Clinical Endocrinology (2003)

59

, 431–436

© 2003 Blackwell Publishing Ltd

431

Blackwell Publishing Ltd.

Effect of acute reduction of free fatty acids by acipimox on growth hormone-releasing hormone-induced GH secretion in type 1 diabetic patients

Paula Alvarez*, Luisa Isidro*, Roberto Peinó, Alfonso Leal-Cerro†, Felipe F. Casanueva§, Carlos Dieguez¶ and Fernando Cordido*

,

‡

*

Department of Endocrinology, Hospital Juan Canalejo la Coruña,

†

Department of Endocrinology, Virgen del Rocio, Sevilla and

‡

Department of Medicine, University of La Coruña, La Coruña, Departments of

§

Medicine and

¶

Physiology, University of Santiago, Santiago, Spain

(Received 20 December 2002; returned for revision 4 February 2003; finally revised 25 February 2003; accepted 15 April 2003)

Summary

BACKGROUND

In type 1 diabetes mellitus (DM1), highGH basal levels and exaggerated responses to severalstimuli have been described. Acipimox is an antilipol-ytic drug that produces an acute reduction of free fattyacids (FFA). The aim of this study was to evaluate theeffect of the reduction of plasma FFA with acipimox,alone or in combination with GHRH, on GH secretionin DM1.

METHODS

Six type 1 diabetic patients were studied(three women, three men), mean age of 30 ±±±±

2·1 years,body mass index (BMI) 23·1 ±±±±

1·5 kg/m

2

. As a controlgroup, six normal healthy subjects of similar age, sexand weight were studied. Each patient and controlreceived GHRH [1 µµµµ

g/kg intravenously (i.v.) at min 180],acipimox (250 mg orally at min 0 and 120) and GHRHplus acipimox on three separated days. Subjects servedas their own control. Blood samples were taken atappropriate intervals for determination of GH, FFA andglucose.

RESULT

In control subjects, the GH area under thecurve (AUC; µµµµ

g/ l ××××

120 min) was for acipimox-treated1339 ±±±±

292 and 1528 ±±±±

330 for GHRH-induced secre-tion. The GH AUC after the administration of GHRHplus acipimox was 3031 ±±±±

669, significantly greaterthan the response after acipimox alone (

P

< 0·05) orGHRH alone (

P

< 0·05). In diabetic patients, the GH

AUC was for acipimox-treated 2516 ±±±±

606 and 1821±±±±

311 for GHRH-induced secretion. The GH AUC afterthe administration of GHRH plus acipimox was 7311±±±±

1154, significantly greater than the response afteracipimox alone (

P

< 0·05) or GHRH alone (

P

< 0·05). TheGH response after acipimox was increased in diabeticwhen compared with normal (

P

< 0·05), with a GH AUCof 1339 ±±±±

292 and 2515 ±±±±

606 for normal subjects anddiabetic patients, respectively. The GH response afteracipimox plus GHRH was increased in diabetic whencompared with normal (

P

< 0·05), with a GH AUC of3031 ±±±±

669 and 7311 ±±±±

1154 for normal subjects anddiabetic patients, respectively. The administration ofacipimox induced a FFA reduction during the entiretest.

CONCLUSIONS

Reduction of free fatty acids with acip-imox is a stimulus for GH secretion in DM1. The com-bined administration of GHRH plus acipimox inducesa markedly increased GH secretion in type 1 diabeticpatients when compared with normal subjects. Thesedata suggest that patients with DM1 exhibit a greaterGH secretory capacity than control subjects, despitethe fact that endogenous FFA levels seems to exerta greater inhibitory effect on GH secretion in thesepatients.

Growth hormone (GH) secretion is mainly dependent on theinteraction between GHRH, Ghrelin, the recently isolatedendogenous ligand of the GH-secretagogues (GHS)-receptorand somatostatin (Cuttler

et al

., 1996; Giustina & Veldhuis, 1998;Dieguez & Casanueva, 2000). In addition several neurotrans-mitters, peripheral hormones and metabolic signals influence GHsecretion (Cutler

et al

., 1996; Giustina & Veldhuis, 1998).In type 1 diabetes mellitus (DM1), high GH basal levels and

exaggerated responses to several stimuli have been described. Innormal subjects, hyperglycaemia inhibits pituitary GH responseto provocative stimuli. However, DM1 patients, despite elevatedblood glucose levels, show exaggerated GH responses to severalphysiological and pharmacological provocative tests (Hansen,1970; Lorenzi

et al

., 1980; Krassowski

et al

., 1988; Almqvist

et al

., 1999), and they have mean 24-h GH levels higher thanthose of normal subjects (Asplin

et al

., 1989; Edge

et al

., 1990).

Correspondence: Dr Fernando Cordido. Servicio de Endocrinología, Hospital Juan Canalejo, Xubias de Arriba 84, 15006 La Coruña, Spain. Fax: +34 981178001. E-mail: fernando_cordido@canalejo.org

432

P. Alvarez et al.

© 2003 Blackwell Publishing Ltd,

Clinical Endocrinology

,

59

, 431–436

Abnormal GH secretion has been suggested as a causative factorin the development of the microangiopathic complications of dia-betes (Lorenzi

et al

., 1980; Salardi

et al

., 1986; Edge

et al

., 1990;Blankestijn

et al

., 1993). Abnormal GH secretion may contributeto the altered metabolic control of diabetes, although chroniceffects of elevated GH levels on the metabolism and control ofdiabetes are unknown (Hansen

et al

., 1970; Lorenzi

et al

., 1980;Krassowski

et al

., 1988; Asplin

et al

., 1989; Edge

et al

., 1990;Miller

et al

., 1992; Carroll

et al

., 2000).Diabetes is associated with a generalized impairment in lipid

metabolism and with increased plasma free fatty acid (FFA)levels (Basu

et al

., 2001; Boden

et al

., 2001), although othershave found normal baseline plasma FFA in diabetic patients(Coiro

et al

., 1999). A classic feedback relationship has beenpostulated between GH and the FFA. GH has a direct lipolyticeffect on adipose tissue, leading to the release of glycerol, FFAand ketone bodies (Vance, 1993). Pharmacological reduction ofFFA is associated with GH release (Dieguez & Casanueva, 1995).Acipimox is an antilipolytic drug that blocks lipolysis, is devoidof side-effects and produces an acute reduction of FFA (Pontiroli

et al

., 1990, 1991).The aim of this study was to evaluate whether increased GH

secretion in type DM1 could be due to an impairment of theinhibitory effect exerted by circulating FFA.

Patients and methods

Six type 1 diabetic patients were studied (three women, threemen), mean age of 30

±

2·1 years, body mass index (BMI) of23·1

±

1·5 kg/m

2

. The duration of diabetes was 8·4

±

2·5 years.Mean level of haemoglobin A

1c

at the time of evaluation was7·6

±

0·6% (normal < 5·5%). The patients were receiving two orthree injections of insulin daily. None had clinical or laboratoryevidence of nephropathy. No medications other than insulin werebeing used by the patients before and during the study period.As a control group six normal healthy subjects of similar age,sex and weight were studied. All subjects provided informed con-sent and approval for this study was obtained from the hospitalcommittee. The tests were started at 08·00–08·30 h after an over-night fast, with the subjects recumbent. An indwelling catheterwas inserted in both forearms for separate blood samplingand drug administration. An equilibration period of 30 min wasallowed before baseline samples were obtained. Each subjectunderwent three tests, performed in random order separated byat least 4 days with each subject serving as his own control. Eachpatient received GHRH [1

µ

g /kg intravenous (i.v.) at min 180],acipimox (250 mg orally at min 0 and 120) and GHRH plusacipimox on three separated days. Blood samples were taken atappropriate intervals for determination of GH, FFA and glucose.

Serum GH was measured by a solid-phase, two-site chemilu-minescent enzyme immunometric assay (Immulite, EURO/DPC)

with a sensitivity of 0·01

µ

g / l (conversion factor for SI units,

µ

g / l

×

2·6 = mIU/l) and with intrassay coefficients of variationof 5·3%, 6·0% and 6·5% for low, medium and high plasma GHlevels, respectively. FFA levels were determined by an enzymaticcolourimeteric method (NEFA-HA, Wako, Zaragoza, Spain).IGF-I was determined by radioimmunoassay (Nichols Institute,San Juan Capistrano, CA, USA). All samples from a given subjectwere analysed in the same assay run. Hormone levels arepresented as absolute values or as the mean GH peak. The areaunder the secretory curve (AUC) were calculated by a trapezoidalmethod.

Statistical analyses were performed by Wilcoxon rank sumbetween related groups and by Mann–Whitney tests betweendifferent groups. Results are expressed as mean

±

SEM.

P

< 0·05was considered significant.

Results

In normal subjects, mean baseline FFA (mmol/ l) were0·54

±

0·11, 0·55

±

0·12 and 0·59

±

0·11 previous to GHRH,GHRH plus acipimox and acipimox alone, respectively, notsignificantly different on the 3 days of testing. In DM1 patients,mean baseline FFA (mmol/ l) were 0·68

±

0·10, 0·70

±

0·12 and0·66

±

0·12 previous to GHRH, GHRH plus acipimox and acip-imox alone, respectively, not significantly different on the 3 daysof testing nor significantly different from normal subjects.

The administration of acipimox induced an FFA reductionduring the entire test. In normal subjects, the AUC (mmol/l

×

120 min) after placebo plus GHRH treatment (107

±

15·2)was significantly higher than after acipimox pretreatmentplus GHRH (25·6

±

7·8;

P

< 0·05) or acipimox plus placebo(23·9

±

8·4;

P

< 0·05). In type DM1 patients, the AUC (mmol/l

×

120 min) after placebo plus GHRH treatment (136·5

±

28·1)was significantly higher than after acipimox pretreatmentplus GHRH (21·8

±

7·1;

P

< 0·05) or acipimox plus placebo(24·2

±

9·1;

P

< 0·05).In control subjects, mean baseline glycaemia (mg/dl) were

92

±

5, 91

±

6 and 94

±

6 previous to GHRH, GHRH plus acip-imox and acipimox alone, respectively, not significantly differenton the 3 days of testing. In DM1 patients mean baseline glycae-mia (mg/dl) were 145

±

13, 154

±

33 and 137

±

12 previous toGHRH, GHRH plus acipimox and acipimox alone, respectively,not significantly different on the 3 days of testing and signifi-cantly higher than in normal subjects (

P

< 0·05). In control sub-jects, mean nadir glycaemia (mg/dl) were 90

±

3, 86

±

4 and88

±

5 after GHRH, GHRH plus acipimox and acipimox alone,respectively. In DM1 patients, mean nadir glycaemia (mg/dl)were 150

±

28, 144

±

30 and 132

±

10 after GHRH, GHRH plusacipimox and acipimox alone, respectively, not significantly dif-ferent on the 3 days of testing and significantly higher than innormal subjects.

Effect of plasma FFA reduction in diabetes mellitus type 1

433

© 2003 Blackwell Publishing Ltd,

Clinical Endocrinology

,

59

, 431–436

Mean IGF levels (

µ

g / l) were 244

±

19, 248

±

23 and 245

±

20in the control group previous to GHRH, GHRH plus acipimox andacipimox alone, respectively, and 155

±

23, 170

±

13 and 168

±

15in the diabetics previous to GHRH, GHRH plus acipimox andacipimox alone, respectively. Baseline serum IGF-I levels in thethree different days of testing were significantly lower in the diabeticpatients than in the control group (

P

< 0·05). Results of diabeticsor controls were not significantly different on the 3 days of testing.

In control subjects, mean GH (

µ

g / l) peak level after acipimoxwas 15·8

±

3·7. Mean GHRH-induced GH secretion peak was23·7

±

5·1. The GH AUC (

µ

g / l

×

120 min) in control subjectswas for acipimox-treated 1339

±

292 and 1528

±

330 for GHRH-induced secretion.The administration of acipimox plus GHRHproduced a mean GH peak secretion of 43·1

± 9·8, significantlygreater than the response after acipimox alone (P < 0·05) orGHRH alone (P < 0·05). The GH AUC after the administrationof GHRH plus acipimox was 3031 ± 669, significantly greaterthan the response after acipimox alone (P < 0·05) or GHRHalone (P < 0·05; Fig. 1).

In diabetic patients, mean GH (µg/l) peak level after acipimoxwas 25·1 ± 6·8. Mean GHRH-induced GH secretion peak was25·5 ± 3·7. The GH AUC (µg/l × 120 min) in diabetic patientswas for acipimox-treated 2516 ± 606 and 1821 ± 311 for GHRH-induced secretion. The administration of acipimox plus GHRHproduced a mean GH peak secretion of 89·9 ± 9·6, significantlygreater than the response after acipimox alone (P < 0·05) orGHRH alone (P < 0·05). The GH AUC after the administrationof GHRH plus acipimox was 7311 ± 1154, significantly greaterthan the response after acipimox alone (P < 0·05) or GHRHalone (P < 0·05; Fig. 2).

We compared the GH AUC in normal subjects and diabeticpatients. The GH response to GHRH was similar in both groups,with a AUC (µg/l × 120 min) of 1528 ± 320 and 1821 ± 311 forcontrols and type 1 diabetic patients, respectively. In contrast,the GH response after acipimox was increased in type 1 diabeticpatients with a GH AUC of 1339 ± 292 and 2515 ± 606 for nor-mal subjects and type 1 diabetic patients, respectively (P < 0·05).The GH response after acipimox plus GHRH was increased intype 1 diabetic patients with a GH AUC of 3031 ± 669 and7311 ± 1154 for normal subjects and type 1 diabetic patients,respectively (P < 0·05; Fig. 3).

Fig. 1 Mean ± SE serum GH levels after the administration of GHRH (�), acipimox (�) and GHRH plus acipimox (Ο AC + GHRH) in normal subjects.

Fig. 2 Mean ± SE serum GH levels after the administration of GHRH (�), acipimox (�) and GHRH plus acipimox (� AC + GHRHR) in type 1 diabetic patients.

Fig. 3 Mean ± SE GH Area under the curve (AUC) after the administration of GHRH, acipimox (AC) and GHRH plus acipimox in normal subjects (�) and type 1 diabetic patients( ).

434 P. Alvarez et al.

© 2003 Blackwell Publishing Ltd, Clinical Endocrinology, 59, 431–436

One control subject and one diabetic patient experienced amild transient flushing after acipimox. No side-effects werereported in the other tests.

Discussion

In agreement with previous data we found that reduction of cir-culating FFA levels with acipimox leads to a marked increase inbasal GH secretion and GH responses to GHRH. Furthermore,our results have shown in type 1 diabetic patients, that FFA reduc-tion with acipimox per se stimulates GH secretion and markedlyincreased GHRH-induced GH secretion. This response was notonly additive but a clear potentiation of both stimuli when com-pared with normal subjects.

GH secretion is tightly linked to metabolic alterations as wellas to variations in the intake or availability of lipids, amino acidsand carbohydrates (Dieguez & Casanueva, 1995). Studies withacipimox, a nicotinic acid analogue that blocks lipolysis andis devoid of side-effects (Pontiroli et al., 1990, 1991), haveprovided the best tool for further understanding the role of FFAdepression in GH regulation. It has been shown that acipimoxmediated plasma FFA depression per se stimulates GH secretionin normal subjects and potentiates the response to other GHreleasing stimuli (Peino et al., 1996). The impaired GHRH-induced GH secretion in obese patients (Cordido et al., 1989,1993) is partially reversed by acipimox in normal obese patientsbut not in obese patients with hypopituitarism (Cordido et al.,1996, 1998).

Patients with DM1 show exaggerated responses to differentprovocative stimuli such as exercise, clonidine, levodopa, orGHRH (Hansen et al., 1970; Lorenzi et al., 1980; Krassowskiet al., 1988; Almqvist et al., 1999). Twenty-four-hour integratedserum GH concentrations are also elevated in DM1, because ofincreases in both the frecuency and peak amplitude of GH secre-tory pulses (Asplin et al., 1989; Edge et al., 1990). IGF-I levelsare either normal or reduced and there is evidence of a reversibledefect in IGF-I generation in these patients (Horner et al., 1981;Press et al., 1984; Asplin et al., 1989). In type 1 diabetes elevatedcirculating GH concentrations are, at least partially, caused by adelayed plasmatic clearance, probably due to a reduced GH clear-ance by its receptor-mediated mechanism. However, there is nodoubt about the presence of a secretory defect (Catalina et al.,2002). This disrupted GH secretion may have relevant physio-pathological implications. Several experimental studies suggestthe possible relationship between abnormal GH regulation andthe development of diabetic microvascular disease (Alzaid et al.,1994; Muchaneta et al., 1994; Sharp, 1995; Feld & Hirschberg,1996). Moreover, this altered GH secretion may play a potentialrole in mediating the metabolic derangement of DM1 (Presset al., 1984). Therefore, long-term correction of GH/IGF-I axisabnormalities of DM1 may be an important consideration in the

development of therapeutic modalities aimed not only at improv-ing metabolic control but also at preventing the pathologicalsequelae associated with DM1.

At present, the underlying mechanisms responsible for soma-totropinergic axis alterations in patients with DM1 are still notwell understood. The primary cause of the altered GH secretionof type 1 diabetes could be an altered hypothalamus, abnormalpituitary function, or a perturbation of the peripheral signals act-ing at either the pituitary or hypothalamic level. Although not auniversal finding, some previous studies have found that DM1patients exhibited a GH response, after GHRH administration,greater than that of normal subjects (Krassowski et al., 1988;Schaper et al., 1990). These augmented responses to GHRHcould not be demonstrated by various other authors (Press et al.,1984; Kopelman et al., 1988). Although unclear at present, thesediscrepancies could be due to the differences in the patients char-acteristics, i.e. sex, BMI, adecuate metabolic control, etc.

In patients with IDDM, there is an increase in the frequencyof GH pulses and an elevation of interpulse GH concentrations(Asplin et al., 1989). These results and other data from the liter-ature (Horner et al., 1981; Giustina et al., 1990; Miller et al.,1992; Villas-Boas et al., 1997) have suggested that the derangedGH secretion observed in these patients is likely to be due, atleast in part, to a reduced hypothalamic somatostatinergic tone.We have shown with this new stimulus, FFA reduction withacipimox, an increased response in type 1 diabetic patients. In thisgroup of patients FFA reduction with acipimox, per se, stimulatesGH secretion and markedly increases GHRH-induced GHsecretion. The response was not only additive, but rather a clearpotentiation of GH secretion was observed with the combinedstimulus. In contrast, in normal subjects the response of acipimoxand GHRH was only additive (Peinó et al., 1996). The GHresponse to the combined stimulus, acipimox plus GHRH, wasincreased in diabetic patients when compared to normal subjects.Some of our data could be explained by a diminished negativefeedback exerted by circulating IGF-I levels upon the GH secre-tion by the pituitary. We found lower plasma IGF-I levels indiabetic patients than in controls. This indicates a partial lack ofinhibiting control upon somatotroph GH secretion. Nevertheless,we think that decreased IGF-I levels do not explain why the GHresponse to the combined stimulus (GHRH plus acipimox) wasnot additive, but rather a clear potentiation of the response wasobserved when compared with controls, specially when thisgroup of patients exhibited a GHRH-induced GH secretionsimilar to that of normal subjects. These data suggest that endog-enous FFA play an important inhibitory role in GH secretion indiabetic patients and that, contrary to our hypothesis, FFA trans-duction mechanisms at pituitary somatotroph level are preservedin DM1. This raises the question regarding the mechanismsresponsible for altered GH secretion in this setting. There areexperimental data that suggest a possible pituitary defect on GH

Effect of plasma FFA reduction in diabetes mellitus type 1 435

© 2003 Blackwell Publishing Ltd, Clinical Endocrinology, 59, 431–436

secretion in diabetes, in fact in diabetic rats the expression of theGH receptor at pituitary level was reduced (Busiguina et al.,2000). DM1 patients have an elevated GH response to GHRP-6and GHRH. The combined administration of these two peptideshas an additive effect and constitutes a very powerful stimulusfor GH secretion in DM1 subjects. The combined administrationof acipimox plus GHRH induced a GH secretion even greaterthan the response after GHRH and GHRP-6 (Catalina et al.,1998). As FFA act at pituitary level, these data indicate that thesomatotroph cell is hyper-responsive to FFA depression in dia-betic patients, although the presence of an adequate responseindicates that FFA transduction mechanisms at pituitary soma-totroph level are preserved in DM1.

In summary, reduction of FFA with acipimox is a stimulus forGH secretion in DM1. The combined administration of GHRHplus acipimox induces a markedly increased GH secretion in type1 diabetic patients when compared with normal subjects. Thesedata suggest that patients with DM1 exhibit a greater GH secre-tory capacity than control subjects, despite the fact that circulat-ing FFA levels are exerting a powerful inhibitory effect on GHsecretion in these patients.

Acknowledgements

This work was supported by a grant from Xunta de Galicia(PGIDT00PXI000PR) and FIS (Fondo de InvestigaciónSanitaria. PI021479).

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