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Bariatric surgery

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Effects of Gastric Bypass Surgery on Insulin Resistance and Insulin Secretion in Nondiabetic Obese Patients
Miriam Promintzer-Schifferl1, Gerhard Prager2, Christian Anderwald1, Martina Mandl1, Harald Esterbauer3, Soheila Shakeri-Leidenmhler2, Giovanni Pacini4, Marietta Stadler5, Martin G. Bischof1, Bernhard Ludvik1, Aanton Luger1 and Michael Krebs1
Roux-en-Y-Gastric-Bypass (RYGB) reduces overall and diabetes-specific mortality by 40% and over 90%. This study aims to gain insight into the underlying mechanisms of this effect. We evaluated time-courses of glucose, insulin, C-peptide, and the incretin glucagon like peptide-1 (GLP-1) following an oral glucose load. Insulin-sensitivity was measured by a hyperinsulinemic-isoglycemic-clamp-test; glucose-turnover was determined using d-[6,6-2H2] glucose. Examinations were performed in six nondiabetic patients with excess weight before (PRE: BMI: 49.3 3.2 kg/ m2) and 7 months after RYGB (POST: BMI: 36.7 2.9 kg/m2), in a lean (CON: BMI: 22.6 0.6 kg/m2) and an obese control group (CONob) without history of gastrointestinal surgery (BMI: 34.7 1.2 kg/m2). RYGB reduced fasting plasma concentrations of insulin and C-peptide (P < 0.01, respectively) whereas fasting glucose concentrations remained unchanged. After RYGB increase of C-peptide concentration following glucose ingestion was significantly higher compared to all other groups (dynamic-area under the curve (Dyn-AUC): 090 min: POST: 984 115 ngmin/ ml, PRE: 590 67 ngmin/ml, CONob: 440 44 ngmin/ml, CON: 279 22 ngmin/ml, P < 0.01 respectively). Early postprandial increase of glucose concentration was however not affected. GLP-1 concentrations following glucose ingestion were sixfold higher after RYBG than before (P = 0.01). Insulin-stimulated glucose uptake tended to increase postoperatively (M-value: PRE: 1.8 0.5, POST: 3.0 0.3, not significant (n.s.)). Endogenous glucose production (EGP) was unaffected by RYGB. Hepatic insulin resistance index improved after RYGB and was then comparable to both control groups (PRE: 29.2 4.3, POST: 12.6 1.1, P < 0.01). RYGB results in hyper-secretion of insulin and C-peptide, whereas improvements of insulin resistance are minor and seem to occur rather in the liver and the adipose tissue than in the skeletal muscle.
Obesity (2011) 19, 14201426. doi:10.1038/oby.2011.92

IntroductIon

Obesity is a refractory condition associated with increased morbidity and mortality. So far, surgical intervention is the only treatment that induces sustained weight loss and has been shown to reduce overall mortality (1,2). However, the mechanisms leading to this reduction of mortality are not sufficiently studied and understood. Mortality after Roux-en-Y-Gastric-Bypass (RYGB), specified according causes, was decreased by 92% for diabetes and by 56% for coronary artery disease (1). The underlying risk factors of these two diseases such as hyperglycemia, hyperinsulinemia, and insulin resistance are
1

regularly present in the severely obese. Elevated blood glucose concentration is supposed to have a harmful effect on the arterial wall and has been shown to be a risk factor for mortality even in nondiabetic individuals (3). Especially postprandial hyperglycemia is strongly associated with the development of macrovascular complications (4). Also insulin resistance and hyperinsulinemia at fasting and postprandially are independently associated with coronary artery disease (5). Meta-analyses reported complete resolution of diabetes in over 80% of patients who had undergone gastric bypass surgery (6). Weight loss and/or altered bowel physiology bringing along altered secretion of gastrointestinal peptides might contribute

Division of Endocrinology and Metabolism, Department of Internal Medicine III, Medical University of Vienna, Vienna, Austria; 2Department of Surgery, Medical University of Vienna, Vienna, Austria; 3Department of Medical and Chemical Laboratory Diagnostics, Medical University of Vienna, Vienna, Austria; 4Metabolic Unit, Istituto di Ingegneria Biomedica del CNR, Area di Ricerca di Padova, Corso Stati Uniti 4, Padua, Italy; 53rd Medical Department, Hospital Hietzing, Vienna, Austria. Correspondence: Michael Krebs (michael.krebs@meduniwien.ac.at) Received 16 March 2010; accepted 24 March 2011; published online 14 April 2011. doi:10.1038/oby.2011.92

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to this effect on glucose metabolism. In particular the postprandial response of the incretin glucagon like peptide-1 (GLP-1) has been observed to be significantly influenced by RYGB (7). However, the effects of bariatric surgery on insulin resistance are unclear, since contrasting results have been reported. Restoration of normal insulin sensitivity before occurrence of substantial weight loss was reported by some (8), whereas other studies described no substantial improvement of insulin sensitivity despite profound weight loss (9). Thus it is unclear whether the postoperative alterations in glucose metabolism including insulin sensitivity occur in proportion to the weight lost (10) or are mainly caused by altered gastrointestinal anatomy. We, therefore, measured peripheral insulin sensitivity employing gold standard methods before and after RYGB. To facilitate interpretation of the results an obese control group without history of gastrointestinal surgery matched for BMI to the patients after RYGB was included in addition to normal weight controls. Many previous studies on the effects of RYBP-surgery have been performed in patients with type 2 diabetes. However, it is well established that glucose toxicity contributes not only to impaired insulin secretion but also to insulin resistance (11). Insulin resistance secondary to hyperglycemia has been estimated to account for ~40% of the reduction in insulin stimulated glucose disposal observed in patients with diabetes mellitus (12). Furthermore, antidiabetic medication influences glucose homeostasis in multiple ways. To study the effects of RYGB-surgery per se we have avoided these confounders by including nondiabetic subjects only.
Methods and Procedures Glucose concentrations, insulin, and C-peptide secretion were measured after an oral glucose load (oral glucose tolerance test, OGTT) before (visit 1, PRE) and 7 months after (visit 3, POST) RYGB surgery in nondiabetic patients with excess weight. Insulin resistance was determined employing hyperinsulinemic isoglycemic clamp test before (visit 2, PRE) and also 7 months after (visit 4, POST) RYGB in these patients. We compared the patients on the one hand to a lean, healthy control group (CON) that was matched for age and gender andto get a more detailed understanding of the glucose homeostasis after RYGBwe also compared the patients after RYGB to a obese nondiabetic control group (CONob) without history of gastrointestinal surgery. CONob was matched to POST for BMI, age, and gender. The study was performed according to the declaration of Helsinki and the protocol was approved by the local ethical board. All study participants gave written informed consent. study participants Nondiabetic obese (BMI >40 kg/m2) study participants planning to undergo RYGB surgery were recruited. Exclusion criteria included history of diabetes, current or former medication known to influence the glucose homeostasis, severe disease, pregnancy, and breastfeeding as well as obesity caused by an endocrine disorder. Study participants were tested for Cushings syndrome and thyroid dysfunction. Both, lean and obese control groups were recruited by local advertising. Visit 1 OGTT. After an overnight fast we performed a 75 g OGTT (GlucoDrink 75; Roche Diagnostics, Vienna, Austria) with frequent blood-sampling (0, 10, 20, 30, 40, 60, 90, 120, 150, and 180 min) for
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determinations of plasma glucose, insulin, and C-peptide concentrations in all study participants. Only patients who were nondiabetic according to the definition of the ADA (13) were included into this study. Blood samples for determination of GLP-1 were collected at 0 and 120 min in PRE and POST. Anthropometric data were registered.
Visit 2 5.2 0.9 days after visit 1, a hyperinsulinemic isoglycemic clamp test was performed. After an overnight fast, two catheters (Vasofix; Braun, Melsungen, Germany) were inserted in the left and right antecubital vein for blood sampling and infusions, respectively. A primed-continuous infusion (bolus: 4 mg kg lean body weight; remaining period: 0.04 mg/min lean body weight) of d-[6,6-2H2]glucose (98% enriched; Cambridge Isotope Laboratories, Andover, MA) was started at 120 min for measurement of endogenous glucose production (EGP) (14). Blood samples for determination of EGP were drawn at 120, 5, 0, 80, 100, and 120 min of the clamp. The clamp goal was determined from the mean of three plasma glucose measurements before the clamp. If the calculated clamp goal was outside 80 and 100 mg/dl, 80 and 100 mg/dl, respectively, were taken as the clamp goal. The hyperinsulinemic-isoglycemic clamp-test was started with primed-continuous insulin (Actrapid; Novo Nordisk, Bagsvaerd, Denmark) infusion (40 mU insulinmin1m2 body surface area). Plasma glucose was measured every 5 min and maintained at the clamp goal by infusing variable amounts of d-glucose, enriched with d-[6,6-2H2]glucose. For calculation of M-values blood was collected every 20 min. Free fatty acids (FFA) were determined at baseline and during standardized hyperinsulinemia. Bariatric surgery All patients underwent laparoscopic Gastric Bypass in a standardized antegastric, antecolic way. The alimentary limb measured 150 cm, the biliopancreatic limb 75 cm. The Gastrojejunostomy was performed with a circular stapler (Orvil 21 mm; Covidien, Dublin, Ireland), the JejunoJejunostomy with a linear 45 mm stapler (ENdoGIA; Covidien). Visits 3 and 4 29.0 3.3 weeks after bariatric surgery measurements of anthropometric data and OGTT were redone in the same way as described above. Clamp test was also repeated 12.8 3.2 days after the OGTT as described above. Plasma metabolites and hormone measurements Plasma glucose concentrations were measured using the glucose oxidase method (Glucose Analyzer II; Beckman, Fullerton, CA). Plasma immunoreactive insulin and C-peptide were analyzed by commercially available radioimmunoassays (insulin; Pharmacia, Uppsala, Sweden: C-peptide; Cis, Gif-Sur-Yvette, France) and plasma FFA concentrations with a microfluorimetric assay (Wako, Richmond, VA). For determination of GLP-1, DPP IV inhibitor of Linco Research, St Charles, Missouri was added to the blood samples and concentrations of active GLP-1 were assessed by ELISA (cat. no. EGLP-35K; Millipore, Billerica, MA). Gas chromatography/mass spectrometry Mole percent excess of plasma and infusate d-[6,6-2H2]glucose was measured by gas-chromatography/mass spectrometry as described (15). calculations EGP and M-values, both given in mg glucosemin1kg1, were calculated as described (16,17). Area under the curve (AUC) was calculated according to the trapezoide rule. Dynamic AUC (Dyn-AUC) was calculated by subtracting the product of the basal value multiplied by time from AUC. Hepatic insulin resistance index was calculated by multiplying fasting insulin concentrations with fasting EGP as described by Gastaldelli et al. (18) and in analogue by multiplying fasting C-peptide concentration with EGP. Hepatic insulin extraction
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table 1 anthropometric data and clinical characteristics of study participants before (Pre) and after (Post) roux-en-Y-GastricBypass (rYGB) and of the lean (con) and the obese (conob) control group
PRE (n = 6) Age Gender BMI (kg/m )
2

POST (n = 6) 43.3 2.1 5 f/1 m

CON (n = 6) 43.1 4.7 5 f/1 m

CONob (n = 6) 47.6 3.1 5 f/1 m 34.7 1.2f 111.9 4.2f 112.2 3.3 5.9 0.6 133.0 40.9 219.3 24.1

42.5 2.1 5 f/1 m 49.3 3.2 151.8 7.9 5.8 0.1 123.8 16.6 191.2 10.4
a,b,c

36.7 2.9 121.0 9.4 5.4 0.1 93.7 3.6

22.6 0.6 81.2 3.0 102.3 4.4 5.3 0.1 77.8 11.2 179.0 6.7

Waist circumference (cm) Hip circumference (cm) HbA1c (%) Triglycerides (mg/dl) Cholesterol (mg/dl)

136.5 5.9a,b,c
a,b,c

111.0 8.9d
e

147.5 7.4e

Significances (P < 0.05) indicated as follows: aPRE vs. POST; bPRE vs. CON; cPRE vs. CONob; dPOST vs. CON; ePOST vs. CONob; fCONob vs. CON. HbA1c, hemoglobin A1c.

(HIE) (as percentage of the secreted hormone) was approximated by a function of (1-(AUCinsulin/AUCC-peptide)) as previously described (19). HOMA was calculated as a measure for basal insulin sensitivity (20). Adaptation index (21) that describes the ability of the beta cell to adapt its own insulin secretion to the prevailing insulin resistance was calculated as the product of M-value and the suprabasal AUCC-peptide. This index provides a further figure of the cell function.
statistics Statistical analyzes were performed using SPSS 15.0. Data was checked for normal distribution using KolmogorovSmirnov test and graphically. Differences between the groups were calculated using ANOVA and Bonferronis Multiple comparison test. Differences were considered statistically significant at P < 0.05. All data are given as means s.e.m. results study participants

a
200 150

Glucose

PRE POST CON CONob

mg/dl

100 50 0 0 60 120 180

Time

b
8,000 6,000 4,000 2,000 0

Anthropometric data and clinical characteristics of the four groups are displayed in Table 1.
Fasting glucose metabolism Fasting concentrations of glucose, insulin, C-peptide, FFA. Before

surgery fasting concentration of glucose was comparable between PRE and CON (PRE: 98.3 4.7 mg/dl, CON: 84.4 2.9 mg/dl, not significant (n.s.), (Figure 1a)), while fasting plasma concentrations of C-peptide, insulin, and FFA were significantly higher in PRE (C-peptide: PRE: 4.0 0.4 ng/ml, CON: 1.3 0.1 ng/ml (Figure 2a); insulin: PRE: 28.5 4.6 U/ml, CON: 6.6 0.7 U/ml (Figure 3a), both P < 0.01, FFA: PRE: 688 66.6 mol/l, CON: 398.3 67.9 mol/l, P < 0.05, (not shown)). Compared to PRE fasting plasma concentrations of C-peptide and insulin were lower post surgery (C-peptide: PRE: 4.0 0.4 ng/ ml, POST: 2.6 0.3 ng/ml, P < 0.01 (Figure 2a); insulin: PRE: 28.5 4.6 U/ml, POST: 10.1 1.2 U/ml, P < 0.01 (Figure 3a)), whereas fasting plasma glucose concentrations and FFA were comparable before and after RYGB (glucose: PRE: 98.3 4.7 mg/ dl, POST: 84.2 2.4 mg/dl, n.s. (Figure 1a); FFA: PRE: 688 66.6 mol/l, POST: 773 42.5 mol/l, data not shown). mated by HOMA significantly decreased postoperatively (PRE: 5.92 1.0, POST: 1.9 0.2, P < 0.05) and was then comparable to the lean controls (CON: 1.2 0.1, P > 0.05).
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PRE

POST

CON

CONob

Dyn AUC 090 min

Figure 1 Time courses of blood glucose concentrations during the oral glucose test (a) before (PRE) and after (POST) Roux-en-Y-GastricBypass (RYGB) as well as of the lean (CON) and obese controls (CONob). (b) In the first 90 min after oral glucose ingestion dynamicarea under the curve of blood glucose concentration was not altered after RYGB. *P < 0.05 PRE vs. CON, P < 0.05 POST vs. CON. PRE, before RYGB; POST, 7 months after RYGB; CON, lean, healthy control; CONob, obese control.

Insulin sensitivity in the fasting state. Basal insulin resistance esti-

Hepatic insulin resistance index was significantly higher in PRE compared to POST and CON (PRE: 29.2 4.3, POST: 12.6 1.1, CON: 10.0 0.8, P < 0.01 respectively (Figure 4)). Hepatic insulin resistance index in POST was comparable to the BMI matched control group (CONob: 12.48 4.0, n.s. vs. POST (Figure 4)). When hepatic insulin resistance index was calculated with C-peptide, which, in contrast to insulin, is not cleared by the liver, a similar effect of surgery on hepatic insulin resistance was evident (Hep IR (EGP fasting C-peptide): PRE: 5.4 0.7, POST: 3.6 0.2, P < 0.05).
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a
C-Peptide 20 15 ng/ml 10 5 0 0 60 Time 120 180 PRE POST CON CONob

EGP in the fasting state was comparable between PRE and POST (PRE: 1.3 0.1, POST: 1.4 0.1 mg/kg/min, n.s.) and also not different between POST and CONob (CONob: 1.3 0.1 mg/kg/min n.s. vs. POST).
Insulin-stimulated glucose metabolism OGTT. After an oral load of 75 g of glucose early (090 min)

1,250 1,000 750 500 250 0 PRE

POST

CON

CONob

Dyn AUC 090 min

Figure 2 Time courses of C-peptide concentrations during the oral glucose test (a) before (PRE) and after (POST) Roux-en-Y-GastricBypass (RYGB) as well as of the lean (CON) and obese controls (CONob). (b) In the first 90 min after oral glucose ingestion C-peptide concentration was significantly higher after Roux-en-Y-Gastric-Bypass (RYGB) than before. P < 0.01 PRE vs. POST, *P < 0.05 PRE vs. CON, P < 0.001 POST vs. CON, P < 0.001 POST vs. CONob. PRE, before RYGB; POST, 7 months after RYGB; CON, lean, healthy control; CONob, obese control.

Insulin 200 150

U/ml

PRE 100 50 0 0 60 Time 120 180 POST CON CONob

b
12,000 #

and late (90180 min) postprandial glucose concentrations were significantly different between obese patients before surgery and lean controls (Dyn-AUC: 090 min: PRE: 5804 585 mgmin/dl, CON: 1855 684 mgmin/dl, P < 0.05, Dyn-AUC: 90180 min: PRE: 4228 799 mgmin/dl, CON: 87.5 422 mgmin/dl, P < 0.01 respectively (Figure 1a,b)). After RYGB early postprandial increase of plasma glucose was identical to PRE (Dyn-AUC: 090 min: PRE: 5804 585 mgmin/dl, POST: 6304 1358 mgmin/dl, n.s. (Figure 1a,b)), whereas plasma glucose concentrations tended to be lower in the late postprandial period, but were however not significantly different (Dyn-AUC: 90180 min: PRE: 4,228 799 mgmin/dl, POST: 3,358 934 mgmin/ dl, n.s. (Figure 1a)). C-peptide secretion within 90 min after glucose ingestion was significantly more pronounced after RYGB surgery than before (Dyn-AUC: 090 min: PRE: 590 67 ngmin/ml, POST: 984 115 ngmin/ml, P < 0.01 (Figure 2a,b)). Increase of insulin concentration after glucose load tended to be higher in POST than in PRE, however not statistically significant (Dyn-AUC: 090 min: PRE: 8733 1480 Umin/ml, POST: 9803 1295 Umin/ml, n.s. (Figure 3a)). Comparing study participants after RYGB with a BMI matched control group without gastrointestinal surgery (CONob) postprandial increase of plasma glucose concentration was comparable (Dyn-AUC: 090 min: CONob: 5133 782 mgmin/dl, POST: see above, n.s. (Figure 1a,b)). Increase of C-peptide and insulin within the first 90 min of the OGTT was significantly higher after bariatric surgery compared to the BMI matched control group (Dyn-AUC: 090 min: C-peptide: CONob: 440 44 ngmin/ml, POST: see above, P < 0.001 (Figure 2a,b), insulin: CONob: 4,181 826 Umin/ ml, POST: see above, P < 0.01 (Figure 3a,b)).
Hyperinsulinemic isoglycemic clamp test. M-value tended to increase after gastric bypass surgery however the difference did not reach statistical significance (PRE: 1.8 0.5, POST: 3.0 0.3 mg/kg/min, n.s. (Figure 5)). Comparing the study participants after gastric bypass surgery to the BMI matched control group M value was not significantly different (Figure 5) (CONob: 4.1 0.7 mg/kg/min, n.s.). Also when the M-value was normalized to the body surface area, results were identical (PRE: 98.8 26.1, POST: 143.3 9.8, CONob: 192.8 35.1, CON: 403.6 60.2 mg/min/body surface area, PRE vs. POST and POST vs. CONob: n.s.). Insulin mediated suppression of FFA was more pronounced after RYGB than before reaching borderline statistical significance (PRE: 556.0 22.9 mol/l, POST: 246.0 6.1 mol/l, P = 0.06).
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6,000

0 PRE POST CON CONob Dyn AUC 090 min

Figure 3 Time courses of insulin concentrations during the oral glucose test (a) before (PRE) and after (POST) Roux-en-Y-Gastric-Bypass (RYGB) as well as of the lean (CON) and obese controls (CONob). (b) In the first 90 min after oral glucose ingestion dynamic-area under the curve of insulin concentration was not significantly higher after RYGB than before. #P < 0.01 PRE vs. CON, %P < 0.05 PRE vs. CONob, P < 0.001 POST vs. CON, P < 0.01 POST vs. CONob. PRE, before RYGB; POST, 7 months after RYGB; CON, lean, healthy control; CONob, obese control.
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Hepatic insulin resistance index 40 30 20 10 0 # %

concentrations before and after surgery (0 min: PRE: 3.2 0.3, POST: 4.0 0.5 pmol/l, n.s.).
dIscussIon

PRE

POST

CON

CONob

Figure 4 Hepatic insulin resistance significantly improved after Rouxen-Y-Gastric-Bypass (RYGB) and was then comparable with the lean controls. P < 0.01 PRE vs. POST, #P < 0.01 PRE vs. CON, %P < 0.05 PRE vs. CONob. PRE, before RYGB; POST, 7 months after RYGB; CON, lean, healthy control; CONob, obese control.
M value 15 # mg/kg/min 10 @

0 PRE POST CON CONob

Figure 5 Insulin sensitivity, determined by the clamp test, did not improve significantly after Roux-en-Y-Gastric-Bypass (RYGB). #P < 0.01 PRE vs. CON, P < 0.01 POST vs. CON, @P < 0.01 CONob vs. CON. PRE, before RYGB; POST, 7 months after RYGB; CON, lean, healthy control; CONob, obese control.

EGP in the fasting state in the last hour of the clamp test was comparable between PRE and POST (PRE: 0.4 0.2, POST: 0.6 0.0 mg/kg/min, n.s.).and also not different between POST and CONob (CONob: 0.4 0.1 mg/kg/min, n.s. vs. POST).
hIe

HIE increased after RYGB and was then comparable to CON and CONob (PRE: 78.3 2.7, POST: 85.3 0.9, CON: 88.7 0.6, CONob: 86.5 1.4 %, PRE vs. POST and CONob: P < 0.05, PRE vs. CON, P < 0.01). The coefficient of correlation of HIE and hepatic insulin resistance index (PRE: r = 0.3, POST: r = 0.46) as well as M-value (PRE: r = 0.1, POST: r = 0.62) was higher after surgery than before.
adaptation index

Adaptation Index as measure of -cell function in relation to insulin resistance was comparable before and after surgery and virtually identical to the obese control group (PRE: 1586 516, POST: 3656 706, CON: 6204 808, CONob: 3749 808, all n.s., only PRE vs. CON, P < 0.001).
GlP-1

After RYGB postprandial GLP-1 concentrations were sixfold higher than before surgery (120 min: PRE: 4.0 0.5 pmol/l, POST: 24.8 4.2 pmol/l, P = 0.01) with comparable basal
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We investigated the impact of Roux-en-Y Gastric Bypass surgery on insulin resistance, insulin and C-peptide secretion as well as glycemia in nondiabetic obese patients. These parameters were also studied in the same way in lean and in obese controls, the latter of which were matched for BMI to the patients after RYGB surgery. As expected and in accordance to other studies (10,22) basal insulin, and C-peptide concentrations were lower after RYGB surgery. We observed, however, in the first hour after oral glucose load a hyper-secretion of insulin and C-peptide in the postsurgery group with significantly higher dynamic AUC and peak concentration of C-peptide than before surgery and the BMI matched controls. Interestingly, only the peak and the dynamic AUC of C-peptide but not insulin reached statistical significance. Insulin but not C-peptide is removed by ~50% by the liver during the first passage (23) and abnormalities in hepatic insulin clearance are integral to type 2 diabetes and obesity (24). Weight loss in obese individuals has been shown to lead to an increase in insulin clearance (24). As downregulation of the insulin receptors is caused by prolonged increases in portal insulin levels (24) lower insulin concentrations after weight loss might reverse the downregulation and lead to higher insulin clearance. In our study calculated HIE was higher after RYGB surgery than before surgery and was then in the same range as seen in the lean and obese controls. Besides weight loss also glucose ingestion increases hepatic insulin uptake presumably through signals from the gut, since intraportal glucose infusion does not provoke this effect (24). Higher hepatic insulin clearance was also seen after weight loss induced by different techniques of bariatric surgery (25,26). Furthermore, a reduction in HIE was reported in insulin resistant states (24). In our study correlation between HIE and both the hepatic insulin resistance index and the M-value was stronger after surgery than before. Thus, weight loss, altered bowel physiology and also improved insulin sensitivity after RYGB surgery might contribute to the higher HIE after RYGB surgery. Hyper-secretion of insulin and C-peptide following glucose stimulation after gastric bypass, as observed in the present study, has been reported previouslyeven in context with symptomatic, hyperinsulinemic hypoglycaemia (25,27). One possible explanation is an inappropriately high insulin secretory rate per -cell after RYGB as the -cell mass in patients after RYGB and obese controls is comparable (28). Furthermore, nuclear diameters of -cells were found to be closely associated with BMI and size of the diameters after surgery was not appropriate for the postsurgery BMI but rather for the presurgery BMI. This is of special interest as nuclear diameters of endocrine cells are linked with their secretory activity (29,30). The underlying causes of these findings and the observed hyperinsulinemia are under discussion. Animal studies imply that the entero-insular axis is sensitive to changes in nutrient transit and absorption (31). Concentrations of the insulinotropic hormone GLP-1,
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which is released in the distal ileum and colon, were found to be increased after RYGB in several studies (32,33) as well as in our patients following glucose or meal ingestion. However, in addition to GLP-1 the entero-insular axis comprises numerous known and probably also unknown hormones that influence glucose metabolism and might, therefore, contribute to altered insulin secretion after gastrointestinal surgery. A long known and common complication of gastric resections is the dumping syndrome. Again, alterations in transit time and composition of nutrients in the intestine leads to changes in appearance of glucose in the blood which, in turn, will affect postprandial insulin secretion. Service et al. (34) concluded that enhanced insulin secretion after RYGB might be simply a component of the dumping syndrome and only severe hyperinsulinemic hypoglycemia suggests a new pathological syndrome. Also failure to adaptively decrease insulin secretion after RYGB might contribute to the increased insulin concentration (24). This is reflected by the adaptation index, which, describing the ability of the -cell to modulate insulin release in relation to changes in insulin resistance, was unchanged after RYGB and comparable to the BMI matched controls in our study. We assessed insulin sensitivity in the fasting and the postprandial state. In the fasting state steady state conditions are present and fasting glucose concentration is determined by hepatic glucose production, insulin sensitivity of the liver, the prevailing insulin concentration (35) and noninsulin-stimulated glucose uptake. Thus, insulin sensitivity in the fasting state mainly reflects hepatic insulin action (20). Basal insulin sensitivity estimated by HOMA (20) was significantly lower postoperatively which is in line with previous reports (36,37). As to the hepatic insulin resistance, fasting EGP was unaffected by RYGB surgery. However, when taking into account the different prevailing fasting insulin concentrations before and after surgery by applying the hepatic insulin resistance index, hepatic insulin resistance improves after RYGB. To exclude that a possible postoperative increase in insulin extraction leads to overestimation of the improvement in hepatic insulin resistance, we have also calculated this index using fasting C-peptide, which is not extracted by the liver and yielded similar results. Improved hepatic insulin resistance bringing along comparable EGP in the presence of lower insulin concentrations may therefore also account for the unchanged fasting glucose concentration after RYGB. Several studies have demonstrated a strong relationship between hepatic insulin resistance and hepatic triglyceride content (38). The mechanism by which hepatic steatosis causes insulin resistance in the liver is not completely clear. Remarkably, it has been shown that already a modest weight loss of <10% of the body weight reverses hepatic steatosis and hepatic insulin resistance but had no effect on peripheral insulin resistance (38). In the postprandial state, i.e., under insulin stimulated conditions, skeletal muscle is primarily responsible for the removal of glucose which is almost exclusively stored as glycogen (39). In the present study, insulin stimulated glucose uptake measured
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by the hyperinsulinemic isoglycemic clamp test, did not significantly increase after RYGB and remained ~3.5 times below the insulin stimulated glucose uptake of lean controls. Mingrone et al. (10) compared RYGB and biliopancreatic diversion and reported that after RYGB the gain in insulin sensitivity is predicted by proportional weight loss whereas after biliopancreatic diversion insulin sensitivity was quickly normalized before a significant weight loss occurred (40,41). Taken together, the severe insulin resistance in skeletal muscle observed in morbidly obese subjects is not readily normalized by RYGB in contrast to glucose induced insulin secretion and hepatic insulin resistance. Insulin resistance in obesity is manifested through heterogeneous mechanisms, including fatty acid flux (42). We observed no change of plasma FFA concentration after RYGB in the fasting state. During the clamp, however, the insulin-induced suppression of FFA after RYGB was more distinct than before RYGB with FFA concentrations less than half the amount than before RYGB. Possible explanations include that the hormonesensitive lipase is activated when insulin levels drop and thus counteract the expected decrease of FFA after bariatric surgery (43). On the other hand, the lack of a significant increase of FFA after bariatric surgery might be associated with higher insulin sensitivity of the adipose tissue (43) which would be in concordance to our findings during the clamp. A definite conclusion however cannot be derived from our data. In summary, it was shown that RYGB surgery decreases mortality of coronary artery disease by 56% (1). Interestingly, hyperglycemia and hyperinsulinemia, which are regarded as eminent risk factors for macrovascular disease (4,5), were not normalized after RYGB in our study. Glycemia was neither affected at fasting condition nor postprandially by RYGB, hyperinsulinemia improved only at fasting. In conclusion, our study shows a hyper-secretion of C-peptide and insulin following oral glucose ingestion in nondiabetic obese study participants after Roux-en-Y Gastric Bypass. Improvement in insulin resistance was small which hints that insulin resistance of the liver and the adipose tissue was more positively affected by RYGB than insulin resistance of the muscle.
acknowledGMents
The authors are indebted to Maria Ozsvar-Kozma and A. Hofer for technical help. M.P.-S. was employed as Post-Doc on a grant from the Austrian Society of Internal Medicine (josef Skoda-Award), which was competitively awarded to M.K. M.K. and H.E. are supported by the WWTF (Vienna Science and Technology Fund), G. Prager is supported by the Educational Grant.

dIsclosure
The authors declared no conflict of interest.
2011 The Obesity Society

reFerences
1. 2. 3. Adams TD, Gress RE, Smith SC et al. Long-term mortality after gastric bypass surgery. N Engl J Med 2007;357:753761. Sjstrm L, Narbro K, Sjstrm CD et al.; Swedish Obese Subjects Study. Effects of bariatric surgery on mortality in Swedish obese subjects. N Engl J Med 2007;357:741752. Balkau B, Shipley M, Jarrett RJ et al. High blood glucose concentration is a risk factor for mortality in middle-aged nondiabetic men. 20-year follow-up in
1425

articles
Bariatric surgery
the Whitehall Study, the Paris Prospective Study, and the Helsinki Policemen Study. Diabetes Care 1998;21:360367. Temelkova-Kurktschiev TS, Koehler C, Henkel E et al. Postchallenge plasma glucose and glycemic spikes are more strongly associated with atherosclerosis than fasting glucose or HbA1c level. Diabetes Care 2000;23:18301834. Karabulut A, Iltumur K, Toprak N et al. Insulin response to oral glucose loading and coronary artery disease in nondiabetics. Int Heart J 2005;46:761770. Buchwald H, Estok R, Fahrbach K et al. Weight and type 2 diabetes after bariatric surgery: systematic review and meta-analysis. Am J Med 2009;122:248256.e5. le Roux CW, Welbourn R, Werling M et al. Gut hormones as mediators of appetite and weight loss after Roux-en-Y gastric bypass. Ann Surg 2007;246:780785. Greco AV, Mingrone G, Giancaterini A et al. Insulin resistance in morbid obesity: reversal with intramyocellular fat depletion. Diabetes 2002;51: 144151. Burstein R, Epstein Y, Charuzi I et al. Glucose utilization in morbidly obese subjects before and after weight loss by gastric bypass operation. Int J Obes Relat Metab Disord 1995;19:558561. Muscelli E, Mingrone G, Camastra S et al. Differential effect of weight loss on insulin resistance in surgically treated obese patients. Am J Med 2005;118:5157. Krebs M, Roden M. Nutrient-induced insulin resistance in human skeletal muscle. Curr Med Chem 2004;11:901908. Cline GW, Magnusson I, Rothman DL et al. Mechanism of impaired insulinstimulated muscle glucose metabolism in subjects with insulin-dependent diabetes mellitus. J Clin Invest 1997;99:22192224. ADA. Diagnosis and classification of diabetes mellitus. Diabetes Care 2004; Suppl 1:S5S10. Hother-Nielsen O, Henriksen JE, Holst JJ, Beck-Nielsen H. Effects of insulin on glucose turnover rates in vivo: isotope dilution versus constant specific activity technique. Metabolism 1996;45:8291. Krebs M, Krssak M, Nowotny P et al. Free fatty acids inhibit the glucosestimulated increase of intramuscular glucose-6-phosphate concentration in humans. J Clin Endocrinol Metab 2001;86:21532160. DeFronzo RA, Tobin JD, Andres R. Glucose clamp technique: a method for quantifying insulin secretion and resistance. Am J Physiol 1979;237: E214E223. Anderwald C, Bernroider E, Krssak M et al. Effects of insulin treatment in type 2 diabetic patients on intracellular lipid content in liver and skeletal muscle. Diabetes 2002;51:30253032. Gastaldelli A, Miyazaki Y, Pettiti M et al. Separate contribution of diabetes, total fat mass, and fat topography to glucose production, gluconeogenesis, and glycogenolysis. J Clin Endocrinol Metab 2004;89:39143921. Stadler M, Anderwald C, Karer T et al. Increased plasma amylin in type 1 diabetic patients after kidney and pancreas transplantation: A sign of impaired beta-cell function? Diabetes Care 2006;29:10311038. Wallace TM, Matthews DR. The assessment of insulin resistance in man. Diabet Med 2002;19:527534. Ahrn B, Pacini G. Impaired adaptation of first-phase insulin secretion in postmenopausal women with glucose intolerance. Am J Physiol 1997;273:E701E707. Bikman BT, Zheng D, Pories WJ et al. Mechanism for improved insulin sensitivity after gastric bypass surgery. J Clin Endocrinol Metab 2008;93:46564663. Duckworth WC. Insulin degradation: mechanisms, products, and significance. Endocr Rev 1988;9:319345. Valera Mora ME, Scarfone A, Calvani M, Greco AV, Mingrone G. Insulin clearance in obesity. J Am Coll Nutr 2003;22:487493. 25. Jimenez J, Zuniga-Guajardo S, Zinman B, Angel A. Effects of weight loss in massive obesity on insulin and C-peptide dynamics: sequential changes in insulin production, clearance, and sensitivity. J Clin Endocrinol Metab 1987;64:661668. 26. Letiexhe MR, Scheen AJ, Grard PL, Desaive C, Lefbvre PJ. Insulin secretion, clearance and action before and after gastroplasty in severely obese subjects. Int J Obes Relat Metab Disord 1994;18:295300. 27. Patti ME, McMahon G, Mun EC et al. Severe hypoglycaemia post-gastric bypass requiring partial pancreatectomy: evidence for inappropriate insulin secretion and pancreatic islet hyperplasia. Diabetologia 2005;48: 22362240. 28. Meier JJ, Butler AE, Galasso R, Butler PC. Hyperinsulinemic hypoglycemia after gastric bypass surgery is not accompanied by islet hyperplasia or increased beta-cell turnover. Diabetes Care 2006;29:15541559. 29. Norman JT, Bohman RE, Fischmann G et al. Patterns of mRNA expression during early cell growth differ in kidney epithelial cells destined to undergo compensatory hypertrophy versus regenerative hyperplasia. Proc Natl Acad Sci USA 1988;85:67686772. 30. Studer H, Derwahl M. Mechanisms of nonneoplastic endocrine hyperplasiaa changing concept: a review focused on the thyroid gland. Endocr Rev 1995;16:411426. 31. Manfredini G, Ermini M, Scopsi L, Bonaguidi F, Ferrannini E. Internal biliary diversion improves glucose tolerance in the rat. Am J Physiol 1985;249:G519G527. 32. Strader AD, Vahl TP, Jandacek RJ et al. Weight loss through ileal transposition is accompanied by increased ileal hormone secretion and synthesis in rats. Am J Physiol Endocrinol Metab 2005;288:E447E453. 33. Toft-Nielsen M, Madsbad S, Holst JJ. Exaggerated secretion of glucagonlike peptide-1 (GLP-1) could cause reactive hypoglycaemia. Diabetologia 1998;41:11801186. 34. Service GJ, Thompson GB, Service FJ et al. Hyperinsulinemic hypoglycemia with nesidioblastosis after gastric-bypass surgery. N Engl J Med 2005;353:249254. 35. Abdul-Ghani MA, Matsuda M, DeFronzo RA. Strong association between insulin resistance in liver and skeletal muscle in non-diabetic subjects. Diabet Med 2008;25:12891294. 36. Isbell JM, Tamboli RA, Hansen EN et al. The importance of caloric restriction in the early improvements in insulin sensitivity after Roux-en-Y gastric bypass surgery. Diabetes Care 2010;33:14381442. 37. Lima MM, Pareja JC, Alegre SM et al. Acute effect of roux-en-y gastric bypass on whole-body insulin sensitivity: a study with the euglycemichyperinsulinemic clamp. J Clin Endocrinol Metab 2010;95:38713875. 38. Petersen KF, Dufour S, Befroy D et al. Reversal of nonalcoholic hepatic steatosis, hepatic insulin resistance, and hyperglycemia by moderate weight reduction in patients with type 2 diabetes. Diabetes 2005;54:603608. 39. Shulman GI, Rothman DL, Jue T et al. Quantitation of muscle glycogen synthesis in normal subjects and subjects with non-insulin-dependent diabetes by 13C nuclear magnetic resonance spectroscopy. N Engl J Med 1990;322:223228. 40. Guidone C, Manco M, Valera-Mora E et al. Mechanisms of recovery from type 2 diabetes after malabsorptive bariatric surgery. Diabetes 2006;55:20252031. 41. Camastra S, Manco M, Mari A et al. beta-cell function in morbidly obese subjects during free living: long-term effects of weight loss. Diabetes 2005;54:23822389. 42. Schenk S, Saberi M, Olefsky JM. Insulin sensitivity: modulation by nutrients and inflammation. J Clin Invest 2008;118:29923002. 43. Salinari S, Bertuzzi A, Iaconelli A, Manco M, Mingrone G. Twenty-four hour insulin secretion and beta cell NEFA oxidation in type 2 diabetic, morbidly obese patients before and after bariatric surgery. Diabetologia 2008;51:12761284.

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5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.

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