Tugas Mandiri Biokimia 2

EFEK DOSIS-RESPONS DARI ASAM LEMAK BEBAS PADA METABOLISME ASAM

AMINO DAN UREAGENESIS

Oleh:

AUDINA THALIA

021511133066

ILMU BIOKIMIA 2 DEPARTEMEN BIOLOGI ORAL

FAKULTAS KEDOKTERAN GIGI

UNIVERSITAS AIRLANGGA

Semester Ganjil - 2016/2017

 BAB 1

PENDAHULUAN

1.1 Latar Belakang

Asam lemak bebas atau Free Fatty Acid (FFA) adalah bahan bakar penting dan

memiliki efek untuk menghemat protein, khususnya selama puasa dan saat kondisi stress.

Namun, tidak pasti apakah efek ini menguntungkan sepanjang rentang fisiologis atau hanya

terjadi pada konsentrasi FFA yang sangat tinggi. Perubahan sekunder kadar hormone dan

ketonesis yang berperan juga tidak jelas terjadi. Oleh karena itu makalah ini bertujuan untuk

mendeskripsikan dosis-respons hubungan antara metabolisme asam amino dan beredarnya

konsentrasi FFA pada tingkat hormon tertentu.

Adanya FFA yang menyebar dapat menurunkan konsentrasi asam amino dan

menghambat aliran fenilalanin seluruh tubuh dan konversi fenilalanin-to-tirosin. Konsentrasi

FFA dari 0 sampai 2 mmol/L menunjukkan bahwa FFA mengerahkan sumber protein mereka

untuk melestarikan efek fisiologis diatas kisaran jangkauan >1,5 mmol/L.

Asam lemak bebas melayani berbagai fungsi penting; sebagai penyedia energi fisiologis

utama selama puasa (Cahill et al. 1966), sebagai konstituen membrane sel, dan dalam beberapa

kasus sebagai kunci regulator ekspresi gen (Distel et al. 1992). FFA juga merangsang sekresi

insulin (Nolan et al. 2006), menghambat sekresi hormone pertumbuhan (Lanzi et al. 1999) dan

menganggu pelepasan glucagon (Olofsson et al. 2004)

Selain itu, gangguan fisiologis sederhana tingkat FFA dapat menyebabkan resistensi

insulin dan tipe 2 diabetes (Roden et al. 1996). Penelitian sebelumnya menyarankan bahwa
peningkatan konsentrasi FFA dan bada keton memiliki efek protein-sparing (Tessari et al. 1986),

dan studi dalam kekurangan hormone pertumbuhan menunjukkan bahwa FFA mampu

menurunkan metabolisme yang terjadi diseluruh tubuh dan menurunkan fungsi protein otot (Shi

et al. 2003). Terutama pada kondisi puasa, sirkulasi FFA akan menurun dengan cara otot

acipimox meningkatkan pemecahan protein dan produksi urea-nitrogen dan ekskresi yang dapat

meningkatkan FFA (Norrelund et al. 2003).

1.2 Tujuan

Untuk mengetahui efek dosis-respons dari asam lemak bebas pada metabolisme asam

amino dan ureagenesis.

1.3 Metode

Dengan menggunakan 8 pria sehat sebagai dan dipelejari 4 kali (6 jam basal, 2 jam

penghambat glukosa). Lipolysis endogen diblokir dengan acipimox dan intralipid dimasukkan di

berbagai tingkat (0, 3, 6 atau 12 L/kg dalam 1 menit) untuk mendapatkan 4 tingkat yang berbeda

dari peredaran FFA. Hormone pertumbuhan endogen, insulin, dan sekresi glucagon diblokir oleh

somatostatin dan diganti eksogen 15N-Fenilalanin, 2H4-tirosin dan 13C-urea yang diresapi terus

menerus untuk menilai omset protein dan ureagenesis.

 BAB 2

PEMBAHASAN

Peningkatan kadar FFA menghambat perputaran protein, ditunjukkan dengan elevasi

jangka pendek FFA yang menghasilkan fenilalanin yang berkurang dan konsentrasi tirosin yang

berkurang Seluruh omset fenilalanin di dalam tubuh, daerah lengan, dan fenilalanin-to tirosin

mengalami degradasi selama hiperinsulinemia. Selain itu, output urea hati dan omset urea

seluruh tubuh tidak terpengaruh oleh peningkatan jangka pendek lipid eksperimentat saat

hormon tidak berproduksi.

Dalam penelitian pada jurnal, konsentrasi FFA yang diinginkan dengan cara Intralipid

yang bersamaan dengan infus heparin dapat dicapai. Hasil aktivitas lipase dihidrolisis cepat oleh

trigliserida dengan peningkatan berikutnya dalam konsentrasi FFA badan keton yaitu metabolit

FFA utama. Oleh karena itu beberapa studi menunjukkan peningkatan konsentrasi setelah infus

intralipid (Jeejee et al. 1976, Boden & Chen 1999). Dalam penelitian, tingkat infus intralipid

tertinggi mengakibatkan peningkatan sekitar 2 kali lipat dalam konsentrasi 3-OH-butirat,

sedangkan kadar FFA dalam keadaan fisiologis keton tubuh selama konsentrasi tidak mengalami

perubahan. Klem somatostatin, berperan sebagai efektif blok glucagon yang berperan dalam

pembentukan keton tubuh (Miles et al. 1982)

Konsentrasi hasil keton tubuh yang rendah, memungkinkan penilaian independen dari

efek intinsik FFA pada metabolisme asam amino. Hasil penelitian menunjukkan bahwa

meskipun tubuh memiliki keton yang rendah atau tidak berubah konsentrasinya, seluruh tubuh

dan fenilalanin akan terbentuk dan serapannya akan dikurangi dengan meningkatkan FFA.
Proteinuria yang memiliki efek hemat dari FFA sebagian besar akan dimediasi melalui badan

keton.

Klasik studi oleh Cahill et al. (1966) menunjukkan bahwa puasa berkepanjangan akan

meningkatkan FFA dan keton dalam tubuh akan meningkat, ditambah dengan penurunan

ekskresi nitrogen urea. Selanjutnya menghubungkan stimulasi metabolisme lipid protein-sparing

dengan pemberian intravena 3-OH butyrate untuk subyek obesitas dengan tujuan menurunkan

sekresi kemih nitrogen (Pawan & Semple 1983). Studi oleh Tessari et al. (1986) dan Nair et al.

(1988) melaporkan bahwa penurunan FFA meningkatkan fluks leusin, tingkat oksidasi pada

subjek yang berpuasa dan administrasi yang mengakibatkan 3-OH-butirat berkurang dan sintesis

protein otot meningkat.

FFA yang tinggi akan menghambat degradasi asam amino (dinilai dengan fenilalanin-to-

tirosin ditekan) selama hiperinsulinemia. Dalam penelitian ini, fenilalanin seluruh omset tubuh

mengalami penurunan pada konsentrasi FFA yang tinggi. Kontras dengan penelitian sebelumnya,

dimana tidak ditemukan efek FFA pada omset fenilalanin meskipun terlihat secara signifikan

penurunan lengan rilis fenilalanin otot (Walker et al. 1993). Ada kemungkinan bahwa FFA

menurun, dan fenilalanin melepaskan diri dari otot rangka. Pengamatan menunjukkan bahwa

FFA mengurangi degradasi protein dengan cara mengurangi omset asam amino baik pada

seluruh tubuh atau di otot saja.

Mekanisme sitosol yang menghubungkan peningkatan FFA, terjadi penurunan degradasi

protein dan asam amino yang terbentuk masih belum jelas. Akumulasi sitosol dari FFA memiliki

efek katabolic yaitu FFA dapat mengaktifkan MAP p38 kinase di miosit jantung (Miller et al.

2005) dan hepatosis (Collins et al. 2006)

 Urueagenesis tidak dipengaruhi oleh peningkatan jangka pendek FFA. Ureagenesis diatur

oleh perubahan insulin / rasio glucagon dengan meningkatkan konsentrasi keton. Dalam konteks

ini perlu digarisbawahi bawah konsentrasi insulin pada hati, glucaon dan badan keton jauh lebih

tinggi dalam keadaan alami dibandingkan dengan eksperimental set up. Efek biologis protein-

sparing dari FFA yang penting untuk kelangsungan hidup dalam kondisi metabolic stres, seperti

kalori kurang murni (puasa, kelaparan dan malnutrisi) dan pengeluaran energi meningkat

dikombinasikan dengan kekurangan kalori (penyakit kritis, peradangan kronis, operasi dan

latihan berkepanjangan). Secara umum, kondisi ini dicirikan oleh perubahan sirkulasi hormon

metabolisme biasanya tingkat insulin rendah dan kadar hormon stres yang tinggi dan dalam

konsentrasi metabolit dan fluks (Coker & Kjaer 2005)

 BAB 3

KESIMPULAN

Free Fatty Acid yang beredar pada tubuh akan menurunkan konsentrasi asam amino dan

menghambat kerja asam amino serta pemasukan fenilalanin dan konversi fenilalanin-to-tirosin.

Free Fatty Acid akan mengerahkan protein yang mereka punya untuk menjaga pertahanan

fisiologis tubuh.
Acta Physiol 2008, 192, 369379

Doseresponse effects of free fatty acids on amino acid

metabolism and ureagenesis

L. C. Gormsen,1 J. Gjedsted,2,3 S. Gjedde,1 H. Nrrelund,1 J. S. Christiansen,1 O. Schmitz,4

J. O. L. Jrgensen1 and N. Mller1
1 Medical Department M (Endocrinology & Diabetes), Aarhus University Hospital, Aarhus, Denmark
2 Department of Anesthesiology, Aarhus University Hospital, Aarhus, Denmark
3 Immunoendocrine Research Unit, Aarhus University Hospital, Aarhus, Denmark
4 Department of Pharmacology, Aarhus University, Aarhus, Denmark

Received 11 May 2007, Abstract

revision requested 10 July 2007,
revision received 7 August 2007,
accepted 21 September 2007
Correspondence: L. Gormsen,
Medical Department M, Aarhus
University Hospital, Nrrebrogade
42, DK-8000 Aarhus C, Denmark.
E-mail:
lars.christian.gormsen@ki.au.dk
Aim: Free fatty acids (FFAs) are important fuels and have vital protein-
sparing effects, particularly during conditions of metabolic stress and fasting.
However, it is uncertain whether these beneficial effects are evident
throughout the physiological range or only occur at very high FFA concen-
trations. It is also unclear whether secondary alterations in hormone levels
and ketogenesis play a role. We therefore aimed at describing doseresponse
relationships between amino acid metabolism and circulating FFA concen-
trations at clamped hormone levels.
Methods: Eight healthy men were studied on four occasions (6 h basal, 2 h
glucose clamp). Endogenous lipolysis was blocked with acipimox and
Intralipid was infused at varying rates (0, 3, 6 or 12 lL kg)1 min)1) to obtain
four different levels of circulating FFAs. Endogenous growth hormone,
insulin and glucagon secretion was blocked by somatostatin (300 lg h)1) and
replaced exogenously. 15N-phenylalanine, 2H4-tyrosine and 13C-urea were
infused continuously to assess protein turnover and ureagenesis.
Results: We obtained four distinct levels of FFA concentrations ranging from
0.03 to 2.1 mmol L)1 and 3-hydroxybutyrate concentrations from 10 to
360 lmol L)1. Whole-body phenylalanine turnover and phenylalanine-
to-tyrosine degradation decreased with increasing FFA levels as did insulin-
stimulated forearm fluxes of phenylalanine. Phenylalanine, tyrosine and urea
concentrations also decreased progressively, whereas urea turnover was
unperturbed.
Conclusion: Circulating FFAs decrease amino acid concentrations and
inhibit whole-body phenylalanine fluxes and phenylalanine-to-tyrosine con-
version. Our data cover FFA concentrations from 0 to 2 mmol L)1 and
indicate that FFAs exert their protein conserving effects in the upper
physiological range (>1.5 mmol L)1).
Keywords acipimox, amino acid turnover, free fatty acids, Intralipid,
somatostatin, urea turnover.

Free fatty acids (FFAs) serve a variety of essential of gene expression (Distel et al. 1992). FFAs also
functions; as a major provider of physiological energy stimulate insulin secretion (Nolan et al. 2006), inhibit
needs during fasting (Cahill et al. 1966), as constituents growth hormone secretion (Lanzi et al. 1999) and may
of cell membranes, and in some cases as key regulators interfere with glucagon release (Olofsson et al. 2004).

 2007 The Authors

Journal compilation  2007 Scandinavian Physiological Society, doi: 10.1111/j.1748-1716.2007.01771.x 369
Effects of FFAs on protein metabolism L C Gormsen et al.
In addition, even modest perturbation of physiological
FFA levels may lead to insulin resistance and type 2
diabetes (Roden et al. 1996, Dresner et al. 1999,
Baldeweg et al. 2000).
As regards protein metabolism previous studies have
suggested that elevated concentrations of FFAs and
ketone bodies have protein-sparing effects (Tessari
et al. 1986, Nair et al. 1988), and studies in growth
hormone-deficient patients have indicated that FFAs
decrease whole-body and muscle protein breakdown
(Shi et al. 2003). These effects are particularly pro-
nounced under conditions of fasting; studies in fasted
healthy subjects have shown that lowering of circulat-
ing FFAs by means of acipimox increases muscle
protein breakdown and urea-nitrogen production and
excretion by close to 50% and that this can be
reversed by experimentally elevating FFAs (Norrelund
et al. 2003).
As outlined above, FFAs decrease both regional
muscle and whole-body protein degradation and sev-
eral studies have investigated the impact of varying
lipid concentrations on amino acid turnover. These
investigations have been carried out using a variety of
lipid intermediates (Intralipid/heparin (Jeejee et al.
1976, Walker et al. 1993, Keller et al. 2002, Freyse
et al. 2003, Norrelund et al. 2003), glycerol (Keller
et al. 2002), or ketone bodies (Pawan & Semple 1983,
Nair et al. 1988), infusion lengths have varied consid-
erably from short-term (Jeejee et al. 1976, Walker
et al. 1993, Keller et al. 2002) (hours) to days (Jeejee
et al. 1976), and infusion rates have been designed to
achieve either modest (Jeejee et al. 1976, Pawan &
Semple 1983, Nair et al. 1988, Walker et al. 1993) or
370 Journal compilation  2007 Scandinavian Physiological Society, doi: 10.1111/j.1748-1716.2007.01771.x
Acta Physiol 2008, 192, 369379
supraphysiological levels of circulating lipids (Keller
et al. 2002, Freyse et al. 2003, Norrelund et al. 2003).
In addition, some protocols (Nielsen et al. 2002,
Norrelund et al. 2003) have employed acipimox to
control endogenous lipolysis. It is therefore still
unclear whether the protein-sparing effect of circulat-
ing lipids is present throughout the physiological
range. The purpose of the present study was therefore
to test the hypothesis that there is a progressive decline
in whole-body and muscle amino acid turnover with
increasing FFA concentrations. To circumvent pertur-
bations in insulin, glucagon and GH levels, we infused
somatostatin and replaced these hormones at fixed
rates. FFA levels spanning the physiological range
(02 mmol L)1) were obtained by infusing Intralipid
and heparin at variable rates.
Methods
Subjects
Eight healthy, weight stable men (24  1 year,
84  9 kg BW, 25  3 kg m)2 BMI) with normal
routine biochemistry were studied on four occasions,
separated by at least 1 month. Informed consent was
obtained from participants after oral and written
information about the study. The study was conducted
according to the Declaration of Helsinki II and
approved by the local Ethical Committee. The study
design and subjects have previously been used to assess
the impact of FFAs on ghrelin levels (Gormsen et al.
2006) and glucose/lipid metabolism (Gormsen et al.
2007).
Figure 1 Study protocol. Subjects were
studied for a total of 8 h (6 h basal and
2 h hyperinsulinaemic-euglycaemic
clamp) on all four study days. Four
different levels of free fatty acids were
obtained by infusing Intralipid/heparin at
variable rates. Endogenous growth
hormone, insulin and glucagon secretion
was blocked by somatostatin and sub-
sequently replaced at fixed rates.
 2007 The Authors
Acta Physiol 2008, 192, 369379
Study protocol
After an overnight fast, subjects were studied for 8 h
from 07:00 to 15:00 hours on four occasions with
varying lipid (Intralipid 20%, Fresenius Kabi) infusions:
0 (saline), 3, 6 and 12 lL kg)1 min)1 (Fig. 1).
Blood sampling. Three intravenous catheters (Venflon;
Viggo AB, Helsingborg, Sweden) were inserted in a left
antecubital vein, a right dorsal hand vein and a right
antecubital vein. The right hand was placed in a heated
box for sampling of arterialized blood. Blood samples
were collected in triplicate from 330360 min
(BASAL) and 450480 min (CLAMP).
Pancreatico-pituitary clamp and lipid infusion. Infusion
of heparin (Heparin SAD; 70 mIE kg)1 min)1), Intral-
ipid and somatostatin (300 lg h)1) was initiated at
t = 0 min together with replacement of GH (Norditro-
pin; Novo Nordisk, Bagsvaerd, Denmark) (2 ng
kg)1 min)1) and glucagon (Glucagen 1 mg mL)1,
0.8 ng kg)1 min)1; Novo Nordisk). Insulin (Novo Nor-
disk) was infused at 0.08 mU kg)1 min)1 for the initial
6 h of the study and continued at 0.6 mU kg)1 min)1
during the 2 h clamp. A constant infusion of somato-
statin has previously been shown (Sherwin et al. 1977)
to result in a biphasic plasma glucose response with an
initial decline after 12 h followed by a subsequent
increase after 3 h. To avoid early hypoglycaemia,
identical 20% glucose infusions were therefore given
from t = 60 to t = 140 min on all occasions. Acipimox
(Olbetam 250 mg; Pfizer, Ascoli Piceno, Italy) was
administered per oral at t = 0 min.
Protein turnover. After priming the amino acid pool with
bolus injections of [15N]phenylalanine (0.7 mg kg)1),
[15N]tyrosine (0.3 mg kg)1), [13C]urea (390.6 mg) and
[2H4]tyrosine (0.5 mg kg)1), continuous infusions of
[15N]phenylalanine (0.7 mg kg)1 h)1), [13C]urea
)1 )1 )1
2
(42 mg h ) and [ H4]tyrosine (0.5 mg kg h ) were
initiated at t = 0 and maintained for the duration of
the study day. Enrichments of [15N]phenylalanine,
[15N]tyrosine, and [2H4]tyrosine were measured by mass
spectrometry as their t-butyldimethylsilyl ether derivates
under electron ionization conditions, and concentrations
of phenylalanine and tyrosine were measured using
L-[2H8]phenylalanine and L-[13C6]tyrosine as internal
standards (Nair et al. 1995). Enrichment of plasma urea
was measured in its bistrimethylsilyl derivative by gas
chromatography-mass spectrometry (GC-MS) as previ-
ously described (Jahoor & Wolfe 1987). We used a
Hewlett-Packard G1722A GC-MS (Hewlett-Packard,
Palo Alto, CA, USA) and monitored signal intensities of
mass-to-charge ratios 231.1 and 232.1 (M and M + 1).
The chemical, isotopic and optical purity of all isotopes
 2007 The Authors
Journal compilation  2007 Scandinavian Physiological Society, doi: 10.1111/j.1748-1716.2007.01771.x
L C Gormsen et al. Effects of FFAs on protein metabolism
was tested before use and solutions were prepared under
sterile conditions and were shown to be free of bacteria
and pyrogen.
Forearm method
Forearm blood flow was measured in the non-dominant
forearm using venous occlusion plethysmography with
mercury-in-silastic strain gauges (Greenfield et al. 1963).
In brief, an arterial occlusion cuff around the wrist was
continuously inflated to suprasystolic pressures
(250 mmHg) during measurements (510 min at a time),
while a venous occlusion cuff around the upper arm is
inflated to 40 mmHg for 7.5 s out of every 15 s,
providing 1 blood flow measurement every 15 s. Forearm
blood flow is expressed as mL 100 mg)1 tissue min)1.
Calculations of whole-body amino acid and urea kinetics
The equations of Thompson et al. (1989) were used for
measurements of whole-body phenylalanine and urea
kinetics. Phenylalanine flux (QPhe), tyrosine flux (QTyr)
and urea flux (Qurea) were calculated as follows:
 
Ei
Qi 1
Ep
where i is the rate of tracer infusion (lmol kg)1 h)1)
and Ei and Ep are enrichment of the tracer infused and
plasma enrichment of the tracer at isotopic plateau
respectively. The rate of phenylalanine conversion by
hydroxylation to tyrosine (Ipt) was calculated as fol-
lows:
15 NTyrei QPhe
Ipt Qtyr  
15 NPheei IPhe QPhe
where [15N]Tyrei and [15N]Pheei are the isotopic
enrichments of the respective tracers in plasma and IPhe
is the infusion rate of [15N]phenylalanine (lmol
kg)1 h)1).
Forearm metabolism
In the forearm study, phenylalanine balance (PheBal)
was calculated as follows using Ficks principle:
PheBal Phea  Phev  F
where Phea and Phev are arterial and deep venous
phenylalanine concentrations and F is blood flow in the
forearm. Regional phenylalanine kinetics were calcu-
lated, using the equations described by Nair et al.
(1995). The forearm protein breakdown represented by
phenylalanine rate of appearance (Ra Phe) was calcu-
lated as follows (Copeland & Nair 1994):
371
Effects of FFAs on protein metabolism L C Gormsen et al.
 
PheEa
Ra Phe Phea  1  F
PheEv
where PheEa and PheEv represent phenylalanine isotopic
enrichment in arteries and veins. The local rate of
disappearance, which represents the muscle protein
synthesis rate, was calculated as:
Rd Phe PheBal Ra Phe
where Rd Phe is phenylalanine synthesis rate.
Urinary nitrogen secretion rate
Urinary nitrogen secretion rate (UNSR) was calculated
as urinary excretion rate (E), corrected for accumula-
tion (A) in total water and for the fractional intestinal
loss (L): UNSR = (E + A)/(1 ) L), where E = (urine
flow, L h)1) (urinary urea ) N, mmol L)1), A =
(change in blood urea ) N, mmol L)1 h)1) (total
water, L). Loss (L) was estimated to be 0.14.
Measurements
Serum FFA were determined using a commercial kit
(Wako Chemicals, Neuss, Germany) and plasma glu-
cose was measured in duplicate on a glucose analyser
(Beckman Instruments, Palo Alto, CA, USA).
An immunofluorometric assay (DELFIA; Wallac,
Turku, Finland) was used to measure serum GH. Insulin
and C peptide were determined by commercial kits
(DAKO, Glostrup, Denmark; Immunoclear, Stillwater,
MN, USA). Whole blood glycerol, lactate and 3-hydroxy-
butyrate (3-OH-butyrate) were analysed by autofluori-
metric enzymatic methods (Harrison et al. 1988).
Statistics
Results are expressed as mean  SEM (parametric data)
or median (range) (nonparametric data). Statistical
comparisons between study days were assessed by
repeated-measures analysis of variance (anova). Post
hoc comparisons were performed with paired t-tests or
the Wilcoxon sign rank test if data were not normally
distributed. If the distribution of the data was skewed,
data were transformed before applying anova and
t-tests. All data presented in tables have been
re-transformed in order to make values immediately
recognizable. P < 0.05 was considered significant.
Results
Circulating hormones and metabolites
Hormones. Our pancreatico-pituitary clamp combined
with exogenous substitution of major hormones at fixed
372 Journal compilation  2007 Scandinavian Physiological Society, doi: 10.1111/j.1748-1716.2007.01771.x
Acta Physiol 2008, 192, 369379
rates ensured comparable levels of insulin, glucagon,
GH, cortisol, insulin-like growth factor (IGF)-I and
IGF-II under all study conditions (Table 1).
FFAs, glycerol and 3-OH-butyrate. As expected and
reflecting their post-absorptive state, participants had
comparable FFAs at baseline [FFABaseline (mmol L)1):
0.49  0.05 vs. 0.40  0.04 vs. 0.55  0.06 vs.
0.48  0.03, anova P = 0.15). Antilipolysis with acip-
imox suppressed FFA levels (50 lmol L)1) and by
infusing intralipid and heparin at variable rates, we
obtained 4 significantly different levels of FFAs
throughout all study days: [FFA (mmol L)1): 0.03 
0.00 vs. 0.49  0.04 vs. 0.92  0.08 vs. 2.06  0.33,
anova P < 0.001]. Interestingly, hyperinsulinaemia
decreased FFA levels on three study days even under
conditions of antilipolysis with acipimox. As expected,
no further reduction in FFA levels was observed on the
study day where no Intralipid was infused. Glycerol
concentrations increased in conjunction with FFA
levels, although this did not reach the level of
significance. In contrast, 3-OH-butyrate concentrations
were significantly increased at very high levels of FFAs
(Fig. 2).
Glucose. Plasma glucose levels decreased rapidly on all
study days following initiation of the somatostatin
infusion as described previously (Norrelund et al.
2003). To avoid hypoglycaemia, 20% glucose was
therefore infused from t = 30 to t = 60 at a rate of
1560 mL h)1. At the end of the basal period, plasma
glucose was comparable on all study days
(6.5 mmol L)1). As intended, glucose was clamped
at 5 mmol L)1 during the hyperinsulinaemic-eugly-
caemic clamp.
Protein metabolism
Phenylalanine turnover. A stepwise elevation of circu-
lating FFAs elicited a significant decrease in whole-body
protein turnover as assessed by phenylalanine flux, both
during basal [Qphe BASAL (lmol kg)1 h)1): 37.2  1.1
vs. 37.5  1.2 vs. 35.4  1.6 vs. 33.2  1.4, anova
P = 0.02) and hyperinsulinaemic clamp conditions
[Qphe CLAMP (lmol kg)1 h)1): 36.3  1.1 vs. 35.8 
1.1 vs. 33.8  1.6 vs. 31.7  1.2, anova P = 0.04)
(Fig. 3). Post hoc comparisons between individual study
days revealed a significant decrease in phenylalanine
turnover from near-normal levels of FFAs to
supraphysiological levels, but by visual inspection of
the figure it appears likely that an inverse linear
relationship exists between the level of FFAs and
phenylalanine turnover. In addition, phenylalanine to
tyrosine conversion was suppressed during the clamp
when FFA levels were elevated to 1.5 mmol L)1. By
 2007 The Authors
Acta Physiol 2008, 192, 369379 L C Gormsen et al. Effects of FFAs on protein metabolism
Table 1 Circulating hormones and metabolites (arterial)

Lipid infusion rate (lL kg)1 min)1)

anova
0 3 6 12 P-value

Hormones
Insulin (pmol L)1)
Basal 26 2 37  7 27  3 36  6 0.21
Clamp 190  11 191  14 197  11 195  7 0.82
C-peptide (pmol L)1)
Basal 169  54 177  48 165  65 191  54 0.51
Clamp 156  61 152  50 149  54 159  53 0.87
Total IGF-1 (lg L)1)
Basal 175  14 189  22 185  13 205  20 0.10
Clamp 167  13 176  20 184  18 193  19 0.12
Total IGF-2 (lg L)1)
Basal 706  34 743  36 694  20 755  28 0.10
Clamp 700  33 714  40 710  22 730  29 0.85
GH (lg L)1)
Basal 0.50  0.08 0.59  0.13 0.52  0.12 0.40  0.03 0.60
Clamp 1.07  0.56 0.42  0.04 0.47  0.06 0.38  0.03 0.16
Cortisol (nmol L)1)
Basal 199 9 236  36 264  56 221  24 0.60
Clamp 246  37 199  27 251  41 206  17 0.56
Glucagon
Basal 65 8 50  7 56  5 46  5 0.08
Clamp 65 7 50  6 56  6 43  6 0.10
Metabolites
Glucose (mmol L)1)
Basal 6.37  0.40 6.66  0.44 6.30  0.39 7.00  0.31 0.39
Clamp 4.81  0.26 4.97  0.23 5.00  0.14 5.48  0.26 0.14
FFAs (mmol L)1)
Basal 0.03  0.00 0.49  0.04 a 0.92  0.08 b 2.06  0.33 c <0.001
Clamp 0.02  0.00 0.34  0.03 a 0.68  0.09 b 1.73  0.28 c <0.001
3-OH-butyrate (lmol L)1)
Basal 10 3 20  4 123  24b 357  93 <0.01
Clamp 7 1 14  4 46  17 197  77 0.05
Phenylalanine (mg L)1)
Basal 8.70  0.38 8.09  0.34 7.87  0.33 6.50  0.28 <0.01
Clamp 8.24  0.35 6.96  0.33 6.62  0.24 5.55  0.23 <0.001
Tyrosine (mg L)1)
Basal 14.53  1.20 14.64  0.98 12.97  0.64 11.88  1.03 0.07
Clamp 12.63  0.95 11.95  0.86 10.59  0.50 9.93  1.03 <0.05
Urea (mmol L)1)
Basal 5.09  0.40 4.99  0.43 4.77  0.34 4.45  0.39 0.09
Clamp 4.85  0.42 4.65  0.42 4.38  0.31 4.05  0.39 0.03

Values are mean  SEM (n = 8). Lower-case letters (a, b and c) refer to post hoc comparisons (pairwise t-test) between study days.
a = P < 0.05 vs. lipid infusion rate 0 lL kg)1 min)1, b = P < 0.05 vs. lipid infusion rate 3 lL kg)1 min)1, c = P < 0.05 vs. lipid
infusion rate 6 lL kg)1 min)1. IGF, insulin-like growth factor; GH, growth hormone; FFA, free fatty acid.

contrast, we failed to detect a significant effect of FFAs
on tyrosine flux (data not shown).
Forearm protein metabolism. Results from the forearm
study are presented in Figure 4. Basal phenylalanine rate
of appearance [Phe Ra BASAL (lmol kg)1 min)1):
5.0  0.8 vs. 4.1  0.2 vs. 3.1  0.7 vs. 4.3  0.5,
anova P = 0.18] and rate of disappearance [Phe Rd
BASAL (lmol kg)1 min)1): 3.3  0.5 vs. 2.7  0.2 vs.
1.9  0.7 vs. 2.8  0.6, anova P = 0.36] were non-
significantly inversely related to the level of FFAs in
the physiological range between 0 and 1 mmol L)1.
Under hyperinsulinaemic clamp conditions the general
pattern of decreasing phenylalanine flux throughout the

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Journal compilation  2007 Scandinavian Physiological Society, doi: 10.1111/j.1748-1716.2007.01771.x 373
Effects of FFAs on protein metabolism L C Gormsen et al.
physiological range of FFAs was observed; however,
differences between study days were now significant [Phe
Ra CLAMP (lmol kg)1 min)1): 4.1  0.5 vs. 3.4  0.2
vs. 2.5  0.5 vs. 3.5  0.5, anova P = 0.03], [Phe Rd
CLAMP (lmol kg)1 min)1): 3.1  0.3 vs. 2.4  0.3 vs.
1.7  0.3 vs. 2.5  0.2, anova P = 0.03].
Net exchange of phenylalanine across the forearm
muscle bed was not significantly affected by FFA levels
during either basal [phenylalanine muscle balance
(lg 100 mL)1 min)1): )1.71  0.30 vs. )1.35  0.16
vs. )1.14  0.19 vs. )1.42  0.19, anova P = 0.23)
or insulin stimulated conditions [phenylalanine
374 Journal compilation  2007 Scandinavian Physiological Society, doi: 10.1111/j.1748-1716.2007.01771.x
Acta Physiol 2008, 192, 369379
Figure 2 Lipid metabolites. P-values
stated in the text refer to repeated-mea-
sures anova with free fatty acid (FFA)
level as the term of interest. Post hoc
comparisons between study days are
stated in Table 1. All error bars are
SEM (n = 8).
muscle balance (lg 100 mL)1 min)1): )1.00  0.28 vs.
)0.97  0.18 vs. )0.75  0.21 vs. )0.98  0.12,
anova P = 0.66).
Urea turnover
Urea synthesis was assessed by a primed-constant
13C-urea infusion as well as by UNSR. As the urea pool
has a slow turnover, we did not attempt to measure
ureagenesis during the clamp. As depicted in Figure 5 left
panel, short-term perturbation of FFA levels did not
result in measurable differences in basal 13C-urea turn-
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Acta Physiol 2008, 192, 369379 L C Gormsen et al. Effects of FFAs on protein metabolism

Figure 3 Whole-body protein turnover. Repeated-measures anova with free fatty acid (FFA) levels as the term of interest was
used to assess whether FFAs impact on whole-body protein metabolism. If anova yielded a P-value <0.05, a post hoc comparison
(paired t-test) between study days was performed (indicated with P-values above connecting bars). All error bars are SEM (n = 8).

over [urea Ra (lmol kg)1 h)1): 389  64 vs. 361  28
vs. 330  29 vs. 326  45, anova P = 0.24] or basal
UNSR [UNSR (mmol h)1): 29.7  2.7 vs. 25.6  3.0 vs.
28.6  3.4 vs. 30.6  4.4, anova P = 0.48].
Discussion
In the current study we tested the hypothesis that
increasing FFA levels inhibit protein turnover. Our data
clearly show that short-term elevation of FFAs results in
progressively reduced circulating phenylalanine and
tyrosine concentrations, whole-body phenylalanine
turnover, regional forearm phenylalanine release and
uptake, and phenylalanine-to-tyrosine degradation dur-
ing hyperinsulinaemia. Moreover, our data show that
hepatic urea output and whole-body urea turnover is
unaffected by short-term experimental lipid elevations,
when hormone levels are clamped.
In our study we accomplished the desired FFA
concentrations by means of concomitant Intralipid
and heparin infusions; in this model, increased lipopro-
tein lipase activity results in rapid hydrolysis of trigly-
cerides with an ensuing increase in FFA concentrations.
Ketone bodies are major FFA metabolites and it is
therefore not surprising that some studies have demon-
strated increased concentrations after intralipid infusion
(Jeejee et al. 1976, Boden & Chen 1999). However, this
is not a consistent finding (Walker et al. 1993, Norrel-
und et al. 2003). In our study, only the highest
Intralipid infusion rate resulted in an approximately
twofold increase in 3-OH-butyrate concentrations,
whereas FFA levels in the physiological range left
ketone body concentrations unaltered. In our view, this
finding can be ascribed to the somatostatin clamp, as
somatostatin effectively blocks glucagon release, which
is instrumental in ketone body formation (Miles et al.
1982). We infused glucagon systemically at a fixed rate
and portal glucagons levels by inference must have been
low. As a result ketone body concentrations also
remained low, allowing independent assessment of the
intrinsic effects of FFA on amino acid metabolism. Our
results show that despite low or unaltered ketone body
concentrations, whole-body and regional phenylalanine
release and uptake is reduced by elevated FFAs. This

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Journal compilation  2007 Scandinavian Physiological Society, doi: 10.1111/j.1748-1716.2007.01771.x 375
Effects of FFAs on protein metabolism L C Gormsen et al. Acta Physiol 2008, 192, 369379

Figure 4 Forearm metabolism. Repeated-measures anova with free fatty acid (FFA) levels as the term of interest was used to assess
whether FFAs impact on regional phenylalanine release and uptake. If anova yielded a P-value <0.05, a post hoc comparison
(paired t-test) between study days was performed (indicated with P-values above connecting bars). All error bars are SEM (n = 8).

finding contrasts some previous papers (Nair et al.
1988, Norrelund et al. 2003) suggesting that the pro-
tein-sparing effect of FFAs is largely mediated via
ketone bodies.
Classic studies by Cahill et al. (1966) showed that
prolonged fasting results in progressively increasing
FFA and ketone body levels coupled with a decrease in
urea nitrogen excretion. Further linking stimulation of
lipid metabolism to protein-sparing, it has been dem-
onstrated that intravenous administration of 3-OH-
butyrate to obese subjects markedly decreases urinary
nitrogen secretion (Pawan & Semple 1983). Later
studies by Tessari et al. (1986) and Nair et al. (1988)
reported that lowering of FFAs increases leucine fluxes
and oxidation rates in fasted dogs and that administra-
tion of 3-OH-butyrate to humans decreases leucine
oxidation and increased muscle protein synthesis. In
addition it has been shown that infusion of lipofundin
(Keller et al. 2002) (a lipid emulsion equivalent to
Intralipid) decreases leucine oxidation, although this is
not a consistent finding (Freyse et al. 2003). Our data
support that high FFA levels inhibit amino acid degra-
dation (assessed by suppressed phenylalanine-to-tyro-
sine conversion) during hyperinsulinaemia, but our
choice of tracer (15N-phenylalanine) precluded us from
measuring oxidation directly.
In our experimental setting, whole-body phenylalanine
turnover decreased at FFA concentrations in the high

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376 Journal compilation  2007 Scandinavian Physiological Society, doi: 10.1111/j.1748-1716.2007.01771.x
Acta Physiol 2008, 192, 369379
Figure 5 Urea metabolism. UNSR, urinary nitrogen secretion rate. All error bars are SEM (n = 8).
physiological range. This observation contrasts that of a
previous study in which the authors found no effect of
FFAs on phenylalanine turnover despite a significant
decrease in forearm muscle phenylalanine release
(Walker et al. 1993). This discrepancy most likely reflects
different study designs, as we infused intralipid for 6 h as
opposed to 3 h employed in that study. It is possible that
elevating FFAs for 3 h causes decreased phenylalanine
release from skeletal muscle, but that this fails to translate
into measurable changes in whole-body turnover,
because of the limited period of observation. That FFAs
most likely affect whole-body amino acid turnover is
supported by several studies reporting an inverse rela-
tionship between whole-body proteolysis and FFA levels
(Tessari et al. 1986, Nielsen et al. 2002). As regards local
muscle protein metabolism we observed decreased
muscle phenylalanine Ra and Rd during the clamp in
the FFA range between 0 and 1.0 mmol L)1; this effect
seemed to level off at FFA levels around 2.0 mmol L)1.
On the whole, the above data together with our present
observations indicate that FFAs reduce protein degrada-
tion by decreasing amino acid turnover both at the whole-
body level and in muscle.
The cytosolic mechanism that links increasing FFA
levels to decreased protein degradation and amino acid
release remains unclear. Most lines of evidence point to
the contrary, i.e. that cytosolic accumulation of FFAs
primarily has catabolic effects. Thus, it has recently
been demonstrated that FFAs can activate the p38 MAP
kinase pathway in cardiac myocytes (Miller et al. 2005)
and hepatocytes (Collins et al. 2006). Activation of this
stress-sensitive pathway results in expression of genes
known to be associated with impaired insulin action,
and as insulin has profound anabolic effects in skeletal
muscle tissue (Fukagawa et al. 1985), increased, rather
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Journal compilation  2007 Scandinavian Physiological Society, doi: 10.1111/j.1748-1716.2007.01771.x
L C Gormsen et al. Effects of FFAs on protein metabolism
than decreased, protein breakdown would be expected.
However, activation and transcription of genes is a
time-dependent process and it is therefore possible that
short-term infusion of lipids as in the current study
does not result in detectable alterations in gene expres-
sion and subsequent protein expression.
In this study, urea turnover was unperturbed by
elevated FFAs. The urea pool is large and has a slow
relative turnover, and it is possible that the time span of
the lipid infusion was too short to lead to a detectable
down-regulation of urea synthesis. However, short-term
perturbations (2 h) of amino acid turnover may result in
a decrease in urea synthesis of a magnitude great
enough to be detected with the 13C-urea tracer tech-
nique (Jahoor & Wolfe 1987). It therefore remains a
distinct possibility that ureagenesis is unaffected by
short-term elevations of FFAs. On the other hand, it is
also possible that ureagenesis is regulated by alterations
in insulin/glucagon ratios or by increasing ketone body
concentrations. In this context it should be underlined
that hepatic concentrations of insulin, glucagon and
ketone bodies are much higher under natural circum-
stances compared to our experimental set-up.
Biologically the protein-sparing effects of FFA are
important for survival under conditions of metabolic
stress, such as pure caloric deprivation (fasting, starva-
tion and malnutrition) and increased energy expendi-
ture combined with caloric deprivation (critical illness,
chronic inflammation, surgery and prolonged exercise).
In general, these conditions are characterized by
alterations in circulating metabolic hormones typi-
cally low insulin levels and high stress hormone levels
and in metabolite concentrations and fluxes (Coker &
Kjaer 2005, McGuinness 2005, Cahill 2006, Nygren
2006).
377
Effects of FFAs on protein metabolism L C Gormsen et al.
Our experimental design partly differs from these
conditions, in terms of selection of young healthy adults
studied in the basal state, relatively short duration of
FFA elevation and clamping of hormone and metabolite
concentrations with somatostatin and acipimox. Our
results therefore cannot be readily extrapolated to the
physiological and clinical situations outlined above. It
has for example been shown that during fasting
lowering of FFA decreases ureagenesis and whole-body
and muscle protein breakdown substantially (Norrel-
und et al. 2003).
In summary, our data show that whole-body amino
acid turnover and insulin stimulated phenylalanine-
to-tyrosine conversion are suppressed when FFAs are
elevated above 1.5 mmol L)1. In addition, muscle
amino acid uptake and release is decreased at FFA
concentrations 1 mmol L)1 during a hyperinsulinae-
mic glucose clamp. Although we failed to observe any
effects on urea synthesis, these mechanisms may
contribute to the overall protein-sparing effects of
FFA.
Conflict of interest
There is no conflict of interest.
This work was supported by grants from The Danish Diabetes
Association and from the A. P. Mller and Hustru Chastine
McKinney Mllers Foundation. The excellent technical assis-
tance of Ms Lone Svendsen, Elsebeth Hornemann, Elin
Carstensen and Ms Susanne Srensen is highly appreciated.
The study was supported by grants from the FOOD Study
Group/Ministry of Food, Agriculture and Fisheries & Ministry
of Family and Consumer Affairs, Denmark.
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