Leptin Deficient Ob&Sol;Ob Mice and Diet-Induced Obese Mice

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ORIGINAL ARTICLE Leptin deﬁcient ob/ob mice and diet-induced obese mice responded differently to Roux-en-Y bypass surgery

Z Hao1, H Münzberg1, K Rezai-Zadeh1, M Keenan2, D Coulon2,HLu3, H-R Berthoud1 and J Ye1

OBJECTIVE: Weight regain contributes to the therapeutic failure in 15–20% of type 2 diabetic patients after Roux-en-Y gastric bypass surgery (RYGB), and the mechanism remains largely unknown. This study was conducted to explore the mechanism of weight regain. RESEARCH DESIGN: Wild-type (WT) diet-induced obese (DIO) mice were used to mimic human obesity, and ob/ob mice were used for leptin deficiency-induced obesity. Two groups of mice were compared in weight regain for 10 months after RYGB. Weight loss, food intake, fecal energy loss and energy expenditure were monitored in the study of weight regain. Fasting insulin, insulin tolerance and homeostatic model assessment-insulin resistance were tested for insulin sensitivity under the weight regain. Weight loss from RYGB and calorie restriction was compared for the impact in insulin sensitivity. RESULTS: In WT mice, RYGB induced a sustained weight loss and insulin sensitization over the sham operation in this 10-month study. However, RYGB failed to generate the same effects in leptin-deficient ob/ob mice, which suffered a weight regain over the pre-surgery level. In ob/ob mice, body weight was reduced by RYGB transiently in the first week, recovered in the second week and increased over the baseline thereafter. Weight loss was induced by RYGB relative to that of sham mice, but the loss was not sufficient to keep body weight below the pre-surgery levels. In addition, insulin sensitivity was not improved by the weight loss. The response to RYGB was improved in ob/ob mice by 2 weeks of leptin treatment. Weight loss from calorie restriction had an equivalent effect on insulin sensitization compared with that of RYGB. CONCLUSION: Those data demonstrate that ob/ob mice and DIO mice responded differently to RYGB surgery, suggesting that leptin may be one of the factors required for RYGB to prevent weight regain and diabetes recurrence. International Journal of Obesity (2015) 39, 798–805; doi:10.1038/ijo.2014.189

INTRODUCTION establishes a new lower level of body weight that is actively Roux-en-Y gastric bypass surgery (RYGB) is among the most defended after RYGB. This function is classically ascribed to the effective bariatric surgeries in producing sustained decrease in hypothalamus, where leptin and other feedback signals regulate body weight and remission of type-2 diabetes.1,2 In addition, RYGB energy balance by orchestrating the necessary changes in energy 17 improves most of the deleterious comorbidities associated with intake and expenditure. It is not clear if leptin contributes to the severe obesity.2 Despite intensive efforts, the critical mechanisms defense of post-surgery low body weight. responsible for these beneficial effects of RYGB have not yet been Leptin resistance contributes to hyperphagia and hypometabo- clearly identified. Ultimately, RYGB must change patterns of lism in obesity. Serum leptin is increased by obesity and reduced 5,18 humoral and neural signaling between the gastrointestinal system by RYGB, suggesting an increase in leptin sensitivity. However, and other organs (such as the brain, liver, adipose tissue and so it is largely unknown if leptin has a role in the control of weight on) involved in the energy regulation.3,4 Much attention has been regain after RYGB.4 In two early studies of leptin receptor-deficient given to changes in circulating gut hormones, such as glucagon- Zucker obese rats, RYGB induced weight loss and insulin like peptide 1 (GLP-1),5,6 peptide tyrosine tyrosine (PYY)7,8 and sensitization immediately post surgery.19,20 However, the long- ghrelin9 in mechanistic studies. However, recent findings in term effect of RYGB was not examined in these studies for weight various rodent models have provided relatively little support for regain. their critical roles in the effects of RYGB,10–14 although synergistic The present study was designed to test the impact of leptin in actions between these hormones and other factors have not been the long-term effects of RYGB with a focus on weight regain and excluded. glycemic control. We have recently established a valid mouse It has also become clear that the negative energy balance after RYGB model, characterized by a small gastric pouch, sustained RYGB does not result from an inability to increase energy intake. If suppression of body weight below the pre-surgical level, and low RYGB rodents were properly stimulated, food intake can easily be mortality.10,21 Here, we take advantage of this model to study doubled leading to substantial weight regain.10,15,16 Also, 15–20% weight regain and glycemic control for 10 months after RYGB in of RYGB patients return to pre-surgical levels of food intake and diet-induced obese (DIO) mice and leptin-deficient ob/ob. In regain body weight in spite of their rearranged gut.1 One addition, leptin-replacement therapy was tested in ob/ob mice for interpretation of these observations is that successful surgery 2 weeks at 25 weeks after RYGB.

1Pennington Biomedical Research Center, Louisiana State University System, Baton Rouge, LA, USA; 2Louisiana State University Agricultural Center, Baton Rouge, LA, USA and 3Department of Endocrinology & Metabolism, The Fifth Affiliated Hospital of Sun Yat-sen University, Zhuhai, Guangdong, China. Correspondence: Dr J Ye, Antioxidant and Gene Regulation Lab, Pennington/LSU, Pennington Biomedical Research Center, Louisiana State University System, 6400 Perkins Road, Baton Rouge, LA 708080, USA. E-mail: [email protected] Received 25 August 2014; revised 8 October 2014; accepted 18 October 2014; accepted article preview online 28 October 2014; advance online publication, 25 November 2014 Leptin in RYGB surgery Z Hao et al 799 MATERIALS AND METHODS limb and the biliopancreatic limb were ~ 5–6 cm in length. The intestine Obese mice was arranged in an ‘S’ position to avoid intestinal obstruction before All experiments were approved by the Institutional Animal Care and Use closing of the abdominal cavity. In sham operation, the perigastric Committee of Pennington Biomedical Research Center. Animals were ligaments were cut, and a 3-mm incision was made in the stomach closed housed under conventional conditions at the Pennington Animal Facility. with a titanium clip. The jejunum was transected 2 cm distal to the In the study design, wild-type (WT) male C57BL/6J mice were used in DIO ligament of Treitz, and the two cut ends were anastomosed. Standard group and chow-fed group (lean control). DIO mice were generated by aseptic procedures were used throughout. In the first 24 h after the feeding 6-week-old mice with a high-fat diet (HFD, D12331 diet, 58% operation, the mice were put in regular shoe box cages on a heating calories from fat, Research Diets Inc.) for 14 weeks. Then, the DIO mice pad at 35 °C. The mice were given 0.8 ml per mouse of saline − were divided into three subgroups (n = 7): DIO+RYGB, DIO+sham and DIO subcutaneously and carprofen (5 mg kg 1, sc) for analgesia immediately +weight matched. The lean control mice (n = 7) were fed regular chow diet after surgery. The mice had access to water and solid diet right (11% calorie from fat, 5001 LabDiet) ad libitum. After surgery, DIO mice after surgery. Post-surgical survival rates were 90% in DIO mice and 80% were fed a medium-fat diet (25% calorie from fat, 5015 LabDiet) to reflect in ob/ob mice. diet condition in patients. DIO+weight-matched group were subject to food restriction at 14 weeks of HFD feeding to induce an identical weight loss to that of DIO+RYGB group. Forty C57BL/6 mice were used to Physiological parameters generate the models and 28 mice were left after excluding unqualified Body weight, body composition and food intake were measured in this DIO mice and dead mice in surgery. Male ob/ob mice (26 mice) in C57BL/6 study as described elsewhere.21 The body weight was determined daily in gene background were purchased from the Jackson Laboratory at the first 2 weeks and then weekly after the surgery. Body composition was – 4 7 weeks in age and fed the regular chow diet ad libitum before and determined using nuclear magnetic resonance in each cohort at times after the surgery. indicated in Figure 1. Food intake was determined in single-housed mouse manually or using the metabolic chamber for 1–2 weeks. Surgical procedures RYGB surgery was performed in DIO mice at 14 weeks on HFD with a body Energy expenditure weight of 46 ± 5 g. The surgery was performed in ob/ob mice at 6 week (35 g) and 10 week (50 g) in age. The surgery operation was conducted as Indirect calorimetry measurement was performed with the Comprehensive described in detail earlier.21 In brief, animals were fasted 4–6 h before the Laboratory Animal Monitoring System (Columbus Instruments, Columbus, 22 operation and anesthesia was administrated with isoflurane inhalation. In OH, USA) for individually housed mice as described in detail earlier. After RYGB surgery, the anterior and posterior left gastric vessels, as well as the 96 h of adaptation, the data for oxygen consumption (VO2), carbon dioxide esophageal vessels, were ligated and cut. A small gastric pouch at 5% of production (VCO2), respiratory exchange ratio and spontaneous physical the total gastric volume was generated and anastomosed with the activity were simultaneously recorded and analyzed.22 The data of day and jejunum by 16–18 interrupted stiches with 11–0 nylon suture. The Roux night are presented.

WT mice ob/ob mice (6-wk old group) ob/ob mice (10-wk old group) Chow Ctrl Sham 70 90 70 RYGB Wt match NMR 60 NMR 80 ITT 60 NMR and hormone 70 50 50 60 40 40 50 40 30 30 Food EE 30 Sham 20 20 Sham Intake EE EE Food 20 Leptin

ITT Body Weight (g) RYGB (g) Weight Body Body Weight (g) Body Weight 10 RYGB 10 Choice 10 Injection 0 0 0

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BW Change (%) -30% * 5 5 -40% Week 1 Week 3 Week 8 0 0 -50% Chow Ctrl Sham RYGB Wt match Sham RYGB Figure 1. Body weight and fat content after RYGB. (a) Body weight of WT mice (DIO). The mice were divided into four groups: chow diet control (Chow Ctrl), Sham-operated, RYGB and weight matched (Wt match). The mice were fed HFD before surgery and the breeder chow (medium fat) diet after surgery. RYGB was performed in the mice around 14 wks on HFD. Body weight was monitored weekly in the ﬁrst 4 wks and then biweekly for 39 wks. Body composition and energy expenditure (EE) were tested at 7 wks. Food choice test was conducted at 16 wks, where the mice had free access to the HFD and the regular chow diet for 6 wks. ITT was tested at 35 wks (N = 7 Chow Ctrl, 7 Sham, 7 RYGB, 7 Wt match). (b) ob/ob mice (6-wk-old group, n = 5). RYGB was performed in the mice at 6 wks in age. Body weight was monitored for 39 wks with composition test at 3 wks, food intake at 6–7 wks, and EE at 8–9 wks post surgery. (c) ob/ob mice (10-wk-old group, n = 6). RYGB was performed in the mice at 10 wks in age. The body weight was monitored for 27 wks with ITT at 11 wks, EE at 19 wks and leptin treatment at 25 wks. (d) Percentage weight loss after RYGB. WT and ob/ob mice were compared in percentage weight loss after RYGB at three time points as indicated. (e) Body fat content of WT mice. Body composition was monitored at 8 wks after RYGB (N = 8 Chow, 7 Sham, 8 RYGB, 7 Wt match). (f) Body fat content of ob/ob mice (6-wk-old group). The body composition was measured at 3 wks after RYGB (n = 5). The data are expressed as mean ± s.e. *Po0.05, ***Po0.001 vs before surgery or calorie restriction by t-test.

© 2015 Macmillan Publishers Limited International Journal of Obesity (2015) 798 – 805 Leptin in RYGB surgery Z Hao et al 800 Fecal calorie content observed in the sham ob/ob mice, suggesting that RYGB may Fecal samples were collected for each mouse after surgery and stored in increase ob/ob mouse sensitivity to stressful challenges. These − 80 oC. The caloric content was determined using a Parr 1266 Isoperibol data suggest that in contract to that in WT mice, RYGB could not Bomb Calorimeter (Parr Instrument Company, Moline, IL, USA). Approxi- prevent weight regain in ob/ob mice. mately 1 gm fecal samples were freeze dried to remove all moisture. For Body fat reduction accounts for the surgery-induced weight – each 1 gm fecal sample, an aliquot of ~ 0.2 0.3 grams were processed in loss. Fat mass was measured using nuclear magnetic resonance in the bomb calorimeter. Hexadecane (Sigma-Aldrich, St Louis, MO, USA), a WT and ob/ob mice. DIO mice exhibited 40% reduction in fat mass combustible spiking agent, was added to each sample to ensure complete combustion of the entire fecal sample. A heat of combustion value is 8 weeks after RYGB (Figure 1d). A portion of the reduction was a obtained for each fecal sample in calories per gram after 8 min. The value result of switching from high-to-medium-fat diet after surgery, as was corrected for combustion heat of the spiking agent automatically and about 9% weight reduction was also observed in sham-operated a final gross heat value was used for the fecal calorie. Each sample was run WT mice. HFD was replaced by the medium-fat diet (breeder in duplicate trials in order to obtain an average value. chow) after surgery to reflect diet preference in RYGB patients and to maintain the adiposity. ob/ob mice did not exhibit a reduction Insulin, leptin and insulin tolerance test in fat mass after RYGB as indicated by data in the first cohort Blood was collected from mice through retro-optical bleeding at 6 weeks (Figure 1f). Instead, fat mass was increased over the pre-surgery post surgery after overnight fasting. Serum insulin and leptin were tested levels in both sham and RYGB ob/ob mice. The data suggest that using multiplex kit (MMHMAG-44K, Millipore Corporation, 28820 Single RYGB fails to reduce fat mass below pre-surgery levels persistently Oak Drive, Temecula, California). Insulin tolerance was performed in each in ob/ob mice. cohort at times indicated in Figure 1. The test was conducted with peritoneal injection of insulin at 0.7 U Kg − 1 after 4 h fasting. MOHA-IR was calculated with a formula: homeostatic model assessment-insulin resis- NRYGB increased energy expenditure in WT, but not ob/ob mice tance (mg dl − 1) = glucose × insulin/405. Energy intake, fecal energy loss and energy expenditure were examined at 5–8 weeks after RYGB when the body weight was Leptin injection stabilized at the lower level in DIO mice. Daily food intake per mouse was not significantly reduced by RYGB in DIO mice For long-term leptin effects on RYGB ob/ob mice, leptin was intraper- itoneally injected twice daily over 2 weeks at a dosage 1 mg kg − 1 per day. (Figure 2a). A modest reduction was observed in ob/ob mice after Food intake and body weight was monitored daily using the BioDaq cage RYGB (Figure 2a), but the change is not significant. Fecal calorie system. content was significantly increased by RYGB in both WT and ob/ob mice (Figures 2b and c). Serum leptin was significantly decreased Statistical analysis in WT mice by RYGB or calorie restriction in weight-matched ’ group (Figure 2d). Energy expenditure per lean body mass was In this study, values are presented as mean ± s.e.m. Student s t-test was fi used in most data analysis for body weight, insulin tolerance, energy signi cantly increased by RYGB in WT, but not in ob/ob mice expenditure, substrate utilization and body temperature. Two-way ANOVA (Figures 3a and b). To the contrary, energy expenditure was was performed in the study of leptin sensitivity followed by a post hoc decreased by RYGB in ob/ob mice (Figure 3b). The decrease was analysis. P-values o0.01 were considered significant. observed when the data were normalized with either lean body mass (Figure 3b) or whole-body weight (Figure 3c). The decrease was significant at day and night time when normalized with RESULTS lean mass, but only night time when normalized with whole- RYGB prevented weight regain in WT, but not ob/ob mice body mass. The difference between WT and ob/ob mice In the DIO model that was generated in WT C57BL/6 mice, RYGB suggests that RYGB was not able to induce energy expenditure surgery was performed at 14 weeks on HFD when the body in ob/ob mice. weight was around 50 g. DIO mice exhibited a persistent weight loss after RYGB (Figure 1a). Most weight reduction was observed in RYGB improved insulin sensitivity in WT, but not ob/ob mice fi the rst 2 weeks post surgery and the weight loss was about 35% Insulin sensitivity was examined with fasting insulin and insulin at the end of 3 weeks. At this time, WT mice lost all of the weight tolerance in this study. In WT mice, serum insulin was increased by gain on HFD. Thereafter, the RYGB mice shared an identical body obesity five- to sixfold (Figure 4a, sham). The insulin was restored weight to the chow diet group in the entire 39-week study to the level of lean control mice by RYGB at 6 weeks post surgery without weight regain (Figure 1a). ob/ob mice were tested in RYGB (Figure 4a, RYGB). The same reduction was observed in weight- in two cohorts at different ages, 6 weeks (35 g) and 10 weeks matched DIO mice (Figure 4a, Wt-matched). However, the RYGB (50 g), to determine the impact of pre-surgery body weight in the fi effect was not observed in ob/ob mice (Figure 4b). In insulin surgery effect. In the rst cohort, a transient weight loss was tolerance test, a similar improvement was observed in WT mice observed in the 6-week group with 4 g weight reduction in the fi fi after RYGB or calorie restriction (Figures 4c and d). A signi cant rst week post-surgery (Figure 1b). Interestingly, a persistent improvement was not observed ob/ob mice after RYGB (Figures weight gain was observed in the mice thereafter. The body weight 4e and f). The data suggest that RYGB improves insulin sensitivity became 20% above the pre-surgical levels 8 weeks post-surgery – in WT, but not in ob/ob mice. (Figures 1b d). Compared with sham-operated mice, RYGB mice To test the weight-dependent effect of RYGB on insulin exhibited less weight regain after surgery. In the second cohort, a sensitivity, weight-matched mice were compared with RYGB mice similar pattern of weight regain was observed in 10-week-old ob/ in insulin sensitivity, which was conducted in WT mice (Figure 1). ob mice although the pre-surgery body weight was much higher After calorie restriction, the weight-matched group exhibited (Figure 1c). Percentage weight loss was compared between WT identical improvement in insulin sensitivity to RYGB group in and ob/ob mice at 1, 3 and 8 weeks post surgery. Persistent terms of changes in fasting insulin and insulin tolerance (Figures weight loss beyond pre-surgery levels was only observed in WT 4a–c), suggesting that weight loss from RYGB is critical in the mice, but not in ob/ob mice after RYGB (Figure 1d). ob/ob mice maintenance of insulin sensitivity. exhibited some weight loss after 10 weeks of RYGB in the second cohort, which might be a consequence of stress responses from the tests, such as insulin tolerance test and metabolic chamber Leptin restored RYGB effects in ob/ob mice study. Even though, the body weight remained above the pre- To test leptin in the effects of RYGB, we administrated leptin in surgery levels (Figure 1c). Such a weight reduction was not ob/ob mice (1 mg kg − 1 per day, i.p.) in a 2-week study. Body weight,

Food intake WT mice 25 *** Chow Ctrl Sham RYGB 5 20 4

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1 Serum leptin (pg/ml) 500 Fecal Energy (kcal/g)

0 0 Chow Sham RYGB Wt Sham RYGB match Figure 2. Energy intake and fecal calorie content after RYGB. (a) Energy intake of WT and ob/ob mice. The mice were single housed and food intake was measured for 3 days after 4 day adaptation. The food intake was conducted at 6 wks post surgery and expressed in kcal per day per mouse (N = 5–6). (b) Fecal calorie content in WT mice (N = 5–6). The test was done at 39 wks post surgery with dry feces. (c) Fecal calorie content in ob/ob mice of the 9-wk-old group (n = 5). The test was done at 23 wks post surgery. (d) Serum leptin. The test was conducted in fasting blood at 6 wks after RYGB. The data are expressed as mean ± s.e. ***Po0.001 vs sham control by t-test.

WT mice ob/ob mice (Lean mass) ob/ob mice (Whole body)

Chow Ctrl Sham 14 Sham RYGB 25 Sham RYGB RYGB Wt match 12 20 20 10 15 15 8 6 10 10

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Chow Ctrl Sham 14 Sham RYGB RYGB Wt match 20 Sham RYGB 30 12 *** *** *** * 25 *** *** 10 *** 15 *** *** *** 20 *** *** *** 8

l/hr/kg) 10 15 6 10 ( Kcal/hr/kg )

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EE (Kcal/hr/kg) 5 EE (Kca 5 2

0 0 0 Day Night Day+Night Day Night Day+Night Day Night Day+Night Figure 3. Energy expenditure after RYGB. (a) Energy expenditure of WT mice. The mice were single housed and energy expenditure was normalized with lean body mass (n = 7–8). (b) Energy expenditure in ob/ob mice of the ﬁrst cohort (n = 5). The test was conducted at 8 wks post surgery and the result was normalized with lean body mass. (c) Energy expenditure of ob/ob mice normalized with whole-body weight. The data are expressed as mean ± s.e. *Po 0.05. ***Po0.001 vs sham by t-test.

WT mice ob/ob mice 4000 9000 *** 3500 8000 *** 7000 3000 *** 6000 2500 5000 2000 4000 1500 3000 1000 Insulin (pg/ml) 2000

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WT mice WT mice Chow Ctrl Sham 10,000 150 RYGB Wt match *** 8,000 *** 100 6,000 ** 4,000

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0 0 0' 30' 60' 90' 120' Sham RYGB Figure 4. Insulin sensitivity. (a) Fasting insulin of WT mice. The fasting blood insulin was examined at 6 wks after RYGB. (b) Fasting insulin in ob/ob mice of the 6-wk-old group. (c) Insulin tolerance test (ITT) in WT mice. Insulin was administrated at 0.7 U kg − 1 by i.p. injection. (d) Area above the curve for ITT in WT mice. (e)ITTinob/ob mice (10-wk group). ITT was conducted at 11 wks post surgery. (f) Area above the curve (AAC) of ob/ob mice. The data are expressed as mean ± s.e. **Po0.01, ***Po0.001 vs sham by t-test. In this study, mouse number is n = 7 (WT), and n = 6(ob/ob).

food intake and insulin sensitivity were monitored in the study. similarity with RYGB surgery in humans, including the ~ 35% Reduction in body weight and food intake was observed in both sustained weight reduction and insulin sensitization for up to 39 sham and RYGB mice following leptin treatment (Figures 5a and b). (10 months) weeks (equivalent to ~ 20 years in humans) and low The weight reduction was identical in mass (g) in the two groups. mortality. These effects are coupled with a negative energy However, percentage weight loss was significantly larger in the balance in DIO mice after RYGB. RYGB group because of lower body weight. The reduction in food ob/ob mice failed to exhibit substantial weight loss after RYGB intake was significantly more (51%) in the RYGB group compared surgery. The mice exhibited a transient weight loss of ~ 10% in the with the sham group (Figure 5c). Insulin sensitivity was measured first week post surgery, but the loss disappeared in 2 weeks after by fasting insulin and homeostatic model assessment-insulin the surgery, which was associated with a sustained weight regain. resistance. More Improvement was observed in RYGB mice Although the body weight exceeded pre-surgery level after after the leptin treatment (Figures 5d–f). Fasting blood glucose 3 weeks in RYGB group, the weight regain was much less than was not significantly different in the two groups of mice after the that of sham group, suggesting that RYGB delayed weight gain in leptin treatment (Figure 5e). These data suggest that leptin ob/ob mice. The reduction in nutrient absorption may contribute injection significantly improves ob/ob mouse response to RYGB to the delay in RYGB mice. However, the nutrient reduction was surgery. not sufficient to keep the body weight below the pre-surgery level in ob/ob mice. In clinical practice, weight reduction beyond the pre-surgery level is used to determine the therapeutic effects of DISCUSSION bariatric bypass surgeries.1 A systematic review and meta-analysis Our mouse model of RYGB allowed us to compare ob/ob mice and of large sets of published data suggest that RYGB therapy fails to DIO mice in RYGB surgery. The model is characterized by the close improve glucose metabolism in 15–20% obese patients.1 In light

Weight loss rate Food reduction rate Cumulative food intake

5% 20% 35 Sham RYGB 0% 0% 30 -5% ** * * 25 -20% -10% *** * 20 -15% -40% *** ** *** 15 -20% Sham -60% ** * 10 -25% RYGB Weight change (%) Food Intake (g)

Intake change (%) -80% -30% 5 -35% -100% 0 02468101214 02468101214 Sham RYGB

Fasting inulin Fasting glucose HOMA-IR 3500 90 40 3000 80 70 2500 30 60 2000 50 20 1500 40

30 HOMA-IR 1000 20 10 Serum Insulin (pg/ml) 500 ** Blood Glucose (mg/dl) 10 0 0 0 Sham RYGB Sham RYGB Sham RYGB Figure 5. Leptin effect in ob/ob mice. Leptin was administrated in ob/ob mice (9-wk group) at 25 wks after RYGB. (a) Weight loss rate. The reduction in body weight was calculated in percentage relative to the pre-treatment level. (b) Reduction rate in daily food intake. The reduction was in percentage relative to the pretreatment level. (c) Cumulative food intake. The number represents a sum of daily food intake over the 2 wks of leptin treatment. (d) Fasting blood insulin after the leptin treatment. (e) Fasting blood glucose after the leptin treatment. (f) MOHA-IR in ob/ob mice after the leptin treatment. The data are expressed as mean ± s.e. (n = 5-6). *Po0.05, *Po0.01, ***Po0.001 vs sham by t-test. of this criterion, our data suggest that the leptin signal may be to ob/ob mice as well. In leptin therapy, the more improvement in required for prevention of weight regain in RYGB. insulin sensitivity was coupled with a larger reduction in food RYGB fails to enhance energy expenditure in ob/ob mice. intake in RYGB ob/ob mice, supporting the role of negative energy Leptin, a hormone produced by adipose tissue, inhibits food balance in the control of insulin sensitization. Several recent intake and stimulates energy expenditure. Lack of leptin makes reports suggest that the negative energy balance from calorie ob/ob mouse an excellent model to test leptin in RYGB. Although restriction has an equivalent effect to RYGB on improving insulin the hypothalamic circuitry is altered in ob/ob mice,23 the neuron sensitivity in patients.6–28 Future experiments with different doses response to leptin is not influenced by the alteration24 and leptin of leptin and clamp techniques will be necessary to shed light on is able to restore the metabolic disorders in ob/ob mice.25 Energy the mechanisms of insulin sensitization in ob/ob mice by RYGB. expenditure was increased by RYGB in DIO mice, but not in ob/ob This study suggests that the intact function of leptin circuitry mice, suggesting that lack of leptin is responsible for the low may be required for RYGB effects. The leptin signaling pathway energy expenditure in ob/ob mice. Leptin sensitivity was includes multiple proteins such as leptin receptor, melanocortin 4 decreased by obesity, and improved by RYGB as suggested by receptor (MC4R), agouti-related protein and peptide tyrosine serum leptin alteration in DIO mice in this study. The improvement tyrosine. In the pathway, leptin binds to the receptor in is translated into sustained weight loss in DIO mice, but not in proopiomelanocortin neurons to induce the secretion of ob/ob mice. Leptin is required by RYGB to enhance energy melanocyte-stimulating hormone (α-MSH) and β-MSH. α-MSH expenditure and decrease body weight. activates MC4R to increase cAMP in the neurons for sensation of ob/ob mice failed to show persistent improvement in insulin satiety. Leptin also inhibits expression of neuropeptide Y and sensitivity after RYGB. In ob/ob mice, insulin sensitivity was tested agouti-related protein, which induce food intake. Agouti-related with insulin tolerance test and homeostatic model assessment- protein stimulates appetite through the inhibition of MC4R.29 insulin resistance. Blood glucose was increased instead of Mutation in any of the signaling proteins may lead to functional decreasing after insulin injection in insulin tolerance test deficiency of the leptin signaling pathway. Our study suggests (Figure 4e). The increase is likely a result of stress response under that a defect in the leptin signaling pathway may decrease the severe insulin resistance in ob/ob mice, which prevent insulin efficacy of RYGB surgery. Although leptin mutation is rare in action in the reduction of glucose. Our data suggest that RYGB humans, mutations in the downstream signaling proteins are was unable to improve insulin sensitivity in ob/ob mice. However, often found in severe obese patients.30 MC4R mutation is the improvement was observed after the 14 day leptin- associated with severe obesity31 and loss of MC4R function replacement therapy, suggesting that leptin may be required for attenuates the effects of RYGB surgery.32 The MC4R activity has the improved insulin sensitivity after RYGB. In DIO model, insulin been verified in MC4R knockout mice, which failed to respond to sensitivity was improved to the same level in the RYGB and WT- RYGB surgery.33 In two previous studies of Zucker obese rats matched groups with identical weight loss, suggesting that the (leptin receptor deficient), RYGB decreased body weight below weight loss is critical for the sustained insulin sensitization after pre-surgery level.19,20 It is not clear which of the many differences RYGB. Although the weight-matched strategy was not used in the (species, surgery, diet, and timeline) between this and other study of ob/ob mice, the weight loss-dependent effect may apply studies accounts for the discrepant outcome in body weight.

© 2015 Macmillan Publishers Limited International Journal of Obesity (2015) 798 – 805 Leptin in RYGB surgery Z Hao et al 804 While, 4 weeks may have been too short to detect weight regain 7 Korner J, Inabnet W, Conwell IM, Taveras C, Daud A, Olivero-Rivera L et al. in Zucker rats after RYGB, particularly with a small (about 14%) Differential effects of gastric bypass and banding on circulating gut hormone and weight loss at 4 weeks in one study.20 Leptin may not be the only leptin levels. Obesity (Silver Spring) 2006; 14:1553–1561. factor in the prevention of weight regain. There are other factors 8 Chandarana K, Gelegen C, Karra E, Choudhury AI, Drew ME, Fauveau V et al. Diet and gastrointestinal bypass-induced weight loss: the roles of ghrelin and contributing to the weight control by RYGB, such as bile acid that peptide YY. Diabetes 2011; 60:810–818. 34 signals via the farnesoid X receptor, re-programming of gut 9 Cummings DE, Overduin J, Foster-Schubert KE. Gastric bypass for obesity: 35 glucose utilization and increased levels of circulating peptide mechanisms of weight loss and diabetes resolution. J Clin Endocrinol Metab 2004; tyrosine tyrosine.8 As ob/ob mouse is fragile and easily stressed, it 89:2608–2615. was quite a challenge to keep mortality low after RYGB. Carefully 10 Ye J, Hao Z, Mumphrey MB, Townsend RL, Patterson LM, Stylopoulos N et al. maintaining body temperature and hydration levels during GLP-1 receptor signaling is not required for reduced body weight after RYGB in rodents. Am J Physiol Regul Integr Comp Physiol 2014; 306: R352–R362. surgery and immediate post surgery was crucial. 11 Mokadem M, Zechner JF, Margolskee RF, Drucker DJ, Aguirre V. Effects of In conclusion, our data demonstrate the difference of ob/ob Roux-en-Y gastric bypass on energy and glucose homeostasis are preserved in and DIO mice in response to RYGB surgery. In DIO mice, RYGB two mouse models of functional glucagon-like peptide-1 deficiency. Mol Metab generated a persistent weight loss and insulin sensitization like 2013; 3: 191–201. what have reported in 80–85% obese patients in the clinical 12 Chambers AP, Jessen L, Ryan KK, Sisley S, Wilson-Perez HE, Stefater MA et al. studies. In ob/ob mice, although RYGB attenuated weight gain Weight-independent changes in blood glucose homeostasis after gastric bypass or vertical sleeve gastrectomy in rats. Gastroenterology 2011; 141:950–958. compared with sham operation, it failed to keep the body weight 13 Abegg K, Schiesser M, Lutz TA, Bueter M. Acute peripheral GLP-1 receptor below the pre-surgery level. The weight regain resembles what agonism or antagonism does not alter energy expenditure in rats after Roux-en-Y has been reported in obese patients with MCR4 mutation after gastric bypass. Physiol Behav 2013; 121:70–78. RYGB. Our observations suggest that leptin sensitivity is improved 14 Wilson-Perez HE, Chambers AP, Sandoval DA, Stefater MA, Woods SC, Benoit SC by RYGB. The improvement is translated into sustained weight loss et al. The effect of vertical sleeve gastrectomy on food choice in rats. Int J Obes 37 – in DIO mice, but not in ob/ob mice. Leptin appears to be one of (Lond) 2013; :288 295. 15 Stefater MA, Perez-Tilve D, Chambers AP, Wilson-Perez HE, Sandoval DA, Berger J the endocrine factors required for the therapeutic effects of RYGB et al. Sleeve gastrectomy induces loss of weight and fat mass in obese rats, surgery. Lack of leptin or dysfunction of leptin signaling circuitry but does not affect leptin sensitivity. Gastroenterology 2010; 138: may contribute to the weight regain and diabetes recurrence in 2426–2436 2436 e1-3. 15–20% obese patients after RYGB surgery. This study re-enforces 16 Grayson BE, Schneider KM, Woods SC, Seeley RJ. Improved rodent maternal that weight loss contributes substantially to insulin sensitization in metabolism but reduced intrauterine growth after vertical sleeve gastrectomy. 5 RYGB. These conclusions may help to predict efficacy of RYGB Science Transl Med 2013; : 199ra112. 17 Bjorbaek C, Elmquist JK, Frantz JD, Shoelson SE, Flier JS. Identification of SOCS-3 surgery before the surgery, and explain the weight regain after as a potential mediator of central leptin resistance. Mol Cell 1998; 1:619–625. surgery. 18 Shin AC, Zheng H, Townsend RL, Sigalet DL, Berthoud HR. Meal-induced hormone responses in a rat model of Roux-en-Y gastric bypass surgery. Endocrinology 2010; 151:1588–1597. CONFLICT OF INTEREST 19 Xu Y, Ohinata K, Meguid MM, Marx W, Tada T, Chen C et al. Gastric bypass model The authors declare no conflict of interest. in the obese rat to study metabolic mechanisms of weight loss. J Surg Res 2002; 107:56–63. 20 Meirelles K, Ahmed T, Culnan DM, Lynch CJ, Lang CH, Cooney RN. Mechanisms ACKNOWLEDGEMENTS of glucose homeostasis after Roux-en-Y gastric bypass surgery in the obese, insulin-resistant Zucker rat. Ann Surg 2009; 249: 277–285. 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