Free fatty acids inhibit growth hormone/STAT5 signaling in human

muscle: a potential feed-back mechanism

Short title: FFA inhibits GH signaling in muscle

Niels Møller1, Lars C. Gormsen

1, Ole Schmitz

2, Sten Lund

1, Jens Otto L. Jørgensen

1, Niels Jessen

1

1Medical Department M (Endocrinology & Diabetes), Aarhus University Hospital, Aarhus, Denmark

2Department of Pharmacology, University of Aarhus, Aarhus, Denmark

Address correspondence and reprint requests to:

Niels Jessen, MD, PhD

Medical Research Laboratory and Medical Department M (Endocrinology & Diabetes)

Aarhus University Hospital, 8000 Aarhus C, Denmark

Phone: +458949 1615 Fax: +458949 2150

E-mail: Niels.Jessen@ki.au.dk

DISCLOSURE STATEMENT: The authors have nothing to disclose

This work was supported by Danish Agency for Science Technology and Innovation Grants to NJ and (grant

number: 271-07-0719) the FOOD Study Group/Danish Ministry of Food, Agriculture and Fisheries &

Ministry of Family and Consumer Affair to NM (grant number: 2101-05-0044).

Word count: 1603

J Clin Endocrin Metab. First published ahead of print March 10, 2009 as doi:10.1210/jc.2008-2624

Copyright (C) 2009 by The Endocrine Society

1

Abstract

Context: Stimulation of lipolysis, leading to increased blood concentrations of free fatty acids (FFA), is a

primary effect of growth hormone(GH) and phosphorylation of intracellular STAT5 is a primary mediator of

the effects of GH.

Objective: Based on preliminary results we intended to test whether FFA exert a negative feedback

inhibition of STAT5 phosphorylation in skeletal muscle.

Design and participants: Eight healthy young men were investigated for 8 h on 4 occasions at 4 different

FFA levels in a single blind, randomized manner. Acipimox was used to suppress FFA levels and Intralipid

was infused to obtain appropriate FFA concentrations. Somatostatin was infused to control GH levels and

GH, insulin and glucagon were replaced. Muscle biopsies were taken after 8 h and compared to a 5th biopsy

taken under normal basal conditions.

Setting: University clinical research unit.

Results: GH concentrations remained steady and comparable in all studies and FFA concentrations varied

between 0.01 and 1.71 mmol/l on the 4 occasions (p < 0.05). We observed a dose dependent 40 % decrease

of STAT5 phosphorylation in skeletal muscle with increasing concentrations of FFA.

Conclusions: Our results strongly suggest the existence of a negative feedback loop, whereby effects of GH

may be dampened by FFA inhibition of GH dependent STAT5 phosphorylation. The mechanisms behind and

biological consequences of this finding awaits additional studies.

2

Introduction

Growth hormone (GH) and its principal intracellular signal mediator STAT5 represent an ancestral endocrine

control system, which throughout evolution has served to regulate growth, maintenance of lean body mass

and intermediary metabolism(1;2). GH together with IGF-I have important protein anabolic actions to

stimulate growth and preservation of protein stores of the lean body mass. However, the single most

prominent effect of physiological GH pulse exposure is a marked stimulation of lipolysis. This is evident by

studies showing doubling of free fatty acid plasma concentrations after 2-3 hours, fading after 6-8 after,

paralleled by similar changes in palmitate turnover rates and lipid oxidation rates(3-7).

The GH receptor (GHR) belongs to the class I of the Hematopoietin superfamily of cytokine receptors,

which includes prolactin, erythropoietin, leptin, granulocyte stimulating factor, and several interleukins(8).

GHR has been identified in many tissues including muscle, fat, liver, heart, kidney, brain and the

pancreas(9). Following GH stimulation, receptor-associated Janus kinase (JAK) 2 binds to GHR and

phosphorylates signal transducers and activators of transcription 5 (STAT5) (10). Phosphorylated STAT5

dimerizes and translocates to the nucleus (10). A STAT5b binding site has been characterized in the IGF-I

gene promoter region, which mediates GH-stimulated IGF-I gene activation (11). A study in healthy young

male subjects exposed to an intravenous GH bolus vs. saline(12) recently reported significant STAT5b

tyrosine phosphorylation 30-60 minutes after GH exposure in muscle and fat biopsies compared to saline(12)

together with evidence of less intense STAT5b activation associated with small spontaneous GH bursts. In

addition DNA binding activity by STAT5 assessed by an electrophoretic mobility shift assay (EMSA) was

evident in fat but not muscle tissue samples and significant GH-dependent IGF-I mRNA expression was

detectable in adipose tissue, whereas SOCS-1 and SOCS-3 mRNA expression tended to increase in muscle

and fat, respectively (12). These results have later been confirmed (13).

Thus, acknowledging that increased lipolysis and FFA levels are the main effect of GH and that STAT5 is a

main intracellular effector of GH action, we conducted analysis of STAT5b phosphorylation to exposure of 4

different concentrations of FFA in 8 subjects. This was done to test whether an intramuscular feed back

3

mechanism was activated that would constitute a novel regulating mechanism for GH-signaling. The

hypothesis was in part generated and supported by a preliminary study showing evidence of inhibition of

STAT5b phosphorylation during a short-term FFA surge. To circumvent FFA induced inhibition of GH

secretion the studies were conducted utilizing somatostatin to suppress endogenous GH secretion and

subsequent exogenous GH administration.

4

Materials and Methods

Subjects

The participants were 8 healthy men (24±1 yr, 84±9 kg BW, 25±3 kg/m2 BMI). Baseline characteristics and

data on lipid and glucose metabolism and insulin sensitivity have been published previously(14). All

participants gave their written informed consent after oral and written information concerning the study

according to the Declaration of Helsinki II. The Aarhus County/local Ethical Scientific Committee approved

the study.

Protocol

The subjects were examined on 4 days separated by 1 month. On all examination days, the subjects were

infused with GH for 8 hours (Norditropin, Novo Nordisk, Denmark) (2 ng*kg-1

*min-1

) after an overnight fast

of 10 hours. This infusion rate yields GH-plasma concentrations in a physiological range. Somatostatin (300

µg*h-1

) was co administrated to suppress endogenous GH production. Simultaneously, the subjects were

infused with various infusion rates of a fat emulsion (Intralipid 20%, Fresenius Kabi) consisting of 12 %

palmitic acid (C16:0), 4 % stearic acid (C18:0), 21 % oleic acid (C18:1 n-9), 53 % linoleic acid (C18:2 n-6),

7 % α-linoleic acid (C18:3 n-3 and 3 % others (ref). 4 different infusion rates were used with 1 infusion rate

per examination day. The rates used were: 0 (saline), 3, 6 and 12 µl*kg-1

*min-1

. The subjects were given

replacement therapy with glucagon (GlucaGen 1 mg*ml-1

, Novo Nordisk, 0.8 ng*kg-1*

min-1

). Acipimox

(Olbetam 250 mg, Pfizer) was administered as a single dose to suppress endogenous lipolysis. Heparin was

infused throughout the experiment (Heparin “SAD”, 70 IE*kg-1

*min-1

). Insulin (Novo Nordisk, Denmark)

was given at 0.08 mU*kg-1

*min-1

for the initial 6 hours of the study. During the last 2 hours of the study the

subjects were clamped at a rate of 0.6 mU*kg-1

*min-1

insulin as previously described(14). At t=480 min a

muscle biopsy was obtained from vastus lateralis of the quadriceps femoris muscle with a Bergström canula

and all infusions were discontinued.

5

A biopsy was obtained from the subjects in the fasting state on a separate examination day without any

infusions. This biopsy served as a reference point for the biopsies taken during GH-stimulation.

Muscle homogenization

Frozen muscles biopsies (~50 mg) were homogenized in ice-cold solubilization buffer containing (in mM) 50

HEPES, 137 NaCl, 10 Na4P2O7, 10 NaF, 2 EDTA, 1 MgCl2, 1 CaCl2, 2 Na3VO4, 1% (vol./vol.) NP-40, 10 %

(vol./vol.) glycerol, 2 µg/ml aprotinin, 5 µg/ml leupeptin, 0.5 µg/ml pepstatin, 10 µg/ml anti-pain, 1.5 mg/ml

benzamidine, and 100 µmol/l AEBSF [4-(-2-aminoethyl)-benzenesulfonyl fluoride, hydrochloride], pH 7.4

and samples were rotated for 60 min at 4°C. Insoluble materials were removed by centrifugation at 16,000g

for 20 min at 4°C and protein content on the supernatant was determined.

Western Blot analysis of STAT5 signaling

Aliquots of protein were resolved by SDS-PAGE, transferred to nitrocellulose, and incubated with primary

antibody. Anti-STAT5 and anti-phospho-STAT5 were purchased from Cell Signaling (Beverly, MA).

Horseradish peroxidase conjugated goat anti-rabbit IgG antibody (Pierce Chemical, Rockford, IL) was used

as secondary antibody. Proteins were visualized by BioWest enhanced chemiluminescence (Pierce

Supersignal) and quantified using UVP BioImaging System (UVP, Upland, CA).

Statistics

Results are expressed as mean ± SEM (parametric data). The Shapiro-Wilk test was used to test for normal

distribution. Statistical comparisons between study days were assessed by repeated measurements analysis of

variance (ANOVA). Post hoc comparison was performed by Pairwise Multiple Comparison Procedures

(Student-Newman-Keuls Method) for normal distributed data and Wilcoxon sign rank test for nonparametric

data. P<0.05 was considered significant.

6

Results

Circulating hormones

Growth hormone and FFA levels in plasma at time of the biopsy (t=480 min) are presented in table 1. There

was no difference between GH levels on the 4 examination days. FFA levels mirrored the increased infusion

rates and as previously described 4 significantly different (P<0.05) levels of circulating FFA levels were

obtained (14). The levels at time of the biopsy are presented in table 1. Plasma levels of glucagon, c-peptide,

IGF-I and IGF-II were all similar between examination days.

STAT5 signaling (Fig 1)

Phosphorylation of STAT5 in muscle biopsies obtained after 8 hour GH-stimulation (t=480 min) are

presented in Fig 1. Data are presented as pct of the STAT5 phosphorylation during GH stimulation in the

absence of lipid infusion. Lipid infusion during GH-stimulation decreased STAT5 phosphorylation by ~40%,

an effect which was evident already at the low lipid infusion rate.

STAT5 phosphorylation without GH-stimulation in the basal control biopsy was significantly lower than

during GH-stimulation in the absence of lipid infusion.

Expression levels of STAT5 did not differ among examination days.

7

Discussion

Our results show that increasing concentrations of FFA inhibits STAT5 phosphorylation in muscle in a dose-

dependent manner in the physiological FFA range between 0.01 and 1.7 mM. This finding is novel and may

have important implications for our understanding of GH and FFA physiology and pathology.

Under physiological circumstances one of the most important effects of GH is stimulation of lipolysis.

Inhibition of STAT5 phosphorylation by increased FFA levels may be viewed as a negative feed-back

mechanism serving to control the intracellular GH signal. This would imply that whenever FFA levels are

high e.g. during fasting/protracted exercise or inflammatory illness, growth and protein anabolism is

restricted. The mechanism may also be important in the clinical course of diabetic ketoacidosis, during which

high levels of FFA may dampen the lipolytic effects of GH, thereby limiting the severity of ketosis. The

GH/STAT5 signal constitute a well preserved system, which also stimulates β-cell proliferation and insulin

biosynthesis and it is also feasible that FFA inhibition of this signal may contribute to β-cell lipotoxicity in

type 2 diabetes(15).

It should be underlined that in the present protocols we achieved FFA levels in the physiological range

between close to zero and 1.7 mmol/l and physiological GH levels around 0.4 ng/ml. We employed

somatostatin to control endogenous GH secretion and did not observe any differences in GH levels, strongly

suggesting that the observed effect is caused directly by FFA. Still, since it has been shown that FFA inhibit

GH secretion(16;17), it cannot entirely be excluded that some small GH spikes may have escaped detection

and contributed to STAT5 phosphorylation in the protocols with low FFA levels. In our hands the day to day

variability within a subject in the phosphorylation of STAT5 is only ~15% (Jessen unpublished

observations). Given the low variability and the effects of invasive procedures we abstained from obtaining a

baseline biopsy on each experiment day. It should also be noted that our results do not provide evidence as to

whether FFA inhibits STAT5 phosphorylation in tissues other than muscle (such as adipose tissue and β-

cells). Furthermore although our findings were observed under conditions of hyperinsulinaemia, previous

studies have shown no effect of insulin on STAT5 phosphorylation (13).

8

Previous studies have shown that GH exposure at the cellular level induces increased mitochondrial function,

increased mTOR phosphorylation and increased SOCS3 and IGF-I mRNA expression(13;18). The question

of to which extent these variables are affected by FFA awaits future studies.

In conclusion, GH induced phosphorylation of STAT5 in human skeletal muscle is inhibited by plasma FFA

in a dose dependent manner. This finding provides evidence for a potential novel feed-back mechanism

regulating the lipolytic effects of GH.

9

Acknowledgements

The authors thank Lone Svendsen and Susanne Sørensen for excellent technical assistance.

10

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11

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12

Legends to figure

Fig 1: GH stimulated phosphorylation of STAT5 during lipid infusion. Increased intralipid infusion rates

decreased STAT5 phosphorylation during a constant GH infusion (2 ng*kg-1

*min-1

) to same level as in

unstimulated muscles. Reference is STAT5 phosphorylation in muscle biopsies taken in the absence of GH

stimulation. Data represent mean ± SEM.

13

Table

GH and FFA levels during lipid infusion:

Lipid infusion rate

(µl*kg-1

*min-1

)

0.00 0.03 0.06 0.12

GH (ng/ml)

(Median, 25% and 75%

quartiles)

0.47

(0.35 and 0.68)

0.39

(0.33 and 0.47)

0.41

(0.37 and 0.47)

0.37

(0.33 and 0.46)

Free fatty acid (mM)

(Mean ± SEM)

0.01 ± 0.00 0.33 ± 0.03 0.66 ± 0.09* 1.71 ± 0.29*

pSTAT5

STAT5

GH + + + + -

Lipid infusion rate 0.0 0.03 0.06 0.12 NA

Figure 1

Lipid infusion rate (ml/kg/min)

pS

TA

T5

(p

ct o

f 0

.0 i

nfu

sion

ra

te)

0

20

40

60

80

100

120

140

0.0 0.003 0.006 0.012 Reference

P<0.05

vs. 0.0

GH infusion (2 ng/kg/min)

P<0.05 vs. 0.0