DIALYSIS - TRANSPLANTATION

Acute leptin regulation in end-stage renal failure: The role ofgrowth hormone and IGF-11

DENIS FOUQUE, LAURENT JUILLARD, YVES LASNE, ALBA TABAKIAN, MARTINE LAVILLE, MARIE-ODILE JOLY,and MAURICE LAVILLE

Departments of Nephrology, Biochemistry, and Centre de Recherche en Nutrition Humaine de Lyon, Hopital Edouard Herriot, Lyon,France

Acute leptin regulation in end-stage renal failure: The role ofgrowth hormone and IGF-1.

Background. Leptin, a recently discovered peptide involved innutrient intake and energy expenditure, has been shown to beabnormally regulated in certain conditions such as obesity. Inchronic renal failure, leptin appears to be increased. However,little is known about leptin regulation during chronic renal failure(CRF).

Methods. We measured serum leptin in eight well nourished,chronic hemodialysis patients (seven males, one female) receivinganabolic factors for three days as either recombinant insulin-likegrowth factor-1 (rhIGF-1) or a combination of recombinantgrowth hormone (rhGH) plus recombinant IGF-1, in a randomcross-over trial.

Results. Serum leptin values were in the range of normalvolunteers matched for body mass index. As reported in otherconditions, serum leptin was strongly correlated with patients drybody wt (P 5 0.01) and body fat (P 5 0.0001). Both treatmentsaffected serum leptin in a rapid and opposite manner. RhIGF-1decreased serum leptin from 11.2 6 20.8 (SD) to 4.3 6 3.8 mg/liter(P 5 0.011), whereas the combination of rhGH 1 rhIGF-1increased serum leptin from 7.4 6 9.4 to 21.0 6 32.9 mg/liter (P 50.011). Regression analyses indicated a linear regression betweenserum leptin and insulin variations after treatment.

Conclusions. This study shows for the first time that bothrhIGF-1 and rhGH acutely regulate serum leptin in dialysispatients. Whether leptin changes are explained by the concomi-tant insulin variation should be further studied under renal failureconditions.

Recently, the ob gene product, a 16 kD peptide calledleptin, was identified from obese mice [1]. Deletion muta-tion of this gene leads to absence of circulating leptin,whereas administration of recombinant leptin strongly re-

duces food intake and induces a rapid weight loss in leanand obese mice [2–4]. The site of action of leptin is mainlythought to be the hypothalamic appetite center [5, 6],although leptin receptors have now been reported in pan-creas, liver and kidney [7–9]. Clinical studies have shownthat obese patients present high circulating levels of leptinsuggesting a resistance to the action of leptin [10]. Somedata suggest that this resistance may be peripheral [11],whereas other recent studies involve leptin binding tospecific circulating proteins [12] as well as abnormal cross-ing of leptin through the blood brain barrier [5].

Because chronic renal failure patients often presentmoderate to severe malnutrition and reduced food intake,it seemed of interest to characterize the regulation of thiscompound in end-stage renal disease patients undergoingchonic dialysis therapy. We designed a study to measure themetabolic effects of recombinant growth factors in dialysispatients, and therefore had the opportunity to measureserum leptin and the regulatory effects of insulin-likegrowth factor (IGF)-1 and growth hormone (GH) on serumleptin in those individuals.

METHODS

Patients

Eight well nourished, chronically hemodialyzed patients(seven males, one female; Table 1) were enrolled in ametabolic study after an informed signed consent approvedby the consultative committee for biomedical research atLyon. These patients had been treated by chronic hemodi-alysis for at least four months prior to the study. There wasno acute or chronic inflammatory process and patients didnot take any glucocorticoids or insulin the last eight weeksbefore and during the study. Two patients were on regularerythropoietin treatment that was not modified during thestudy. They were under regular antihypertensive treatmentwhen needed and oral calcium supplements. The patients’usual diet was assessed from a dietary interview at inclu-sion, and was kept constant during hospital stays (Table 1).

1 See Editorial by Dagogo-Jack, p. 997.

Key words: nutrition, leptin, dialysis, chronic renal failure, peptides,obesity, insulin.

Received for publication August 20, 1997and in revised form February 12, 1998Accepted for publication March 27, 1998

© 1998 by the International Society of Nephrology

Kidney International, Vol. 54 (1998), pp. 932–937

932

Patients were asked to remain for four days in a meta-bolic ward on two occasions that were separated by 14 to 28days. During these stays, they underwent a daily blooddrawn and two metabolic studies, one on the first (D1) andthe second on the fourth day (D4). In order to keepmetabolic conditions as stable as possible, patients under-went their routine dialysis sessions on day 0, day 2 (after-noon) and day 4 (evening).

Anthropometry

Anthropometry was collected once for each patient atenrollment in the study. Measurements included the deter-mination of actual body weight corrresponding to the dryweight of patients, that is, the lowest weight obtained at theend of their regular dialysis session. Relative body wt(RBW) was expressed as the ratio of patient dry weight bystandard body wt as determined by Metropolitan Insurancetables [13]. Skinfold thickness measurements were per-formed in triplicate at four different locations: biccipital,tricipital, subscapular and suprailiac. Fat mass was deter-mined from the Durnin and Wormseley tables [14].

Treatment

The study was designed to compare the effects of eitherrecombinant human (rh)IGF-1 administration alone (40mg/kg body wt every 12 hr) or a combination of rhIGF-1 (40mg/kg body wt every 12 hr) and recombinant human growthhormone, rhGH (Genotonorm®; 50 mg/kg body wt perday). Treatments were administered subcutaneously in theanterior quadrant of the quadriceps at 8:00 a.m. (rhIGF-1)and 8:00 p.m. (rhIGF-1 and rhGH). Both recombinanthormones were generously provided by Pharmacia andUpjohn, Sweden.

Serum measurements

Blood was drawn at 7:45 a.m. before breakfast and aftera 10 hour overnight fast. Samples were immediately chilledon ice. After clotting, samples were centrifuged at 3000 g at4°C for 10 minutes and serum was then pipetted forconservation at 270°C until measurements.

Serum leptin was measured in duplicate by RIA (LincoResearch, St. Louis, MO, USA) as previously described[10]. The sensitivity of assay was 0.5 ng/ml. Intra- andinterassay coefficient of variation were 6.2 and 8.3%,respectively. Normal serum leptin values in our laboratorywere 11.0 6 (SD)4.9 and 5.1 6 3.3 ng/ml in female and male,respectively. Serum leptin values for adult patients under-going routine hemodialysis in our unit were found to be16.7 6 17.8 and 12.6 6 11.4 ng/ml in female (N 5 23) andmale (N 5 23), respectively.

Serum IGF-1 was measured by RIA using a rabbitantibody after acid-ethanol extraction and cryoprecipita-tion as previously described [15] in order to eliminate theIGF binding protein degradation products often present inchronic renal failure. Adult normal serum IGF-1 valueswere 283 6 77 mg/liter. Serum GH was measured byimmunoradiometric assay using a mouse monoclonal anti-body (BioMerieux, Marcy l’Etoile, France). The GH assaylimit of detection was 0.16 mg/liter. Intra- and interassaycoefficient of variation were 5.2 and 6.0%, respectively.Serum insulin was measured by immunoradiometric assayusing a mouse monoclonal antibody (ERIA DiagnosticsPasteur, Marnes la Coquette, France). The insulin assaylimit of detection was 0.2 mU/liter. Intra- and interassaycoefficient of variation were 3.8 and 7.5%, respectively.

Statistics

The paired t-test and Wilcoxon rank test were performedfor comparisons between final and baseline values orpercentage variations from baseline when applicable, andfor between treatment comparisons. For correlation searchbetween baseline serum values (that is, insulin, IGF-1, GHand leptin) and various parameters, the mean of the twobaseline measures was used. Statview software (Abacusconcept, CA, USA) was used on a Macintosh computer. AP value less than 0.05 was considered significant.

RESULTS

Table 1 shows the characteristics of patients. Relativebody weigth (RBW) was greater than 90% of standard bodywt. None of them were obese or malnourished as assessedby actual body wt, serum albumin or body mass index(BMI). However, one male patient was at the upper normallimit, had a BMI of 25.5, a RBW of 106% and a fat mass of24.3%. His baseline serum leptin and insulin values were46.2 ng/ml and 13.9 mU/liter, respectively. The only womanin this study had a BMI of 18.8 kg/m2 and a fat mass of23.0%. Her serum leptin and insulin values were 7.8 ng/mland 3.8 mU/liter, respectively. Otherwise, these two pa-tients responded equally to the treatments, expressingsimilar changes to those of the remaining men and there-fore were kept for final analysis.

Serum hormones appear on Table 2. Baseline values forall parameters (D1) were not different for the two basalmeasurements separated by 14 to 28 days. Mean basal

Table 1. Characteristics of the subjects

Age year 41.6 6 12.1Sex ratio (M/F) 7/1Dialysis treatment duration months 30.1 6 23.9

Range 4–74Actual body weight kga 63.7 6 13.2Relative body weight %b 95 6 7Serum albumin g/liter 45.5 6 3.2Body Mass Index 21.6 6 2.2Body fat % 16.1 6 5.0Lean body mass kg 53.3 6 9.9Energy intake kcal/kg per day 36.2 6 7.8Protein intake g/kg per day 1.40 6 0.27

Values are expressed as mean 6 SD.a As dry body weight at the end of the last hemodialysis treatment

preceding the first treatment periodb Relative body weight, calculated from [13]

Fouque et al: Leptin regulation in dialysis patients 933

concentrations of serum leptin were 10.1 6 17.7 ng/ml inmales and 7.7 ng/ml in the female patient and were notdifferent from normal adults (5.1 6 3.3 in males and 11.0 64.9 ng/ml in females, N 5 27). Serum IGF-1 and GH valueswere in the range of normal adults (Table 2). Serum insulinwere in the range of fasting healthy adults for the sevenlean patients (5.0 6 2.2 mU/liter), whereas it was markedlyelevated in the borderline overweight patient (13.9 mU/liter).

As recently described in dialysis patients [16], we foundstatistical correlations by linear regression between base-line leptin and body wt (r 5 0.82, P 5 0.012), BMI (r 50.72, P 5 0.047), percent of body fat mass (r 5 0.74, P 50.03) and most significant between leptin and absolute fatmass (r 5 0.99, P 5 0.0001; Fig. 1). When the borderlineoverweight patient was not considered for analysis, theregression between leptin and weight or BMI lost itssignificance, but was still robust between leptin and percent

of body fat mass (r 5 0.82, P 5 0.025, N 5 7) or absolutefat mass (r 5 0.85, P 5 0.014, N 5 7).

After a three-day rhIGF-1 treatment (40 mg/kg body wtevery 12 hr, total of 5 injections), serum IGF-1 valuesincreased by 103% (P 5 0.011 from baseline; Table 2).When patients received the rhIGF-1 1 rhGH treatment(40 mg/kg rhIGF-1 every 12 hr, total of 5 injections and 50mg/kg rhGH per day, total of 3 injections), serum IGF-1increased by 317% from baseline (P 5 0.01). The incre-ment in serum IGF-1 under the rhIGF-1 1 rhGH combi-nation appeared to become statistically different from theIGF-1 treatment alone at day 3 (data not shown). SerumGH levels increased during the combination of rhIGF-1 1rhGH (P 5 0.011 from baseline), whereas they tend todecrease under rhIGF-1 treatment alone (P 5 0.12, NS).

The values for serum leptin were statistically modifiedfrom baseline by each treatment. Recombinant IGF-1administration decreased serum leptin levels by 25 6 26%from baseline (P 5 0.025), whereas the combination ofrhIGF-1 1 rhGH increased fasting serum leptin by 117 691% from baseline (P 5 0.009; Fig. 2). Serum insulin wasalso markedly altered by both treatments. Under rhIGF-1alone, fasting insulin decreased to 3.6 6 2.9 mU/liter (P 50.011; Table 2). On the contrary, under rhGH 1 rhIGF-1treatment, the serum levels of insulin markedly increased to33.6 6 51.9 mU/liter, that is, by a magnitude of five ascompared to baseline (P 5 0.011; Table 2).

The effects of growth factors on leptin regulation may beassessed from the relationship between the variation inserum leptin and the concomitant change in other serumvalues after treatment at D4. Figure 3 plots the change inserum leptin versus the change in serum insulin duringtreatment. All baseline leptin values were modified in asimilar pattern, that is, the leptin value increased whenserum insulin rose under the combined rhIGF-1 1 rhGHtreatment, whereas serum leptin decreased when seruminsulin was blunted under rhIGF-1 treatment alone (Fig.3). There was a strong, statistically significant correlationbetween the change in serum leptin and the concomitantchange in serum insulin (r 5 0.66, P 5 0.006, Fig. 3). If theborderline overweight patient was not considered for anal-ysis, this relationship was still significant (r 5 0.58, P 50.029).

Fig. 1. The linear regression between fat mass as estimated by skinfoldthicknesses, and fasting serum leptin of eight well nourished, mainte-nance dialysis patients (r 5 0.99, P 5 0.0001).

Table 2. Serum hormones values before and after three days of treatmenta

Treatment

rhIGF-1 80 mg/kg/day/3 days (rhIGF-1 80 mg/kg 1 rhGH 50 mg/kg)/day/3 days

Baseline Day 4 Baseline Day 4

Leptin mg/liter 11.2 6 20.8 4.3 6 3.8b 7.4 6 9.4 21.0 6 32.9b,c

Insulin mU/liter 6.5 6 4.9 3.6 6 2.9b 5.8 6 3.0 33.6 6 51.9b,c

Growth hormone mg/liter 3.4 6 4.8 1.5 6 1.6 2.1 6 1.7 12.3 6 3.9b,c

IGF-1 mg/liter 355 6 164 706 6 294b 357 6 144 1399 6 491b,c

a Values are mean 6 SD. Eight subjects were studied under both rhIGF-1 treatment or rhIGF-1 1 rhGH treatment in a random order.b Differs from the corresponding baseline value: P , 0.025c Differs from rhIGF-1 treatment: P , 0.025, Wilcoxon rank test

Fouque et al: Leptin regulation in dialysis patients934

Comparable analyses were performed for serum growthhormone and serum IGF-1 values. However, no significantrelationship was found between these latter values andserum leptin. In addition, we did not found any relationshipbetween baseline serum leptin (D1) and either baselineserum free fatty acids, serum total cholesterol or serumtriglycerides (data not shown), nor was any relationshipobserved between the absolute or percentage change inthese values after any treatment.

DISCUSSION

Malnutrition is a common feature in end-stage renaldisease [17–19]. Anorexia has been commonly reported inthese patients, and despite intense counseling from thestaff, patients often fail to increase their food intake. Anumber of reports have shown that it is not uncommon torecord energy intake as low as 23 to 25 kcal/kg body wt/dayin maintenance hemodialysis patients [20]. Furthermore,this spontaneous reduction in food intake may appearbefore end-stage renal disease, and two studies reported alinear energy and protein intake decrease with the declineof renal function over time [21, 22].

Recently, a new peptide affecting food intake, namedleptin, has been discovered [1], and due to renal failure itssynthesis or clearance may be impaired. Serum leptin hasbeen reported to be elevated in patients with pre-dialysischronic renal failure [23, 24] and undergoing chronic hemo-or peritoneal dialysis [16, 25–27]. Although the kidney isresponsible for about 80% of leptin clearance in healthyadults [28], little is known on the fate of leptin in chronic

renal failure patients. Stenvinkel, Heimburger andLonnqvist recently pointed out that dialysis patients withhigh serum leptin could be separated from those withnormal leptin based on their fasting serum insulin [24]. Wehad the opportunity to measure this peptide during meta-bolic studies in well nourished, non-obese maintenancehemodialysis patients and looked at the potential role oftwo other peptide hormones, growth hormone and IGF-1,in altering the leptin status in eight well nourished, non-obese maintenance dialysis patients.

The leptin values we report here are within the normalrange of healthy volunteers matched for gender and bodymass index. To further clarify this unexpected finding, welooked at other parameters in these lean patients. Interest-ingly, like Stenvinkel et al [24], we found that the presentpatients, except one, had low to normal fasting seruminsulin levels and their leptin values were in the normalrange. On the contrary, the borderline overweight patienthad elevated fasting leptin and insulin values (46.2 ng/mland 13.9 mU/liter, respectively), confirming the thresholdinsulin value of about 14 mU/liter reported by Stenvinkel etal for elevated leptin in pre-dialysis CRF patients [24].

The relationship between serum leptin and body compo-sition in dialysis patients have been precisely documentedin a recent report by Heimburger et al [16]. Although weused a different method for estimating body fat mass(skinfold thickness measurement), the present results (Fig.1) confirm Heimburger’s findings, showing a strong rela-tionship between body fat, that is, percent body fat, totalbody fat and BMI, and leptin, giving strength to the role ofleptin as a marker of body composition during maintenancehemodialysis [16]. This relationship is still present in alimited range of body fat values if the bigger patient isremoved from analysis (r 5 0.85, P 5 0.01), and also nicely

Fig. 2. The percent change from baseline of serum leptin in eightmaintenance hemodialysis patients after a three-day treatment of eitherrhIGF-1 or a combination of rhIGF-1 1 rhGH administered in a randomorder, separated by 14 to 28 days (*P 5 0.025 vs. baseline; †P < 0.01 vs.baseline and vs. rhIGF-1 alone).

Fig. 3. The linear regression between the percent change from baseline inserum leptin and the concomitant serum insulin variation after treatment(r 5 0.66, P 5 0.006).

Fouque et al: Leptin regulation in dialysis patients 935

confirmed in end-stage renal disease patients having ele-vated fat mass as high as 40 to 50% body wt [16].

Factors regulating serum leptin in dialysis patients

Leptin is acutely regulated by peptidic hormones inend-stage renal disease patients. Indeed, increasing serumIGF-1 values by a magnitude of two after administeringrhIGF-1 during three days lowered serum leptin by about25% (P , 0.03; Fig. 2). Since adipocytes are sensitive toIGF-1 [29], the administration of exogenous rhIGF-1 maydirectly reduce the adipocyte leptin release. This hypothesiscannot be confirmed due to the lack of experimental data.On the other hand, because IGF-1 induces a negativefeedback on GH secretion [30], one may argue that theobserved leptin drop may be secondary to the GH decreaseunder rhIGF-1 treatment. We did not observe a significantdecrease in serum GH, since GH fell in five patients andeither did not change or increased in the other threepatients. In addition, no significant correlation was foundbetween the decrease in serum leptin and serum GH.

The addition of rhGH to rhIGF-1 not only prevented theIGF-1-induced serum leptin decrease, but actually in-creased leptin values in all patients (P 5 0.011, Fig. 2).Thus, rhGH may have had an independant effect on leptinrelease, as reported by Bianda and colleagues in eight adultGH-deficient patients without renal failure [31]. Over thelong-term, this fact may be attributed to GH-inducedchanges in body composition (increase in body wt, seruminsulin and appetite). However, during short-term meta-bolic studies, changes in body composition are unlikely tooccur and the actual results are in line with those of Biandaet al, convincingly showing an increase in serum leptinduring a short-term rhGH treatment [31].

A number of experimental in vitro and in vivo studieshave shown that insulin regulates adipocyte leptin secretion[32, 33]. In humans, a prolonged exposition to insulinincreased serum leptin, whereas a five hour insulin admin-istration did not change the serum leptin value [32]. Ininsulin resistant states, increased serum leptin levels havebeen reported [34]. Since an insulin resistant state is oftenpresent in ESRD patients, an impaired leptin status may bepresent.

The linear regression observed on Figure 3 suggests apotential regulatory effect of insulin on leptin secretionafter three days of treatment, since no correlation wasfound between leptin and either GH or IGF-1 levels, orbetween their concomitant changes after treatment. WhenrhIGF-1 was administered alone, serum leptin significantlydecreased, and was significantly related to the serum insulindecrease. The only other study that reports the actions ofrhIGF-1 on leptin in human showed a 48 hour delaybetween the decrease in C-peptide and serum leptin [31],which is not in favor of an acute insulin regulation of leptinsecretion. Since we did not measure leptin daily, we cannotconfirm the fact that insulin acutely blunts the leptin

release. However, at 72 hours, it is worthwhile to emphasizethe relationship between serum leptin and serum insulin inMHD patients.

The addition of rhGH to rhIGF-1 increased serum leptinand insulin values (Table 2). This GH effect not onlysurpassed the IGF-1-induced leptin reduction, but actuallyincreased the serum leptin by a magnitude of two. Con-comitantly, serum insulin dramatically rose in all patients, awell described consequence of an acute rhGH administra-tion. Since we did not directly administer insulin, we cannotclearly identify the GH effect from that of insulin on leptinrelease. However, in support of a delayed insulin regulationof leptin release, Boden et al recently showed in healthyadults that a constant insulin administration did increaseserum leptin in a dose-dependant manner after 24 hours ofinfusion and was sustained during the rest of the 72 hourstudy [35]. In an in vitro study, Boni-Schnetzler et al showedthat administering rhGH for six days to hypophysectomizedrats did not increase adipocyte leptin mRNA [36]. Inter-estingly, these authors did not observe an increase in seruminsulin in the rhGH treated group of rats. Although theacute regulation of leptin mRNA may not reflect the serumleptin profile, it is possible that, in order to observe aGH-induced leptin release, an increase in serum insulinshould have occurred [36].

In conclusion, according to the available research dataand the present study, serum leptin appears to be a reliableindex of body fat in end-stage renal disease. Elevatedvalues may be seen in patients with insulin resistanceand/or elevated fasting serum insulin, whereas normalvalues are often present in lean CRF patients with lowserum insulin values. Administering recombinant hor-mones such as growth hormone and IGF-1 modify serumleptin, suggesting that there is a conserved metabolicregulation of leptin production in end-stage renal disease.Whether these changes are directly caused by rhIGF-1 andrhGH or the concomitant insulin changes remain to beclarified in the dialysis condition. Furthermore, the role ofinsulin on leptin accumulation should also be studied, sincetreatments may worsen insulin resistance and lead to moreelevated serum leptin levels and a potential anorecticstatus.

ACKNOWLEDGMENTS

This study was supported by the Baxter ExtraMural Grant Program. Wethank Bioanalytical Immunology, Development Pharmacia and Upjohn,Sweden for providing recombinant growth factors. We thank the AURALfor close collaboration. We are indebted to the nurses of the metabolicand dialysis units for excellent patient care. We also express our gratitudeto Ms. Catherine Riouffreyt, Ms. Jocelyne Peyrat and Ms. Corinne Urbainfor technical assistance, and Prof. J.P. Riou for fruitful discussions.

Reprint requests to Dr. Denis Fouque, Department of Nephrology, HopitalEdouard Herriot, 69437 Lyon Cedex 03, France.E-mail: fouque@univ-lyon1.fr

Fouque et al: Leptin regulation in dialysis patients936

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