFD for 14 weeks after VSG and sham surgery were euthanized by CO 2 asphyxiation and cecal contents were collected, immediately. The cecal contents were weighted and stored at -80˚C before DNA extraction. The microbiome genomic DNA was extracted using QIAamp DNA stool Mini Kit (Qiagen, Valencia) according to manufacturer's instruction. Sequencing library preparation, sequencing, and data analysis were performed by the Integrative Genomics Core at City of Hope National Medical Center. The V3 and V5 variable regions of 16S rRNA gene were used for cecal microbiome analysis (11). Briefly, 30 ng of cecal microbiome DNA were used for two rounds of HiSeq2500. For data analysis, paired-end reads for V3 and V5 regions were merged into single reads, respectively. These reads were classified using Ribosomal Database Project (RDP) classifier with confidence level of 0.7, resulting in taxa for each sample. Taxa were retained for at least 50 reads in one sample. Hierarchical clustering was used to visualize distinct taxa using edgeR.

Statistical Analysis
All data are expressed as mean ± s.e.m. Statistical significance was analyzed using unpaired Student's t-test or one-way analysis of variance (ANOVA) with Dunn's post-test. The threshold of statistical significance was set at P < 0.05.