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: [email protected]
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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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*
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