0021-972X/04/$15.00/0
`Printed in U.S.A.
`
`The Journal of Clinical Endocrinology & Metabolism 89(6):3055–3061
`Copyright © 2004 by The Endocrine Society
`doi: 10.1210/jc.2003-031403
`
`Glucagon-Like Peptide 1 Induces Natriuresis in Healthy
`Subjects and in Insulin-Resistant Obese Men
`
`JEAN-PIERRE GUTZWILLER, STEFAN TSCHOPP, ANDREAS BOCK, CARLOS E. ZEHNDER,
`ANDREAS R. HUBER, MONIKA KREYENBUEHL, HEIKE GUTMANN, JU¨ RGEN DREWE,
`CHRISTOPH HENZEN, BURKHARD GOEKE, AND CHRISTOPH BEGLINGER
`Division of Gastroenterology and Department of Research (J.-P.G., S.T., C.B.), and Division of Clinical Pharmacology and
`Toxicology and Department of Research (H.G., J.D.), University Hospital, CH-4031 Basel, Switzerland; Central Laboratory
`and Division of Nephrology (A.B., A.R.H., M.K.), Kantonsspital Aarau, CH-5001 Aarau, Switzerland; Division of Nephrology
`(C.E.Z.), Clinica las Condes, Santiago, Chile; Division of Endocrinology (C.H.), Kantonsspital Luzern, CH-6000 Luzern,
`Switzerland; and Division of Gastroenterology and Department of Internal Medicine (B.G.), Klinikum Grosshardern,
`University Hospital, Ludwig Maximilian University, D-81377 Munich, Germany
`
`Glucagon-like peptide-1-(7–36)-amide (GLP-1) is involved in
`satiety control and glucose homeostasis. Animal studies sug-
`gest a physiological role for GLP-1 in water and salt homeosta-
`sis. This study’s aim was to define the effects of GLP-1 on water
`and sodium excretion in both healthy and obese men.
`Fifteen healthy subjects and 16 obese men (mean body mass
`index, 36 kg/m2) were examined in a double-blind, placebo-
`controlled, crossover study to demonstrate the effects of a 3-h
`infusion of GLP-1 on urinary sodium excretion, urinary out-
`put, and the glomerular filtration rate after an iv 9.9-g salt
`load.
`Infusion of GLP-1 evoked a dose-dependent increase in uri-
`
`nary sodium excretion in healthy subjects (from 74 ⴞ 8 to 143ⴞ
`18 mmol/180 min, P ⴝ 0.0013). In obese men, there was a sig-
`nificant increase in urinary sodium excretion (from 59 to 96
`mmol/180 min, P ⴝ 0.015), a decrease in urinary Hⴙ secretion
`(from 1.1 to 0.3 pmol/180 min, P ⴝ 0.013), and a 6% decrease in
`the glomerular filtration rate (from 151 ⴞ 8 to 142ⴞ 8 ml/min,
`P ⴝ 0.022).
`Intravenous infusions of GLP-1 enhance sodium excretion,
`reduce Hⴙ secretion, and reduce glomerular hyperfiltration
`in obese men. These findings suggest an action at the proximal
`renal tubule and a potential renoprotective effect. (J Clin
`Endocrinol Metab 89: 3055–3061, 2004)
`
`EPIDEMIOLOGICAL STUDIES INDICATE that the prev-
`
`alence of obesity in the United States and other Western
`countries has been steadily rising over the past two decades,
`a trend that has been linked to changing dietary habits and
`lifestyle (1–3). Obesity is associated with a high prevalence
`of hypertension, dyslipidemia, cardiovascular disease, dia-
`betes mellitus, and other chronic diseases (4). The annual
`number of deaths attributable to obesity in the United States
`has been estimated to be 280,000 –325,000 (5).
`One of the major factors responsible for obesity-associated
`morbidity and mortality is an elevated blood pressure. The
`pathogenesis of obesity-induced hypertension has not been
`elucidated. Glomerular hyperfiltration and increased so-
`dium reabsorption in the kidney have been demonstrated
`previously in obese patients and animals (6, 7). The authors
`of these studies suggest a putative factor in obese patients
`(e.g. hyperinsulinemia, the renin angiotensin system, the
`sympathetic nervous system, or an increase in renal inter-
`stitial pressure) as being responsible for an enhanced salt
`reabsorption in the proximal tubule and the loop of Henle.
`The consequence of enhanced salt reabsorption is an increase
`
`Abbreviations: AUC, Area under the curve; CH2O, free water clear-
`ance; GFR, glomerular filtration rate; GLP-1, glucagon-like peptide-1-
`(7–36)-amide; HOMA, homeostasis model assessment; IR, insulin resis-
`tance; PRA, plasma renin activity; TcH2O, free water reabsorption.
`JCEM is published monthly by The Endocrine Society (http://www.
`endo-society.org), the foremost professional society serving the en-
`docrine community.
`
`in blood pressure due to extracellular volume expansion.
`Elevated salt reabsorption at a segment proximal to the mac-
`ula densa would reduce sodium chloride delivery to the
`macula densa and initiate a rise in the glomerular filtration
`rate (GFR) through tubulo-glomerular feedback. An elevated
`GFR would tend to return distal sodium delivery to normal.
`In fact, a few studies in humans have shown that obese
`patients have an increased GFR (8, 9). Another study has
`clearly demonstrated that nondiabetic, nonhypertensive
`obese people have an elevated GFR (10).
`The pro-glucagon-derived glucagon-like peptide-1-(7–36)
`amide (GLP-1) is a gastrointestinal hormone that is released
`in response to the presence of food in the distal small intes-
`tine (11, 12). Its physiological effects include a glucose-
`dependent insulinotropic action on pancreatic ␤-cells and
`inhibition of gastric emptying. The latter effect can be inter-
`preted as being part of the ileal brake mechanism, an endo-
`crine feedback loop that becomes activated when nutrients
`are present in the ileum (12, 13). Furthermore, it has been
`suggested that GLP-1 plays a physiological regulatory role in
`controlling appetite and energy intake in normal volunteers
`and in patients with type 2 diabetes mellitus (14 –16). As a
`result of its biological effects, GLP-1 is currently being con-
`sidered as a potential therapeutic agent for the treatment of
`hyperglycemia associated with type 2 diabetes mellitus
`(17–19).
`In previous studies related to the role of GLP-1 in regu-
`lating food intake, we have observed a significant reduction
`of water intake after administration of GLP-1 (15). This ob-
`
`3055
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`3056 J Clin Endocrinol Metab, June 2004, 89(6):3055–3061
`
`Gutzwiller et al. (cid:127) GLP-1 Reduces Glomerular Hyperfiltration
`
`servation has been confirmed in moderately obese patients
`with type 2 diabetes mellitus (16). Also, preliminary data in
`rats suggest a role for GLP-1 in regulating water and salt
`homeostasis (20). On the basis of this information, the present
`study was designed to investigate, in a randomized, double-
`blind, crossover fashion, the effects of GLP-1 on urinary
`sodium and hydrogen ion excretion and on glomerular fil-
`tration in both healthy volunteers and obese patients.
`
`Subjects and Methods
`
`Subjects
`
`Healthy subjects. Fifteen healthy males aged 25.5 ⫾ 0.5 yr who had a
`normal body mass index of 22.1 ⫾ 0.5 were recruited for the study.
`Each volunteer provided written informed consent. The protocol had
`been previously approved by the Human Ethics Research Committees
`of the University Hospitals of Basel and Aarau, Switzerland. Before
`being enrolled in the study, participants were required to complete a
`medical interview, undergo a full physical examination, and participate
`in an initial laboratory screening. No subject was taking any medication
`or had a history of diabetes, hypertension, or kidney disease. Two
`subjects were excluded after the screening visit because of noncompli-
`ance with the study protocol.
`Obese subjects. Sixteen obese males aged 44.6 ⫾ 3.0 yr who had body mass
`index of 36.5 ⫾ 1.2 were recruited for the study (Table 1). Each patient
`provided written informed consent. The protocol had been previously
`approved by the local Human Ethics Research Committee. Before being
`enrolled in the study, participants were required to complete a medical
`interview, undergo a full physical examination, and participate in an
`initial laboratory screening for type 2 diabetes mellitus. The screening
`included a full physical examination and an initial laboratory screening
`including urinalysis. Patients suffering from any heart disease, mi-
`croalbuminuria, or proteinuria were excluded from the study. Two
`patients had previously diagnosed type two diabetes mellitus, one pa-
`
`TABLE 1. Demographic data at screening of 16 obese subjects
`
`Characteristic
`
`Value
`44.6 ⫾ 3.0
`Age (yr)
`36.5 ⫾ 1.2
`BMI (kg/m2)
`122.6 ⫾ 2.7
`Waist circumference (cm)
`132 ⫾ 3
`Systolic blood pressure (mm Hg)
`74 ⫾ 2
`Diastolic blood pressure (mm Hg)
`5.4 ⫾ 0.2
`Cholesterol (mmol/liter)
`1.7 ⫾ 0.1
`Triglycerides (mmol/liter)
`BMI, Body mass index. Data are expressed as mean ⫾ SEM.
`
`tient suffered from hypertension, and six patients had elevated plasma
`cholesterol levels. One patient was subsequently diagnosed with hy-
`pertension, and one patient was diagnosed with type 2 diabetes. Patients
`were kept on their daily medication throughout the study protocol.
`
`Homeostasis model assessment (HOMA)
`
`Insulin resistance (IR) was determined by the HOMA (21) as follows:
`HOMA-IR ⫽ fasting serum insulin ⴱ fasting serum glucose/22.5, where
`insulin is expressed in ␮U/ml and glucose is expressed in mmol/liter
`(21). IR, as determined by this method, closely correlates with more
`complex techniques, such as the euglycemic clamp method (22).
`The index of insulin secretion was calculated as follows: ␤-cell func-
`tion ⫽ (20 ⫻ insulin)/(glucose ⫺ 3.5), where insulin is expressed in
`␮U/ml and glucose is expressed in mmol/liter.
`
`Protocols
`
`Dose response to GLP-1 in healthy subjects. For the purpose of the study,
`placebo, GLP-1 0.375 pmol/kg䡠min, and GLP-1 1.5 pmol/kg䡠min were
`infused in a random order, with infusions being separated by at least
`7 d. All solutions were administered with a concomitant iv saline load
`(Fig. 1).
`On the day of each study, volunteers had fasted from 2400 h onward
`before coming to the research unit 8 h later. The fasting state was
`assessed in the morning by an ultrasound examination of the gallblad-
`der. At 0800 h, volunteers emptied their bladders, and this was con-
`firmed by ultrasound. Afterwards, subjects were maintained supine for
`the duration of the experiment to avoid activation of the renin-angio-
`tensin system with exercise. Teflon catheters were placed into each
`forearm, one for infusions and one for blood sampling.
`After the baseline blood sample was drawn, an iv infusion of hy-
`pertonic saline was started at a rate of 0.06 ml/kg䡠min for 2 h. Simul-
`taneously, a second infusion of 0.9% saline containing albumin 0.5%
`(placebo) or one of the synthetic GLP-1 doses dissolved in 0.9% saline
`and 0.5% albumin was started and continued for 3 h. The three solutions
`were indistinguishable in appearance and were prepared by a pharma-
`cist who was not directly involved in the study. The physician in charge
`was not aware of the respective treatment, thereby permitting a double-
`blind study design. During the first 2 h ofeach experiment, no fluid
`consumption was allowed; starting with the third hour, volunteers were
`allowed to drink water ad libitum. Food intake was not permitted. At the
`end of each 180-min investigation period, water intake and the quantity
`of urine (ml) were measured; bladder emptying was confirmed by
`ultrasound.
`After starting the hypertonic saline infusion, volunteers scored their
`subjective feelings of thirst on visual analog scales at 30-min intervals
`throughout the experiments, with values ranging from 0 –100 mm. A
`score near 0 mm (at the left) for thirst indicated the subject was not thirsty
`
`FIG. 1. Urine period: urine was collected over 180 min.
`Hypertonic saline: infusion with 2.5% saline was done at
`a rate of 0.03 ml/kg䡠min. Placebo or GLP-1: infusion of
`placebo or GLP-1 (0.375 and 1.5 pmol/kg䡠min in the
`group of healthy subjects; and only the 1.5-pmol/kg䡠min
`dose in the group of obese subjects). Drinking time: vol-
`unteers were allowed to drink during 1 h (after stopping
`hypertonic saline infusion between minutes 120 and
`180). Blood samples: time points of blood sampling dur-
`ing experiment. Visual analog scale for thirst (time point
`of measurement).
`
`MPI EXHIBIT 1149 PAGE 2
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`

`

`Gutzwiller et al. (cid:127) GLP-1 Reduces Glomerular Hyperfiltration
`
`J Clin Endocrinol Metab, June 2004, 89(6):3055–3061 3057
`
`at all, and a score near 100 mm (at the right) indicated he was maximally
`thirsty. This scale has been described and validated elsewhere (17).
`Adverse effects were assessed by the attending physician through close
`observation of the participants.
`
`Effect of GLP-1 in obese subjects. The experimental procedure was similar
`to the design depicted in Fig. 1 with the exception that only one dose of
`GLP-1 (1.5 pmol/kg䡠min) was infused. Treatments were given in ran-
`dom order, with infusions being separated by at least 7 d. All solutions
`were administered with a concomitant iv saline load (Fig. 1).
`On the day of each study, patients had fasted from 2400 h onward
`before coming to the research unit 8 h later. The fasting state was
`assessed in the morning by an ultrasound examination of the gallblad-
`der. At 0800 h, the patients emptied their bladders, which was confirmed
`by ultrasound. Afterwards, to avoid additional, nonstandardized acti-
`vation of the renin-angiotensin system, subjects were maintained supine
`for the duration of the experiment. Teflon catheters were placed into
`each forearm, one for infusions and one for blood sampling. After the
`baseline blood sample was taken, an iv infusion of hypertonic saline
`(2.5% NaCl) was started at a rate of 0.03 ml/kg䡠min for 2 h. The total salt
`load was 9.9 ⫾ 0.3 g, which represents the daily salt load in a liberal
`Western diet. Simultaneously, a second infusion of 0.9% saline contain-
`ing 0.5% albumin (placebo) or 0.9% saline plus synthetic GLP-1 (for an
`infusion rate of 1.5 pmol/kg䡠min) was started and continued for 3 h
`(infusion rate 50 ml/h). The additional salt load through the peptide/
`placebo infusion was equal for both treatments and was approximately
`1.35 g NaCl.
`During the whole experiment, free fluid consumption was allowed.
`Food intake was not permitted. At the end of each 180-min investigation
`period, water intake and the quantity of voided urine (ml) were mea-
`sured, and bladder emptying was again confirmed by ultrasound. Ad-
`verse effects were assessed by the attending physician through close
`observation of the participants and by questioning.
`
`mercially available kit (Schering Schweiz AG, Baar, Switzerland) with
`a detection limit of 2.0 ␮IU/ml. Plasma renin activity (PRA) was mea-
`sured by RIA (DSL-25100; Diagnostic Systems Laboratories, Inc., Web-
`ster, TX). Inter- and intraassay coefficients of variation were 3.0 and
`4.3%, respectively, with a detection limit of 1.8 pg/ml. Plasma concen-
`trations of aldosterone were determined using a commercially available,
`solid-phase RIA (Diagnostic Products Corporation, Los Angeles, CA).
`Inter- and intraassay coefficients of variation at 140 pg/ml were 6.9 and
`5.5%, respectively, with a detection limit of 16 pg/ml.
`
`Laboratory analyses
`
`At the beginning of the study and at 30-min intervals, blood samples
`were drawn for glucose, sodium, osmolality, renin activity, angiotensin
`II, and aldosterone determinations (Fig. 1). Sodium excretion, osmola-
`lity, pH, and creatinine were measured in the urine collected at the end
`of 180 min.
`Glucose concentrations were measured with the hexokinase method.
`Sodium concentrations in plasma and in urine were measured on an
`automated analyzer (DADEBEHRING Corp.), following the manufac-
`turer’s instructions, using an ion-selective electrode method. Osmolality
`in plasma and urine were determined on an osmometer (OM802; Vogel
`Corp.) using the freezing point method.
`PRA was measured by RIA (Diagnostic Systems Laboratories). Inter-
`and intraassay coefficients of variation were 3.0 and 4.3%, respectively,
`with a detection limit of 1.8 pg/ml. Angiotensin II was measured by RIA
`(double-antibody RIA; Bu¨ hlmann Laboratories AG, Allschwil, Switzer-
`land) with a detection limit of 0.7 pg/ml and a coefficient of variation
`of 6%. Plasma concentrations of aldosterone were determined using a
`commercially available, solid-phase RIA (Diagnostic Products Corpo-
`ration). Inter- and intraassay coefficients of variation at 140 pg/ml were
`6.9 and 5.5%, respectively, with a detection limit of 16 pg/ml.
`
`Materials
`
`Calculations
`
`Synthetic human GLP-1 was obtained from Bachem (Bubendorf,
`Switzerland). The peptide content was used in the calculation of the
`doses infused. The infusions were prepared by the University of Basel
`Hospital Pharmacy (Basel, Switzerland) according to good manufac-
`turing practice criteria. The solutions were tested for sterility and
`pyrogenicity.
`
`Glucose, electrolyte, pH, osmolality, and renin analyses and
`measurement of glomerular filtration, solute clearance, and
`solute-free water reabsorption
`
`At the start of the study and subsequently in 30-min intervals, blood
`samples were drawn for glucose, sodium, osmolality, creatinine, vaso-
`pressin, renin activity, angiotensin II, and aldosterone determinations
`(Fig. 1). Sodium, chloride, H⫹, and calcium excretion, osmolality, and
`creatinine were measured in the urine collected during 180 min. Glo-
`merular filtration was assessed by creatinine clearance and solute clear-
`ance by the osmolal clearance.
`Creatinine clearance was calculated using the following formula:
`⫽ Ucr
`⫻ V/Pcr, where Ccr is creatinine clearance, Ucr is urine cre-
`Ccr
`atinine concentration, V is the urine volume collected during 180 min,
`and Pcr is plasma creatinine concentration.
`Using the same formula, osmolal clearance was calculated using urine
`and plasma osmolality.
`Free water reabsorption (TcH2O) was determined using the follow-
`ing formula, which considers plasma and urine osmolality: TcH2O ⫽
`V(Uosm/Posm ⫺ 1), where V is urine volume expressed in liters, Uosm
`is urine osmolality, and Posm is plasma osmolality.
`Glucose concentrations were measured with the hexokinase method.
`Electrolyte concentrations in heparin plasma and in urine were mea-
`sured on an automated analyser (Dimension RXL; DADE-BEHRING
`Corp, Wilmington, DE), following the manufacturer’s instructions, us-
`ing an ion-selective electrode method. pH was measured immediately
`from urine samples using an autoanalyzer (ABL 700; Radiometer,
`Copenhagen, Denmark). Plasma and urine osmolality were determined
`with an osmometer (OM802; Vogel Corp., Bern, Switzerland) using the
`freezing point method. Insulin was determined by RIA using a com-
`
`Solute-free or free water clearance (CH2O) was assessed according to
`the following: CH2O ⫽ V ⫻ (1 ⫺ Uosm/Posm), where V is urine volume,
`and Uosm and Posm are osmolality in urine and plasma, respectively.
`The TcH2O by the kidneys is inversely related to the CH2O and, there-
`fore, can be estimated as follows: TcH2O ⫽ ⫺CH2O.
`
`Statistical analysis
`
`Comparisons between the different infusion periods were made by
`ANOVA for repeated measurements or by paired t tests (two-tailed) as
`appropriate. Paired t tests were used when ANOVA was statistically
`significant using a Bonferroni correction. Otherwise, the Wilcoxon
`signed rank sum test was used. For all calculations, STATA software,
`version 6.0 for Windows 95/98 (Stata Corporation, College Station,
`Texas) was used. Unless otherwise noted, data are expressed as means ⫾
`sem. A significance level of 5% was used throughout.
`
`Results
`Effect of GLP-1 on blood glucose, water intake, and thirst in
`healthy subjects
`Figure 2A shows the physiological effect of GLP-1 on
`blood glucose. The graded doses of synthetic human GLP-1
`reduced glucose concentrations and the glucose/time area
`under the curve (AUC) in a dose-dependent manner. Vol-
`unteers reduced water consumption by 15% during the in-
`fusion of the higher GLP-1 dose; however, the difference was
`not statistically significant (P ⫽ 0.178, ANOVA). Water in-
`gestion was slightly reduced from 1405 ⫾ 110 ml (placebo)
`to 1327 ⫾ 95 ml (GLP-1 infusion rate of 0.375 pmol/kg䡠min)
`and, finally, to 1279 ⫾ 78 ml. GLP-1 infusions did not have
`an influence on the visual thirst analog scales (data not
`shown).
`
`MPI EXHIBIT 1149 PAGE 3
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`

`

`3058 J Clin Endocrinol Metab, June 2004, 89(6):3055–3061
`
`Gutzwiller et al. (cid:127) GLP-1 Reduces Glomerular Hyperfiltration
`
`FIG. 2. Data are means (⫾SEM) of plasma glu-
`cose during treatment with GLP-1 and placebo
`(0.9% saline) in (A) healthy subjects and (B)
`obese persons.
`
`Effects of GLP-1 on sodium, urine volume excretion, and
`water reabsorption in healthy subjects
`
`Renal handling of sodium and free water. Sodium excretion
`increased from 74 ⫾ 8 to 86⫾ 9 and 143 ⫾ 18 mmol (P ⫽
`0.0009, ANOVA), and fractional sodium excretion rose from
`1.6 ⫾ 0.2 to 1.7 ⫾ 0.2 and 2.7 ⫾ 0.3% (P ⫽ 0.0004, ANOVA)
`during the placebo, GLP-1 0.375 pmol/kg䡠min, and GLP-1 1.5
`pmol/kg䡠min infusion periods, respectively. According to
`this pattern, osmolal clearance increased from 4.45 ⫾ 0.42 to
`4.96 ⫾ 0.29 and 7.11 ⫾ 0.67 ml/min (P ⬍ 0.0086, ANOVA).
`Water reabsorption equalled 450 ⫾ 56, 573 ⫾ 34, and 747 ⫾
`70 ml (P ⫽ 0.0158) with the infusions of placebo, GLP-1 0.375
`pmol/kg䡠min, and GLP-1 1.5 pmol/kg䡠min, respectively.
`
`Urine volume increased from 360 ⫾ 38 to 400 ⫾ 28 and 639
`⫾ 68 ml/3 h (P ⫽ 0.0009). Urine volume and sodium excre-
`tion changes by infused GLP-1 were dose dependent. The
`creatinine clearance did not change in healthy subjects under
`the experimental conditions (data not shown). Table 2 sum-
`marizes the results on urinary volume and electrolyte
`outputs.
`
`Effects of GLP-1 on the renin-angiotensin-aldosterone axis
`In healthy volunteers, increasing doses of GLP-1 did not
`affect the time course, the PRA, angiotensin II, or the aldo-
`sterone plasma concentrations. The AUCs are depicted in
`Table 3.
`
`MPI EXHIBIT 1149 PAGE 4
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`

`

`Gutzwiller et al. (cid:127) GLP-1 Reduces Glomerular Hyperfiltration
`
`J Clin Endocrinol Metab, June 2004, 89(6):3055–3061 3059
`
`TABLE 2. Effect of GLP-1 on urine fluid and electrolyte output in healthy subjects
`
`Parameter
`
`Placebo
`
`0.375 pmol/kg 䡠 min
`400 ⫾ 28
`360 ⫾ 38
`Urine volume (ml/180 min)
`746 ⫾ 25
`701 ⫾ 59
`Urine osmolality (mosmol/liter)
`86 ⫾ 9
`74 ⫾ 8
`Sodium excretion (mmol/180 min)
`1.7 ⫾ 0.2
`1.6 ⫾ 0.2
`Fractional excretion of sodium (%)
`Data are mean values ⫾ SEM. P values calculated using ANOVA for repeated measurements.
`
`GLP-1
`
`1.5 pmol/kg 䡠 min
`639 ⫾ 68
`673 ⫾ 28
`143 ⫾ 18
`2.7 ⫾ 0.3
`
`P (ANOVA)
`
`0.0009
`0.2595
`0.0013
`0.0004
`
`TABLE 3. Effect of different doses of GLP-1 on hormone responses of the renin-angiotensin-aldosterone system in healthy volunteers
`
`Parameter
`
`Placebo
`
`GLP-1
`
`P (ANOVA)
`
`1.5 pmol/kg 䡠 min
`0.375 pmol/kg 䡠 min
`17,562 ⫾ 1,114
`17,700 ⫾ 1,302
`17,490 ⫾ 1,185
`0.928
`AUC aldosterone
`1,152 ⫾ 81
`1,319 ⫾ 155
`1,256 ⫾ 150
`0.316
`AUC renin
`729 ⫾ 71
`889 ⫾ 122
`860 ⫾ 92
`0.196
`AUC angiotensin II
`Data are mean values ⫾ SEM of AUCs of the renin-angiotensin-aldosterone system. P values were calculated using ANOVA for repeated
`measurements. Units are expressed as pg*180 min.
`
`TABLE 4. Insulin resistance in 16 obese subjects
`
`Insulin resistance data
`Diabetes mellitus (%)
`IR-HOMA (mU䡠mmol/liter2)
`Index of insulin secretion (%)
`Plasma glucose (mmol/liter)
`Plasma insulin (mU/ml)
`Data are mean values ⫾ SEM at study entry.
`
`Obese subjects
`25
`8.2 ⫾ 0.9
`544 ⫾ 167
`5.5 ⫾ 0.3
`33.8 ⫾ 3.0
`
`Glucose tolerance and IR in the obese population
`The diagnosis of type 2 diabetes mellitus was made in 25%
`of the patients (n ⫽ 4) based on abnormalities in glucose
`tolerance (Table 4).b Of these patients, two were on diet
`therapy alone, one was using a combination of sulfonylureas
`and metformin, and one was on a combination of thiazo-
`lidindiones and metformin; none was on insulin therapy.
`There was a family history of diabetes in one subject, and 31%
`of patients had first-degree relatives with diabetes.
`IR data are presented in Table 4. IR as defined by HOMA
`was present in all nondiabetic patients (Table 4).
`
`Effect of GLP-1 on blood glucose and plasma insulin
`Figure 2B depicts the well-known effect of GLP-1 on blood
`glucose, an effect that is similar in both the obese and healthy
`volunteers. Synthetic human GLP-1 reduced glucose con-
`centrations and the glucose AUC (P ⫽ 0.0001). Insulin release
`was slightly stimulated during GLP-1 administration, as
`shown by an increase in the insulin/time AUC (data not
`shown; P ⫽ 0.006), thereby confirming the peptide’s well-
`known insulin-releasing property.
`
`Renal effects of GLP-1
`
`Glomerular filtration and urine volume. The effect of GLP-1 on
`creatinine clearance is shown in Fig. 3. GLP-1 decreased
`creatinine clearance from (mean ⫾ sd) 151 ⫾ 8 to 142⫾ 8
`ml/min (P ⫽ 0.022). In contrast to the decline in creatinine
`clearance, patients showed a higher urine output with GLP-1;
`urine volume increased from 343 ⫾ 35 to 454 ⫾ 62 ml (P ⫽
`0.028, paired t test) during the collection period of 180 min.
`
`FIG. 3. Data are presented with box-whisker plots. With GLP-1 in-
`fusion, the GFR decreased from 151 ml/min (placebo) to 142 ml/min
`(GLP-1; *, P ⫽ 0.022, paired t test) in obese persons.
`
`This improvement was achieved with enhanced solute ex-
`cretion as shown by an increase in the osmolal clearance, an
`increase in sodium, chloride, and calcium excretion, and an
`increase in tubular water reabsorption.
`Renal handling of solutes and water. Osmolal clearance rose
`from 3.8 ⫾ 0.3 to 4.8 ⫾ 0.5 ml/min (P ⫽ 0.023). Figure 4 shows
`the effects of the GLP-1 infusion on sodium and chloride
`excretion. Sodium excretion increased by 60% (P ⫽ 0.015),
`and fractional sodium excretion rose from 1.4 ⫾ 0.1 to 2.3 ⫾
`0.3% (P ⫽ 0.003). Similarly, chloride excretion improved by
`44% (P ⫽ 0.011). Calcium excretion increased by 60% (P ⫽
`0.011; Fig. 4C), whereas hydrogen excretion was dramatically
`reduced by 75% (P ⫽ 0.013; Fig. 4D). Potassium excretion, on
`the other hand, did not change under treatment with GLP-1
`compared with placebo; potassium excretion reached 23.4 ⫾
`1.4 mmol with GLP-1 and 21.6 ⫾ 1.7 mmol with placebo (P ⫽
`0.24).
`
`MPI EXHIBIT 1149 PAGE 5
`
`

`

`3060 J Clin Endocrinol Metab, June 2004, 89(6):3055–3061
`
`Gutzwiller et al. (cid:127) GLP-1 Reduces Glomerular Hyperfiltration
`
`FIG. 4. Effect of GLP-1 or placebo on
`kidney functions in obese subjects. Data
`are means (⫾ SEM) of sodium excretion
`(A) and chloride excretion (B) in milli-
`moles during treatment with GLP-1 (in-
`fusion rate of 1.5 pmol/kg䡠min) and pla-
`cebo (0.9% saline), respectively. The
`ratios GLP-1 and placebo in sodium and
`chloride excretion are approximately
`the same, and changes were statisti-
`cally significant (*, P ⬍ 0.05). Data are
`means (⫾SEM) for calcium excretion (C)
`and H⫹ excretion (D) in millimoles dur-
`ing treatment with GLP-1 and placebo,
`respectively. The differences were sta-
`tistically significant (*, P ⬍ 0.05). These
`changes implicate a mechanism of
`GLP-1 at the proximal tubular cells.
`
`TcH2O did not change; however, there was a trend toward
`an increase from 457 ⫾ 37 (placebo) to 551 ⫾ 41 (GLP-1)
`ml/180 min (P ⫽ 0.12).
`Effects on renin. During GLP-1 infusion, there was a reduction
`in PRA; the PRA/time AUC diminished from 1588 ⫾ 185 to
`1186 ⫾ 84 pg䡠min/ml (P ⫽ 0.044). The drop in PRA correlated
`slightly with the increase in urinary sodium (data not
`shown).
`
`Discussion
`This study demonstrates that the infusion of synthetic
`human GLP-1 significantly increased natriuresis in healthy
`male subjects and in obese, insulin-resistant men. The effects
`of GLP-1 on the kidney broaden the spectrum of the biolog-
`ical actions of the peptide, in addition to its well-known
`effects on blood glucose and insulin release. We have pre-
`viously reported that food and water intake were diminished
`with continuous GLP-1 infusion in patients suffering from
`type 2 diabetes mellitus (16). Here we have documented that
`GLP-1 infusion dose-dependently increases urinary sodium
`excretion in healthy male volunteers. The natriuretic effect of
`GLP-1 was confirmed in obese, insulin-resistant patients,
`
`25% of whom were suffering from type 2 diabetes mellitus.
`The GLP-1-induced natriuresis was paralleled by enhanced
`chloride and urinary calcium excretion. After sodium, chlo-
`ride is the most prevalent ion in the filtrate, and the proximal
`reabsorption is linked to active sodium transport; likewise,
`a reduction of the proximal reabsorption process of sodium
`will affect chloride reabsorption in a similar manner. Most of
`the filtered calcium is reabsorbed in the proximal tubule and
`the medullary loop of Henle. This transport is almost passive
`and follows the gradients established by sodium, chloride,
`and water reabsorption. A decreased sodium and chloride
`reabsorption in the proximal tubule will lead to diminished
`calcium reabsorption in the same tubular segment. The in-
`crease in urinary calcium excretion in this setting reinforces
`the proximal inhibitory effect of GLP-1 on tubular sodium
`reabsorption. Furthermore, this study showed a diminished
`hydrogen excretion, suggesting a reduction in the sodium
`hydrogen exchange in the proximal tubule. These data im-
`plicate a direct effect of GLP-1 on the Na⫹/H⫹ exchange at
`the proximal tubular cells. Preliminary evidence suggests the
`possibility that GLP-1 receptors are present in human kid-
`neys (Gutmann, H., and J. Drewe, unpublished data).
`
`MPI EXHIBIT 1149 PAGE 6
`
`

`

`Gutzwiller et al. (cid:127) GLP-1 Reduces Glomerular Hyperfiltration
`
`J Clin Endocrinol Metab, June 2004, 89(6):3055–3061 3061
`
`Whereas GLP-1 infusion was followed by natriuresis, po-
`tassium excretion remained constant. The excretion of po-
`tassium is mainly determined by its secretion into the lumen
`of the cortical-collecting tubule under the influence of aldo-
`sterone. In this study, aldosterone release was not signifi-
`cantly altered during GLP-1 administration.
`Obesity and type 2 diabetes mellitus lead to a volume
`expansion caused by a high sodium resorption in the prox-
`imal renal tubules. These effects are linked to the develop-
`ment of hypertension in this group of patients. The mecha-
`nism leading to this phenomenon has not yet been
`elucidated. We propose that diminished GLP-1 release in
`obese individuals (23) and in patients suffering from type 2
`diabetes mellitus could, in part, be responsible for an in-
`creased tubular reabsorption of sodium and, as a conse-
`quence, for volume expansion with the potential risk of de-
`veloping hypertension (24, 25). GLP-1 may, therefore, be the
`peptide that protects the body from sodium excess that can
`occur during meals by enhancing sodium excretion, a con-
`clusion that is supported by our data.
`In conclusion, we have shown that a pharmacological dose
`of GLP-1 increases sodium excretion in the proximal renal
`tubule and decreases glomerular hyperfiltration in obese,
`insulin-resistant men. The reduction of the GFR is possibly
`related, via tubulo-glomerular feedback, to the improvement
`in sodium excretion shown during GLP-1 administration.
`This theory is strengthened by the fact that GLP-1 receptors
`have been proposed to be present in human kidney (Gut-
`mann, H., and J. Drewe, unpublished data). Therefore, we
`infer that this natriuretic effect is mediated directly by GLP-1
`receptors in the renal tissue by the incretin hormone. Our
`functional data suggest a GLP-1 action at the proximal renal
`tubulus cells.
`What are the clinical consequences of our results? GLP-1
`might protect obese patients with IR against volume expan-
`sion, glomerular hyperfiltration, and the development of
`arterial hypertension. Diabetes is associated with hypergly-
`cemia, and hyperglycemia produces hyperfiltration, which
`in the long run is associated with kidney damage. We spec-
`ulate that GLP-1 could reverse this vicious cycle. Several
`GLP-1 analogs are under clinical investigation, and consid-
`ering the data presented in this article, they represent a new
`treatment regimen for patients with type 2 diabetes. The
`renoprotective properties of GLP-1 may be an important
`aspect in the prevention of diabetic nephropathy. In addition
`to its blood glucose-lowering effects through stimulation of
`insulin secretion and its appetite-suppressing effects, GLP-1
`exerts additional beneficial effects outside the entero-insular
`axis. The renal effects of GLP-1 merit further investigation.
`
`Acknowledgments
`
`We express our special thanks to Dr. Rolf Graeni, Kantonales Spital
`Wolhusen, Switzerland, for logistical support, Kathleen Bucher for ed-
`iting the manuscript, and Gerdien Gamboni
`for expert
`technical
`assistance.
`
`Received August 11, 2003. Accepted February 20, 2004.
`Address all correspondence and requests for reprints to: Christoph
`Beglinger, M.D., Division of Gastroenterology, University Hospital CH-
`4031 Basel, Switzerland. E-mail: beglinger@tmr.ch.
`This work was supported by a grant from the Swiss National Science
`Foundation (Nr. 3200-065588.01/1) and by a grant from the European
`Commission (Moebius-IST 1999-1159).
`
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