Niclas Abrahamsson,1 Joey Lau Börjesson,1,2 Magnus Sundbom,3 Urban Wiklund,4

F. Anders Karlsson,1 and Jan W. Eriksson1

Gastric Bypass Reduces Symptomsand Hormonal Responses inHypoglycemiaDiabetes 2016;65:2667–2675 | DOI: 10.2337/db16-0341

Gastric bypass (GBP) surgery, one of the most commonbariatric procedures, induces weight loss and metaboliceffects. The mechanisms are not fully understood, butreduced food intake and effects on gastrointestinalhormones are thought to contribute. We recently ob-served that GBP patients have lowered glucose levelsand frequent asymptomatic hypoglycemic episodes.Here, we subjected patients before and after undergo-ing GBP surgery to hypoglycemia and examined symp-toms and hormonal and autonomic nerve responses.Twelve obese patients without diabetes (8 women, meanage 43.1 years [SD 10.8] and BMI 40.6 kg/m2 [SD 3.1])were examined before and 23 weeks (range 19–25)after GBP surgery with hyperinsulinemic-hypoglycemicclamp (stepwise to plasma glucose 2.7 mmol/L). The meanchange in Edinburgh Hypoglycemia Score during clampwas attenuated from 10.7 (6.4) before surgery to 5.2 (4.9)after surgery. There were also marked postsurgeryreductions in levels of glucagon, cortisol, and catechol-amine and the sympathetic nerve responses to hypogly-cemia. In addition, growth hormone displayed a delayedresponse but to a higher peak level. Levels of glucagon-like peptide 1 and gastric inhibitory polypeptide rose dur-ing hypoglycemia but rose less postsurgery comparedwith presurgery. Thus, GBP surgery causes a resettingof glucose homeostasis, which reduces symptoms andneurohormonal responses to hypoglycemia. Furtherstudies should address the underlying mechanisms aswell as their impact on the overall metabolic effectsof GBP surgery.

Gastric bypass (GBP) surgery for the treatment of morbidobesity induces marked weight loss and metabolic effects.

Excess BMI loss is typically ;70–80% (1), and almost in-stant changes occur in glucose homeostasis postsurgery,including frequent remission of diabetes up to 70% (1).After a meal, the rise in glucose and insulin is ;50%higher, reflecting the faster absorption of glucose postsur-gery. Incretins are also affected; prandial gastric inhibitorypolypeptide (GIP) and glucagon-like peptide 1 (GLP-1) peakat ;2 and 10 times higher levels after surgery, respectively(2).

The sustained weight loss achieved by GBP surgery,superior to that achieved by diet regimens, reflects loweredcaloric intake and possibly altered secretion of gastroin-testinal peptides (2). Comparing eating habits before andafter the GBP surgery, patients lowered the intake withmarked reductions in carbohydrate intake in the form ofsweets, soda, and milk/ice cream. Fear of dumping wassuggested as the main mechanism (3). Dumping can occurwithin 10–30 min of food intake when, in the absence of apyloric function, it quickly enters the intestine and pro-duces an osmotic effect. Fluid is shifted from the circula-tion into the intestine, resulting in a fall of blood pressureand tachycardia. In a rat model, similar food preferenceadaptations evolved with time, indicating that the pref-erence shift was dependent on learning and dumping(4).

In a recent study with continuous glucose monitoring(5), we observed hypoglycemic episodes (glucose,3.3 mmol/L[60 mg/dL]) in 50% of patients who had undergone GBPsurgery, of whom none had experienced symptoms of hypo-glycemia. On average, the patients spent 40 min per 24-hperiod in a hypoglycemic glucose range, mostly (80%)without any symptoms. Goldfine et al. (6) observed a high

1Department of Medical Sciences, Uppsala University, Uppsala, Sweden2Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden3Department of Surgical Sciences, Uppsala University, Uppsala, Sweden4Department of Radiation Sciences, Biomedical Engineering, Umeå University,Umeå, Sweden

Corresponding author: Niclas Abrahamsson, niclas.abrahamsson@medsci.uu.se.

Received 15 March 2016 and accepted 10 June 2016.

© 2016 by the American Diabetes Association. Readers may use this article aslong as the work is properly cited, the use is educational and not for profit, andthe work is not altered. More information is available at http://diabetesjournals.org/site/license.

Diabetes Volume 65, September 2016 2667

OBESITYSTUDIES

frequency of asymptomatic hypoglycemia after a mixed-mealtest in patients after GBP surgery, and another study (7)reported asymptomatic hyperinsulinemic hypoglycemia ina small number of patients who have undergone gastricbanding. Furthermore, in a well-documented study ofeight subjects with massive weight loss after verticalbanded gastroplasty, Guldstrand et al. (8) have demon-strated a reduction in the hypoglycemic response of clas-sic counterregulatory hormones.

The brain relies on glucose as the sole source of energyunless ketone bodies are provided, typically upon starva-tion. Gluco-sensing neurons are placed in the brain (e.g.,in the hypothalamus) and in peripheral tissues, such asthe carotid body, oral cavity, gut, and the hepatic portalvein, conveying signals to the brain (9). Upon perceivedhypoglycemia, the brain acts on the anterior pituitary,pancreas, and adrenal medulla to increase the levels ofcounterregulatory hormones such as growth hormone(GH), glucagon, cortisol, epinephrine, and norepinephrine(9,10). In addition, endocrine cells in the pancreatic isletscan respond directly to surrounding glucose levels. Au-tonomic nerve activation contributes to hormonal re-sponses and also directly influences glucose turnover inliver, muscle, and adipose tissue (11). Furthermore, thesecretion of the incretins GLP-1 and GIP may be in-volved, regulated by vagal signaling and the entericnervous system (12). These systems provide a highlyconserved and powerful integrated defense against se-vere hypoglycemia.

In view of previous findings by our group and others,we hypothesized that the counterregulatory response tohypoglycemia would be attenuated postsurgery. In thecurrent study, we examined symptoms and counterreg-ulatory responses during hyperinsulinemic-hypoglycemicclamps in patients before and after GBP surgery. Further,we analyzed GLP-1 and GIP in view of the upregulation oftheir prandial levels after undergoing GBP surgery (13), aswell as their reported role in enhancing the glucagon re-sponse to hypoglycemia (14).

RESEARCH DESIGN AND METHODS

PatientsMorbidly obese (BMI .35 kg/m2) patients without dia-betes were recruited at the Metabolic Outpatient Clinic ofthe Uppsala University Hospital. Patients accepted forbariatric surgery were consecutively invited to participatein the study. Fifteen patients were enrolled in the study,but three patients discontinued their participation afterthe first clamp, one due to pregnancy and two due to lackof time. The per-protocol cohort thus consisted of eightwomen and four men who were examined 3 months (1–6)before and 4–5 months after surgery using a hyperinsu-linemic-hypoglycemic clamp. All patients underwent lap-aroscopic Roux-en-Y GBP surgery, including a 100-cmRoux limb connected to a small proximal gastric pouchand a 50-cm biliopancreatic limb. The GBP surgery was

preceded by 4 weeks of eating a low-calorie diet, accordingto clinical routine, to reduce liver size and intestinal fat.Baseline characteristics are shown in Table 1.

Table 1—Anthropometric measures and clinicalbiochemistry at start of clamp, presurgery and postsurgery

Presurgery Postsurgery

Age (years) 43.1 (10.8)

Height (m) 1.69 (0.09)

Weight (kg)A 116.5 (15.7) 86.4 (16.2)***

BMI (kg/m2) 40.6 (3.1) 30.1 (4.3)***

Body fat (%)B 43.3 (7.4) 36.0 (7.4)***

Body surface (m2) 2.25 (0.2) 1.96 (0.2)***

Systolic BP initial(mmHg) 126 (12) 121 (12)*

Diastolic BP initial(mmHg) 81 (9) 75 (5)

Heart rate (bpm) 65 (8) 56 (10)***

Hemoglobin (g/L) 140.5 (10.7) 133.6 (10.1)*

Plasma creatinine(mmol/L) 75.8 (12.7) 66.7 (9.8)***

eGFR (mL/min/1.73 m2) 82.2 (5.3) 88.1 (3.2)**

Plasma albumin (g/L) 35.2 (2.7) 34.7 (3.2)

HOMA-IR 6.32 (2.2) 1.96 (1.0)***

Fasting plasmaglucose (mmol/L) 5.9 (0.5) 5.4 (0.4)***

HbA1c [% (mmol/mol)] 5.5 (1.5)(37.4 [4.6])

5.2 (1.3)(32.8 [3.1])***

Serum insulin (mU/L) 22.3 (10.1) 8.1 (4.1)***

Serum C-peptide(nmol/L) 1.4 (0.3) 0.9 (0.2)***

Plasma cholesterol(mmol/L) 4.7 (1.0)* 3.8 (0.8)***

Plasma HDL cholesterol(mmol/L) 1.0 (0.3) 1.0 (0.3)

Plasma LDL cholesterol(mmol/L) 3.1 (0.8)* 2.3 (0.7)***

Plasma triglycerides(mmol/L) 1.7 (0.7)* 1.1 (0.3)**

Glucagon (pmol/L) 13.4 (6.9) 8.6 (5.4)**

Cortisol (nmol/L) 233 (99.8) 199 (49.0)

GH (mg/L) 0.18 (0.3) 1.3 (3.1)

Epinephrine (nmol/L) 0.3 (0.001) 0.3 (0.06)

Norepinephrine (nmol/L) 1.4 (0.7) 1.1 (0.3)

GLP-1 (pmol/L) 33.5 (36.5) 18.7 (13.4)

GIP (pg/mL) 55.9 (27.4) 40.0 (12.0)*

FFAs (mmol/L) 203 (55.8) 219 (64.8)

Glycerol (mmol/L) 60.0 (17.1) 61.6 (27.1)

Values are reported as the mean (SD). BP, blood pressure;eGFR, estimated glomerular filtration rate; IR, insulin resistance.AWeight on the day of surgery (112 kg) was not significantlychanged compared with presurgery weight at clamp investiga-tion. BMeasured with bioimpedance. *P , 0.05; **P , 0.01;***P , 0.001.

2668 Gastric Bypass and Responses to Hypoglycemia Diabetes Volume 65, September 2016

Hypoglycemic ClampA hyperinsulinemic-hypoglycemic clamp was performedafter an overnight fast (water intake allowed). The clampwas modified according to the study by Norjavaara et al.(15). After a priming infusion, insulin was infused at a fixedrate, 80 mU/m2 body surface/min together with a variableglucose (200 mg/mL) infusion to maintain plasma glucose atlevels of 5 mmol/L (90 mg/dL) for 0–60 min, 4 mmol/L(72 mg/dL) for 60–90 min, 3.2 mmol/L (58 mg/dL) for90–135 min, and 2.7 mmol/L (49 mg/dL) for 135–165 min.

At 165 min, the insulin infusion was stopped, with glucoseinfusion continued until a glucose level of .4.0 mmol/L(72 mg/dL) was achieved. Plasma glucose in arterialized ve-nous blood was measured every 5 min during the clamp withthe Contour Glucose Meter (Bayer Healthcare, Leverkusen,Germany). Levels of insulin, C-peptide, GH, glucagon, cortisol,

epinephrine, norepinephrine, GLP-1, and GIP were measuredat 0, 60, 90, 120, 135, 150, and 165 min during clamp, andfree fatty acids (FFAs) and glycerol were measured at 0, 135,and 165 min. Participants were continuously monitored withelectrocardiogram, and recordings were used for heart ratevariability (HRV) assessment.

Symptom ScoreThe Edinburgh Hypoglycemia Score, composed of the11 symptoms statistically derived to most closely associatewith hypoglycemia, was used (16). The 11 hypoglycemicsymptoms contain four autonomic symptoms (sweating,palpitation, shaking, and hunger), five neuroglycopenicsymptoms (confusion, drowsiness, odd behavior, speechdifficulty, and incoordination), and two malaise symptoms(nausea and headache). The participants graded theirsymptoms between 1 (no symptoms) and 7 (maximum

Figure 1—Values are reported as the mean (SD). A: Glucose levels during the different plateaus of the clamp, presurgery and postsurgery.B: Insulin levels during the different plateaus of the clamp, presurgery and postsurgery. C: C-peptide levels during the different plateaus ofthe clamp, presurgery and postsurgery. D: Glucose infusion rate (GIR) per lean body mass (LBM) during clamp, presurgery and postsurgery.All time points differ significantly. Note the marked increased demand for glucose during hypoglycemia during the postsurgery examination.Pre, presurgery; Post, postsurgery.

diabetes.diabetesjournals.org Abrahamsson and Associates 2669

symptoms) at 0, 60, 90, 120, 135, 150, and 165 min, andscores for all 11 symptoms were added.

Biochemical MeasurementsBlood samples were taken at 8:00 A.M. after 10 h of fast-ing. Routine blood chemistry and most hormonal analyseswere performed at the Department of Clinical Chemistryat the University Hospital, Uppsala, Sweden. If not ana-lyzed immediately, samples were frozen at 280°C. Thefollowing analyses were used: insulin (Cobas e; Roche),cortisol (Cobas e; Roche), GH (Immulite XP; SiemensHealthcare Global), and C-peptide (Cobas e; Roche). Cat-echolamine analyses (liquid chromatography) were per-formed at the Laboratory of Clinical Chemistry at theKarolinska Universitetssjukhuset, Stockholm, Sweden).

Specific analyses were performed at the Clinical Di-abetes Research Laboratory: glucagon was measured withELISA (Glucagon #10–1271–01; Mercodia, Uppsala, Swe-den). Total GLP-1 and total GIP were measured withELISA (Merck Millipore, Darmstadt, Germany), FFAs weremeasured with the Free Fatty Acid Fluorometric Assay Kit(Cayman Chemical, Ann Arbor, MI), and glycerol was mea-sured with Free Glycerol reagent (Sigma-Aldrich, St. Louis,MO). HOMA of insulin resistance was calculated as follows:(fasting insulin [in mU/L] 3 glucose [in mmol/L])/22.5.

HRV AnalysesAs a marker of efferent activity in the autonomic nervoussystem, HRV analyses were performed as described pre-viously (17,18). Six of the 12 subjects had complete re-cordings both presurgery and postsurgery that were usedfor the statistical analysis. In the other subjects, at leastone recording was incomplete because of technical issues.The total spectral power (PTOT), the power of the low-frequency component (PLF) (0.04–0.15 Hz), and thepower of the high-frequency component (PHF) (0.15–0.50 Hz), all log transformed, were calculated over con-secutive 5-min periods from the complete recording. PHFmainly reflects the parasympathetic activity, whereas PLFreflects a combination of sympathetic and parasympa-thetic activity, and the PLF/PHF ratio is used as a markerof the balance between sympathetic and parasympa-thetic activity (18). The HRV analysis was performed us-ing Matlab Software (MathWorks, Natick, MA).

Statistical AnalysesStatistical calculations were performed in Statistica(Dell Statistica; StatSoft, Aliso Viejo, CA) except for theanalysis of HRV indices, which were performed using R(version 3.1, 2014, Vienna, Austria). Shapiro-Wilks testswere performed to assess normal distributions, in addi-tion to visual graph inspection. Anthropometric and fastinglaboratory results presurgery and postsurgery were com-pared using Student t tests. Metabolite and hormonelevels during hypoglycemic clamp were analyzed withrepeated-measures ANOVA. Areas under the curve duringthe hypoglycemic period (AUChypo) (90–165 min) were

analyzed by paired t tests. Symptom scores were com-pared using paired t tests.

For HRV analysis, the time-variant changes in HRV dur-ing the complete recording were modeled using generalizedadditive mixed-effects models using thin plate splines (19).Differences between presurgery and postsurgery recordingswere modeled as a binominal categorical variable. Changesduring the euglycemic phase (0–60 min) and hypoglycemicphase (90–165 min) were evaluated by ANOVA for repeatedmeasurements.

EthicsThe Regional Ethics Committee of Uppsala, Sweden, ap-proved this study (Dnr 2013/480). Patients gave writteninformed consent, and the study was conducted according tothe tenets of the Declaration of Helsinki.

RESULTS

Clinical EffectsGBP surgery induced marked metabolic effects, notablylower glucose, insulin, and HbA1c levels (Table 1).

Hypoglycemic ClampThe glucose, insulin, and C-peptide levels during theclamp investigations are depicted in Fig. 1. The mean (SD)glucose levels achieved during the clamp investigations,presurgery versus postsurgery, were as follows: for the5.0 mmol/L target period 5.0 (0.4) mmol/L vs. 5.0 (0.4)mmol/L; for the 4.0 mmol/L target period 4.3 (0.3)mmol/L vs. 4.1 (0.3) mmol/L; for the 3.2 mmol/L timeperiod 3.2 (0.3) mmol/L vs. 3.0 (0.2) mmol/L; and for the2.7 mmol/L target period: 2.9 (0.3) mmol/L vs. 2.8 (0.2)mmol/L (all differences were nonsignificant). The circulat-ing levels of insulin during insulin infusion increased to;200 mU/L before surgery and 150 mU/L after surgery,suggesting an increase in insulin clearance after bariatricsurgery, which is in line with what has been previouslyreported (8,20). The glucose infusion rate was signifi-cantly higher during the postsurgery clamp, indicatingimprovement in insulin sensitivity.

Table 2—Parameters during hypoglycemic clampexamination

Presurgery Postsurgery

DSymptom score 10.7 (6.4) 5.2 (4.9)*

Systolic BP peak (mmHg) 132 (13) 125 (12)*

DSystolic BP (mmHg) 6 (9) 4 (6)

Diastolic BP peak (mmHg) 82 (9) 78 (5)

DDiastolic BP (mmHg) 1 (6) 3 (6)

Peak HR (bpm) 75 (9) 69 (10)***

DHR (bpm) 10 (9) 13 (5)

M-value (mg/kg LBM/min) 2.36 (1.22) 4.19 (1.36)**

Values are reported as the mean (SD). D, rise from beginning topeak during clamp; BP, blood pressure; HR, heart rate; LBM,lean body mass; M-value, mean value during euglycemia;Symptom score, Edinburgh Hypoglycemia Score. *P , 0.05;**P , 0.01; ***P , 0.001.

2670 Gastric Bypass and Responses to Hypoglycemia Diabetes Volume 65, September 2016

Figure 2—Values are reported as the mean (SEM) for counterregulatory hormones, incretin hormones, and HRV during hypoglycemicclamp presurgery and postsurgery. *Significant difference. Target glucose levels during clamp are shown on top. Insets: AUChypo diagrams.A: Glucagon. B: Cortisol. C: Epinephrine. D: Norepinephrine. E: GH. F: HRV analysis; PLF/PHF ratio (log), shaded area is the SEM. Note themarked reduction of the response during postsurgery clamp, reflecting a downregulated sympathetic nervous response. G: Total GLP-1.H: Total GIP. Pre, presurgery; Post, postsurgery.

diabetes.diabetesjournals.org Abrahamsson and Associates 2671

Patients experienced fewer symptoms during hypogly-cemia in the postsurgical clamp. The total mean compositeEdinburgh Hypoglycemia Score at the end of hypoglycemia(165 min) was 24.0 (SD 6.9) before surgery and 18.5 (5.2)after surgery. The hypoglycemia-induced increase in totalsymptom score was halved after surgery (Table 2).

Heart rate increased during both clamps, from 65 to75 bpm presurgery and from 56 to 69 bpm postsurgery,respectively. The mean time to peak heart rate was longerpostsurgery; peaks appeared at 130 min presurgery andat 150 min postsurgery. There were no presurgery versuspostsurgery difference in hypoglycemia-induced increase insystolic or diastolic blood pressure or heart rate (Table 2).

Counterregulatory HormonesCounterregulatory hormone levels are shown in Fig. 2 andTable 3. The hypoglycemia-induced glucagon response wasmarkedly decreased postsurgery. During presurgery exam-ination, the glucagon rise started at 120 min and duringthe postsurgery examination at 135 min. Cortisol levelswere similar during the euglycemic phase of both clamps,and hypoglycemia responses became significant at 135 min

both presurgery and postsurgery but were lower post-surgery than presurgery (Table 3). Epinephrine and nor-epinephrine levels rose at 120 min during the clamppresurgery and later (at 135 min) postsurgery. The epi-nephrine and norepinephrine responses during hypogly-cemia were lower postsurgery. Levels of GH were similarup to 120 min and then increased during the presurgeryclamp. Postsurgery, the GH levels responded later, at135 min, and reached a threefold higher level postsur-gery versus presurgery.

Incretin HormonesPlasma levels of incretin hormones GLP-1 and GIP areshown in Table 3 and Fig. 2. Both hormones rose duringthe hypoglycemic part of the clamps. The responses weresignificantly attenuated postsurgery versus presurgery.

FFAs and GlycerolFFAs and glycerol exhibited equal fasting levels presurgeryand postsurgery. The levels fell, FFAs relatively more thanglycerol, during the hyperinsulinemic-hypoglycemic clamps.The nadir levels were lower postsurgery versus presurgery(Fig. 3 and Table 3).

HRVWhen comparing HRV presurgery and postsurgery, therewere significant increases in PTOT (total variability mea-sure) and spectral components (PLF, PHF) during botheuglycemia and hypoglycemia (Table 4). PLF/PHF ratio, ameasure of sympathetic/parasympathetic balance, showedless of an increase from the euglycemic part of the clamp tothe hypoglycemic part after surgery compared with beforesurgery (Fig. 2). The interbeat R-R interval was longer post-surgery versus presurgery, corresponding to the decrease inheart rate, both during euglycemia and hypoglycemia.

DISCUSSION

In this study, we found that GBP surgery is followed byreduced symptoms and responses in classic counterregulatoryhormones and autonomic nervous outflow during hypogly-cemia. GLP-1 and GIP secretion rose during hypoglycemia,implicating a role of the incretins in counterregulation.

The glucagon, cortisol, catecholamine, and incretinresponses to hypoglycemia were all attenuated postsur-gery and showed a similar pattern with later and lowerresponses to hypoglycemia at the post-GBP examinations.GH, like the other counterregulatory hormones, exhibiteda later response postsurgery compared with presurgery.During the postsurgery clamp, however, the magnitudeof the response was higher than during the presurgeryclamp. This may nonetheless be attenuated, in light ofthe participants’ loss of weight after surgery. GH levelsare suppressed in obesity (21), and Corneli et al. (22)found a mean GH response of ;30 mg/L in subjects witha BMI of ;30 kg/m2 who were subjected to a GH-releasinghormone-arginine test compared with the rise to 15 mg/Lduring postsurgery hypoglycemia in the current study(BMI 30.1 kg/m2).

Table 3—Counterregulatory hormones and lipolytic markersduring hypoglycemic clamp

Presurgery Postsurgery

DGlucagon (pmol/L) 30.4 (19.2) 15.2 (10.2)**

AUChypo glucagon(pmol/L/min) 2,130 (860) 1,080 (480)***

DCortisol (nmol/L) 392 (195) 300 (178)*

AUChypo cortisol(nmol/L/min) 28,940 (6,740) 21,250 (5,610)**

DGH (mg/L) 7.0 (4.2) 14.1 (8.9)*

AUChypo GH(mg/L/min) 271 (122) 436 (281)*

DEpinephrine(nmol/L) 2.8 (1.2) 2.2 (1.0)A

AUChypo epinephrine(nmol/L/min) 130 (62) 89 (36)B

DNorepinephrine(nmol/L) 1.3 (0.8) 0.9 (0.6)

AUChypo norepinephrine(nmol/L/min) 146 (50) 113 (42)*

DGLP-1 (pmol/L) 29 (25) 18 (15)

AUChypo GLP-1(pmol/L/min) 2,620 (2,160) 1,250 (1,190)**

DGIP (pg/mL) 420 (44) 14 (11)*

AUChypo GIP(pg/mL/min) 4,690 (2,440) 2,910 (980)*

AUChypo FFAs(mmol/L/min) 1,720 (830) 800 (790)**

AUChypo glycerol(mmol/L/min) 1,370 (460) 810 (470)**

Values are reported as the mean (SD). D, difference betweeninitial and peak level. AP = 0.09. BP = 0.07. *P, 0.05; **P, 0.01;***P , 0.001.

2672 Gastric Bypass and Responses to Hypoglycemia Diabetes Volume 65, September 2016

Insulin levels during clamps were ;25% lower postsur-gery versus presurgery, and the absolute insulin amountinfused was ;12% lower after surgery (data not shown),indicating increased insulin clearance, and more so if ad-justments to body surface are performed. The differencein insulin concentration was not correlated with changesin hormonal responses (data not shown). The findingsare in accordance with those of previous reports, and amore effective hepatic clearance after bariatric surgery hasbeen proposed (8,20).

The nadir levels of FFAs and glycerol were lowerpostsurgery versus presurgery, reflecting the compositeeffects of increased insulin sensitivity; reduced levelsof counterregulatory hormones, in particular catechol-amines, glucagon, and cortisol; and attenuation of thesympathetic nervous response (see paragraph below).Sympathetic activity in the adipose tissue can stimulatelipolysis directly via norepinephrine release (reviewed inBartness et al. [23]).

The HRV results are in accordance with a reducedsympathetic response to hypoglycemia postsurgery, whichcould contribute to the delayed and attenuated catechol-amine levels and lipolysis. In a previous study (24), a rapidimprovement in vasoreactivity and a reduction of heartrate were observed, indicating a reduced sympathetic ac-tivity after GBP.

Presurgery, GLP-1 levels doubled during the hypogly-cemic phase, an increase of the same order as theresponse to hyperglycemia after a mixed meal in healthycontrol subjects (13). This is, however, less than themarked increase in GLP-1 levels typically seen duringthe postprandial hyperglycemic phase postsurgery (25).GLP-1 level has a role in glucagon secretion, and it mayinfluence the a-cell either directly or via indirect mecha-nisms like insulin or autonomic nervous control (26). Infact, the effects on both insulin and glucagon secretionmay be mediated primarily via nervous system circuits(27). There are data suggesting an effect of GLP-1 analogson increasing glucagon secretion during hypoglycemia(28), an action similar to that of GIP (14). In a previousstudy (14), when GIP was infused in physiological dosesduring hypoglycemia, the glucagon level rose 1.5 timesmore than with saline infusion.

In patients with diabetes treated with insulin, hypogly-cemia is known to lower the threshold for counterregula-tory hormonal responses and symptoms (29). Resetting of

Figure 3—Values are reported as the mean (SEM) levels of lipolytic markers during hypoglycemic clamp presurgery and postsurgery.*Significant difference. Target glucose levels during clamp are shown on top. A: FFAs. B: Glycerol. Pre, presurgery; Post, postsurgery.

Table 4—HRV analysis

Preoperative Postoperative

NormoglycemiaRR (s) 0.92 (0.04) 1.08 (0.01)***PTOT (ms2, log) 3.64 (0.05) 3.79 (0.03)***PLF (ms2, log) 3.11 (0.05) 3.21 (0.03)**PHF (ms2, log) 2.88 (0.12) 3.02 (0.03)***PLF/PHF ratio (log) 0.23 (0.03) 0.19 (0.03)

HypoglycemiaRR (s) 0.89 (0.04) 0.99 (0.01)***PTOT (ms2, log) 3.57 (0.06) 3.77 (0.04)***PLF (ms2, log) 3.01 (0.06) 3.13 (0.04)**PHF (ms2, log) 2.66 (0.07) 2.89 (0.05)***PLF/PHF ratio (log) 0.36 (0.10) 0.25 (0.03)***

Change from normoglycemiato hypoglycemia

RR (s) 20.03 (0.02) 20.08 (0.01)***PTOT (ms2, log) 20.07 (0.06) 20.01 (0.03)PLF (ms2, log) 20.09 (0.07) 20.07 (0.04)PHF (ms2, log) 20.23 (0.07) 20.14 (0.04)*PLF/PHF ratio (log) 0.14 (0.05) 0.07 (0.03)*

Values are given as the mean (SEM). Spectral indices arelog-transformed. Hypoglycemia, 90–165 min; normoglycemia,0–60 min; RR, mean interbeat R-R interval. Results indicatehigher parasympathetic activity during normoglycemia post-surgery and lower sympathetic response to hypoglycemiapostsurgery. *P , 0.05; **P , 0.01; ***P , 0.001.

diabetes.diabetesjournals.org Abrahamsson and Associates 2673

the hypoglycemia threshold is also known to occur in preg-nancy (30) and has been described in healthy subjectsafter exercise (31). Boyle et al. (32) kept healthy sub-jects in a hyperinsulinemic-hypoglycemic clamp (glucose2.9 mmol/L [52 mg/dL]) for 4 consecutive days and reportedthat on day 1 the threshold for cognitive impairment was3.05 mmol/L (55 mg/dL) and for symptoms 3.6 mmol/L(65 mg/dL), whereas it decreased to 2.5 mmol/L (45 mg/dL)for both on the final day. Collectively, previous reportsand the present data illustrate a remarkable ability ofthe brain to adapt to lower glucose levels in differentphysiological settings. The potential adverse effects ofan asymptomatic low glucose level have not been wellestablished.

Our present data show an attenuated hypoglycemicresponse in multiple neuroendocrine pathways after GBPsurgery. This indicates that glucose sensing and possiblyglucose utilization in the brain have been augmented, butthe underlying mechanisms need to be further characterized.Glucokinase activity in ventromedial hypothalamus modu-lates the counterregulatory responses, and an increasedactivity attenuates hormonal responses to hypoglycemia(33). An increase in gene expression for glucokinase,GLUT2, and GLP-1 receptor in the hypothalamus has beenreported in food-restricted rats (34). Possibly, increasedbrain glucose uptake leads to an enhanced satiety signalin proopiomelanocortin neurons in the hypothalamus(35) and contributes to the weight maintenance seen inpatients who have undergone GBP surgery.

The current study is limited by the relatively fewparticipants. Moreover, the hypoglycemia was inducedunder experimental circumstances and not during real-lifeconditions, when the insulin concentration would dropduring hypoglycemia. However, the participants in thecurrent study closely resemble the average patient afterGBP surgery (1,36) in that they were aged 43 years, two ofthree were women, and they had lost ;10 BMI units. Theactivity level of participants was not known.

In conclusion, we found that GBP surgery is followedby attenuated symptoms, counterregulatory hormonal re-sponses, and sympathetic nervous activation during hy-poglycemia. We suggest that such adaptive mechanismscontribute to the occurrence of asymptomatic hypoglycemiaand to the prevention of type 2 diabetes after GBP surgery.

Acknowledgments. The authors thank Jan Hall for invaluable workwith the clamps and Prasad Kamble, Cherno Sidibeh, Caroline Moberg, LovisaNordlinder, and Maria Joao Pereira for invaluable assistance during clamps andlaboratory work (all from the Department of Medical Sciences, UppsalaUniversity). The authors also thank Marcus Karlsson (Department of RadiationSciences, Biomedical Engineering, Umeå University) for analysis of the electro-cardiogram recordings and calculation of the HRV indices.Funding. This study was funded by grants from the Research Fund of theSwedish Diabetes Association, Exodiab, Ernfors Foundation, and ALF (SwedishGovernment Research Fund).Duality of Interest. No potential conflicts of interest relevant to this articlewere reported.

Author Contributions. N.A., F.A.K., and J.W.E. designed the study;researched the data; and wrote, revised, and approved the manuscript. J.L.B.,M.S., and U.W. researched the data and revised and approved the manuscript.N.A. is the guarantor of this work and, as such, had full access to all the data inthe study and takes responsibility for the integrity of the data and the accuracy ofthe data analysis.Prior Presentation. Parts of this study were presented in poster form atthe 76th Scientific Sessions of the American Diabetes Association, New Orleans,LA, 10–14 June 2016.

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