1. Volume 144, Issue 3, Pages 580-590.e7 (March 2013)
Weight-Independent Effects of Roux-en-Y Gastric Bypass on Glucose Homeostasis via Melanocortin-4 Receptors in Mice and Humans 
Juliet F. Zechner, Uyenlinh L. Mirshahi, Santhosh Satapati, Eric D. Berglund, Jari Rossi, Michael M. Scott, Christopher D. Still, Glenn S. Gerhard, Shawn C. Burgess, Tooraj Mirshahi, Vincent Aguirre 
Gastroenterology 
Volume 144, Issue 3, Pages e7 (March 2013)
DOI: /j.gastro
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2. Figure 1
RYGB induces weight loss in DIO mice. (A) Total body weight (left) during postoperative week 6 and expressed as a percentage of preoperative weight (right) in RYGB-treated (red), sham (blue), and CRWM-sham (green) (RYGB, 27; sham, 29; CRWM-sham, 12). (B) Reduced fat and lean mass in RYGB and CRWM-sham mice (n = 4 per group). (C) RYGB did not reduce food intake (n = 11–25 per group). Daily calorie restriction required to weight-match CRWM-sham mice (n = 12) to RYGB mice is shown in green. (D) RYGB reduced feeding efficiency (weight gain per kJ consumed). Feeding efficiency remained substantially reduced after adjusting for fecal energy losses (adjusted feeding efficiency) (n = 11–25 per group). (E) Energy expenditure was increased by 33% after RYGB (n = 11–25 per group). (F) Calorie absorption was reduced slightly after RYGB (n = 8 per group). *P < .05 vs sham, ++P < .05 vs CRWM-sham.
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3. Figure 2
RYGB improves hepatic glucose homeostasis in DIO mice. (A) RYGB reduced fasting glucose (left), fasting insulin (middle), and HOMA-IR (right) (n = 4–11 per group). (B) RYGB improved oral glucose tolerance, (C) reduced glucose-stimulated plasma insulin, and (D) improved insulin tolerance, presented as the percentage of baseline glucose to control for differences in baseline glucose (n = 5–6 per group). (E) RYGB reduced basal endogenous glucose production, gluconeogenesis, and glycogenolysis as measured during week 6 using in vivo NMR metabolic flux analysis (n = 5–9 per group). (F) RYGB increased insulin-stimulated IRS2 tyrosine phosphorylation and protein expression (n = 4 per group). IP, immunoprecipitate; IB, immunoblot. *P < .05 vs sham, ++P < .05 vs CRWM-sham.
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4. Figure 3
MC4Rs in autonomic neurons mediate effects of RYGB on energy expenditure and body weight. (A) RYGB-induced weight loss was attenuated in both MC4R-null (left) and Phox-MC4R mice (middle). In contrast, RYGB induced substantial weight reduction in ChAT-MC4R mice (right). The weight reduction observed in ChAT-MC4R mice was comparable with that seen in DIO mice (Supplementary Figure 3). Body weight is presented as total (bottom) and also is expressed as a percentage of preoperative weight (top), to facilitate comparison of the relative effects of RYGB across genotypes (MC4R-null, 22–24 per group; Phox-MC4R and ChAT-MC4R, 6–22 per group). (B) RYGB failed to increase energy expenditure in MC4R-null and Phox-MC4R mice (MC4R-null, 10–25 per group; Phox-MC4R, 6–22 per group). In contrast, RYGB increased energy expenditure in ChAT-MC4R mice, consistent with their weight loss (ChAT-MC4R mice, 6–22 per group). (C) RYGB reduced body weight of obese MC4R-Het mice by 25% during week 6 (n = 7–10 per group). Consistent with their weight reduction after RYGB, energy expenditure was increased in MC4R-Het mice (n = 7–10). (D) RYGB improved fasting glucose, fasting insulin, and HOMA-IR in MC4R-Het mice (n = 7–10). *P < .05 vs sham.
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5. Figure 4
MC4Rs in parasympathetic vagal motor neurons mediate effects of RYGB on glucose homeostasis independent of changes in body weight. (A–F) Left panels: RYGB significantly reduced fasting glucose in MC4R-null mice, but failed to produce statistically significant improvements in other measures of glucose homeostasis: (B) insulin, (C) HOMA-IR, (D) glucose tolerance, (E) glucose-stimulated plasma insulin, and (F) insulin tolerance, presented as the percentage of baseline glucose to control for differences in baseline glucose (n = 6–14 per group). (A–F) Middle panels: in contrast, RYGB reduced (A) fasting glucose and (B) insulin, and improved (C) HOMA-IR, (D) oral glucose tolerance, and (F) insulin tolerance in Phox-MC4R mice, despite a similar blunted weight reduction as seen in MC4R-null mice. RYGB did not reduce their glucose-stimulated plasma insulin (n = 6–7 per group). (A–F) Right panels: consistent with their substantial weight reduction, (A) fasting glucose, (B) fasting plasma insulin, and (C) glucose-stimulated plasma insulin were reduced and (D) oral glucose tolerance improved in RYGB-treated ChAT-MC4R mice and CRWM-sham ChAT-MC4R mice (n = 6–7 per group). *P < .05 vs sham.
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6. Figure 5
RYGB induces resistance to HFD-induced weight-gain. (A) RYGB-treated MC4R-Het mice maintained after surgery on regular chow (RC) fail to gain weight upon challenge with HFD for 8 days compared with sham mice. In contrast, sham- and RYGB-treated MC4R-null mice gained substantial and equivalent weight (n = 4–11 per group). (B) Food intake was increased on HFD in RYGB-treated and sham-operated mice of both genotypes (n = 4–11 per group). (C and D) Sham-operated MC4R-Het and MC4R-null mice and RYGB-treated MC4R-null mice assimilated all excess consumed energy on HFD as body mass at an increased metabolic efficiency. Energy expenditure is reduced in sham-operated MC4R-Het mice as a compensation for their weight gain on HFD. In contrast, RYGB-treated MC4R-Het mice failed to increase their metabolic efficiency on HFD. In addition, their increased energy expenditure persisted and remained increased vs sham MC4R-Het mice (n = 4–11 per group). *P < .05 RYGB vs sham or HFD vs RC.
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7. Figure 6
MC4Rs mediate beneficial effects of RYGB on glucose homeostasis in humans with rare MC4R variants. (A) Postoperative weight, expressed as a percentage of preoperative body mass index (%BMIs), was equivalent in MC4R(I251L) carriers (green; n = 26), rare variant carriers linked to obesity (red; n = 18), and noncarriers (black; n = 1399) at 2 weeks after surgery (range, 10–20 days). (B) Resolution of type 2 diabetes (T2D), defined as cessation of all antidiabetic medications, in carriers of the MC4R(I251L) variant (green; 8 of 9 patients), carriers of rare MC4R variants (red; 4 of 8), and noncarriers (black; 399 of 597) at 2 weeks, 2 months, and 6 months after RYGB.
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8. Supplementary Figure 1
RYGB induces weight loss in mice (related to Figure 1). (A) Physical activity, as measured during week 5, was unchanged after RYGB (n = 5–6 per group). (B) Fasting serum leptin was comparably reduced in RYGB and CRWM-sham mice (n = 9–11 per group). (C) Body weight, expressed as a percentage of preoperative weight, was reduced substantially in RYGB-treated DIO mice (red) compared with sham (blue) by postoperative week 1 as a result of perioperative stress and calorie restriction. Acute convalescence ensues for the subsequent 3 weeks because sham-operated mice do not fully regain their preoperative weight until week 4 (RYGB, 27; sham, 29). (D) Schematic and photograph of RYGB in mice. After reconstruction, the biliopancreatic (BP) limb and Roux limb each comprise 12.5% of total intestinal length and the common channel (common) comprises 75%. After reconstruction, ingested nutrients pass from the esophagus, through the proximal gastric pouch, and into the Roux limb. Digestive secretions from the excluded gut drain through the BP limb to meet ingested nutrients at the start of the common channel. G-J, gastrojejunal anastomosis. *P < .05 vs sham.
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9. Supplementary Figure 2
RYGB fails to improves glucose homeostasis in skeletal muscle and adipose tissue of DIO mice (related to Figure 2). (A) Peripheral glucose uptake into skeletal muscle (SKM) and white adipose tissue (WAT), as measured using 2-deoxy-glucose (2-DG), was unchanged after RYGB (n = 4–5 per group). (B) Insulin-stimulated insulin receptor substrate-1 (IRS1) tyrosine phosphorylation and IRS1 expression in SKM was unchanged after RYGB (n = 4 per group).
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10. Supplementary Figure 3
RYGB induces equivalent weight loss compared with conventional sham-operated mice and sham-operated mice weight-matched during postoperative recovery (related to Figure 3). To control for differences in susceptibility to perioperative stress (weight loss and calorie restriction) and ensure that the observed differences in energy expenditure and body weight in MC4R models during week 6 were exclusively caused by RYGB-induced differences in energy balance, RYGB-treated and sham-operated mice were weight-matched during postoperative recovery. The group of DIO mice in this figure was generated in parallel with the MC4R mouse models as a positive control for RYGB-induced weight reduction. As seen in this figure, RYGB-treated DIO mice are equivalently weight-reduced by week 6 whether compared with conventional sham-operated mice (provided high-fat diet ad libitum after postoperative recovery; sham) or sham-operated mice that were weight-matched to RYGB-treated mice during recovery (RM-sham) (sham, 4; RM-sham, 6; RYGB, 4). *P < .05 vs sham, #P < .05 vs RM-sham.
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11. Supplementary Figure 4
RYGB improves glucose homeostasis and pyruvate tolerance in Phox-MC4R and ChAT-MC4R mice (related to Figure 4). (A) Despite persistent obesity (left), HOMA-IR is reduced in Phox-MC4R mice compared with MC4R-null mice (right) (n = 5 per group). (B) RYGB reduces glucose excursion after pyruvate administration in DIO, ChAT-MC4R, and Phox-MC4R mice (n = 5–8 per group). *P < .05 vs sham, †P < .05 vs MC4R-null.
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12. Supplementary Figure 5
RYGB-treated mice are resistant to HFD-induced positive energy balance and weight gain (related to Figure 5). (A) Calorie absorption in RYGB-treated MC4R-null and MC4R-Het mice on regular chow (RC) and during acute (8-day) challenge with HFD (n = 5–9 per group). (B) Sham- and RYGB-treated MC4R-null mice gained a substantial and equivalent amount of body weight when challenged with HFD for 3 weeks (n = 10–14 per group). *P < .05 vs RYGB, RC.
Gastroenterology  , e7DOI: ( /j.gastro )
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13. Supplementary Figure 6
RYGB induces equivalent weight loss in carriers and noncarriers of rare MC4R variants (related to Figure 6). (A) Carriers of rare MC4R variants (red, rare) and noncarriers (black, wild type [WT]) show equivalent weight loss after RYGB. The percentage change in body mass index (%BMIs) is shown graphed against the perioperative month; the hashed line at time 0 represents the time of surgery. Purple (carriers) and gray (noncarriers) lines represent best-fit curve analysis of change in %BMIs for each group. (B and C) Because some rare MC4R variants have been reported in nonobese subjects, we further subdivided the patients into those carrying variants found only in obese subjects and those variants that have been identified previously in lean and obese patients. (B) Weight loss among these 2 groups compared with noncarriers. (C) Three-month running average for weight loss in the 3 groups. A list of the rare variants identified in this study is shown in the inset.
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