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2
*
Department of Agriculture, University of Aberdeen, 581 King St., Aberdeen AB24 5UA, Scotland,
Rowett Research Institute, Bucksburn, Aberdeen AB21 9SB, Scotland
2To whom correspondence should be addressed.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • protein metabolism • sheep • branched-chain amino acids • insulin • muscle protein
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, Wray-Cahen et al. 1998
), and the subdued responsiveness of insulin to feeding in
ruminants may account for some of the differences in efficiency of N
utilization between ruminants and nonruminants.
Skeletal muscle protein metabolism is particularly sensitive to insulin
in young and food-deprived animals. For example, in young growing
pigs, protein synthesis is stimulated by administration of insulin, but
the response was 4-fold higher at 7 d compared with 26-d-old pigs
(Wray-Cahen et al. 1998
). Anticatabolic effects of
insulin have also been observed in adult humans, but via a mechanism
involving decreased protein degradation (Fryburg et al. 1995
). Regardless of which component of protein turnover is
modulated, effects of insulin are strongest in the fasted state, as
observed in rats (Garlick and Grant 1988
), sheep
(Oddy et al. 1987
) and humans (Fryburg et al. 1995
). Indeed, limited efforts to demonstrate protein anabolic
responses of skeletal muscle to insulin in the fed state have been
unsuccessful (e.g., Oddy et al. 1987
) unless chronic,
supraphysiologic doses are given (Wolff et al. 1989
).
In nonruminants, infusion of an amino acid mixture, or refeeding,
results in decreased protein degradation and/or increased protein
synthesis in skeletal muscle. While some of these responses may be
direct (Svanberg et al. 1996a
), there may be an
interaction with endocrine status. Most notably, in young
food-deprived rats, branched-chain amino acids
(BCAA)3
have been shown to enhance protein synthesis in skeletal muscle through
increasing the sensitivity of the tissue to insulin by up to 10-fold
(Garlick and Grant 1988
). Furthermore, BCAA have also
been shown to decrease whole-body proteolysis in humans, and this
may again represent a potentiation of the action of insulin in adults
(Ferrando et al. 1995
).
In view of the less episodic absorption of nutrients in ruminants leading to lower and more constant plasma concentrations of insulin, there may be potential for further responses to insulin in these species, even in the fed condition. The primary objective of the present study, therefore, was to ascertain whether insulin, at physiologic levels, increases protein anabolism [using net phenylalanine (Phe) uptake as an index of skeletal muscle protein gain] in the hind limb of fed lambs. In view of the observation in nonruminants that the action of insulin on protein metabolism may be potentiated by the presence of BCAA, a secondary objective was to determine if a similar mechanism operates in ruminants.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Lambs were allowed 2 wk of recovery in heated floor pens after surgery
prior to transfer to metabolism crates where they were fed hourly, by
automatic feeder, a grass hay/barley-based concentrate diet
(Table 1
) at maintenance intake [0.2 MJ metabolizable enerergy/(d · kg
BW); 0.4 g N/(d · kg BW)]. A temporary catheter (0.8 mm
i.d. x 1.2 mm o.d. PVC; Dural Plastics and Engineering, Auburn,
NSW, Australia) was inserted, via a jugular vein, into the right
ventricle of the heart the day preceding the start of the infusion
series. This catheter was used as a source of mixed systemic blood.
Alternatively, if a permanent iliac vein catheter failed prior to use,
a temporary catheter (0.4 mm i.d. x 0.8 mm o.d. polyethylene, Portex,
Hythe, Kent, United Kingdom) was inserted via a lateral saphenous vein
so that the tip of the catheter was sited in the external iliac vein.
This distance was calculated from earlier post mortem examinations.
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). Over the
last 200 min of infusion, five blood samples were withdrawn
continuously from the iliac vein and heart catheters at a rate of 3.5
mL/40 min. Lambs were administered heparin (83.3 IU/min) to
facilitate continuous sampling of blood.
Concurrent with sampling, indocyanine green (150 µg/min, Fluka
Gillingham, Dorset, United Kingdom), in a solution containing 10 g/L of
BSA and 154 mmol/L of NaCl, was infused into the iliac artery to
measure plasma flow (Cherrick et al. 1960
). Indocyanine
green solutions were prepared immediately before use and filter
sterilized as they were administered. Indocyanine green concentration
was determined in plasma from the absorbance at 795 nm.
Plasma Phe concentration was measured in deproteinized plasma by
isotope dilution gas chromatography mass spectroscopy (Lobley et al. 1998
) with L-[1-13C]Phe (Isotec
Inc., Miamisburg, OH) used as internal standard and the fragment ions
at m/z 231 and 233 of the t-butyldimethylsilyl
derivative monitored under electron ionization conditions. Plasma
glucose concentration was measured by the Trinder method (Sigma, Poole,
Dorset, United Kingdom). Immunoreactive plasma insulin concentration by
radioimmunoassay using porcine insulin as standards (Midgley et al. 1969
). Intra- and inter-assay CV were 5.4 and 10.7%,
respectively.
Because the treatments were not replicated equally across periods due to failure of some catheters during an infusion series, data were analyzed using the Residual Maximal Likelihood procedure in Genstat 5 (release 3.2; Lawes Agricultural Trust, IACR Rothamsted, United Kingdom) with lamb and lamb x period as random effects and treatment as the fixed effect. A Wald statistic was generated to test the fixed effect, and predicted means were compared using a two-tailed t test. Differences were considered significant at P < 0.05. This experiment was approved by the Ethical Review Committee of the Rowett Research Institute.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Thus, although iliac vein insulin concentration was doubled and
quadrupled by low- and high-insulin infusion, respectively
(P < 0.05), close to that predicted, insulin
concentration in mixed systemic blood, i.e., taken from the right
ventricle of the heart, was not different among treatments. Similarly,
there were no differences in systemic or iliac vein glucose
concentrations among treatments except for the high-insulin
infusion where there were slight decreases (-13 and -24%,
P < 0.05, systemic and iliac vein, respectively).
However, glucose uptake by the hind limb was not affected significantly
by treatment. Plasma flow to the hind limb was not affected by
treatments and averaged 120 ± 12.5 g/min (Table 2)
.
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).There was no difference between low- and high-insulin infusions,
although Phe retention tended to be higher (P < 0.2)
for the latter. Infusion of BCAA resulted in Phe uptakes that were not
different from that during low insulin, and there was no enhancement in
Phe retention when BCAA were co-infused with insulin.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Young
food-deprived rats also respond to insulin administration, but
maximal responses in muscle protein synthesis are only achieved at
rates of infusion which result in hormone concentrations close to the
upper physiologic limits (Garlick and Grant 1988
). In
adult humans, insulin also has a muscle protein anabolic effect, but
this is accomplished more by decreasing protein breakdown and has only
been observed in a fasted state (Fryburg et al. 1995
).
In young food-deprived lambs, insulin also decreased muscle protein
degradation, but similarly, this was not observed in the fed state
(Oddy et al. 1987
). In contrast, Wolff et al. (1989)
achieved muscle gain in fed lambs, but only when insulin
was infused chronically at supraphysiologic doses. These anomalies,
coupled with the more constant nutrient absorption pattern observed,
provided a rationale for the re-evaluation of the role of insulin
on protein metabolism in ruminants.
In the present study, insulin concentration was raised locally 1- and
3-fold with little systemic perturbation. Other studies in sheep have
infused doses of insulin close-arterially which have overwhelmed
the clearance of the hormone by the tissues and resulted in extensive
recirculation; thus systemic metabolism was affected (Oddy et al. 1987
; Wolff et al. 1989
).
The current study is also unique in that the experiment was performed
on fed animals, albeit at maintenance, using doses of insulin and BCAA
which remained within physiologic limits (Cole et al. 1988
; Lobley et al. 1998
). The majority of
studies examining effects of insulin on protein metabolism have been
performed in postabsorptive or food-deprived nonruminants. Fasting
is used normally because the individual is in a "basal" state where
an even flow of nutrients is provided to tissues and when, arguably,
they are probably more sensitive to insulin. Nonruminants are normally
supplied food as infrequent meals and, in consequence, plasma insulin
varies from low concentrations (in the postabsorptive period just
before a meal) to relatively high values (in the absorptive period
post-prandially). Ruminants, on the other hand, generally have more
constant patterns of nutrient absorption and, thus, insulin
concentration is lower and less variable. The question is whether these
latter concentrations are optimal to promote protein anabolism.
The current data clearly demonstrate that, in the fed state, low doses
of insulin, i.e., within the physiologic range, enhanced net Phe uptake
(and presumably net protein gain). Positive responses to insulin have
been reported in few other ruminant studies. For example,
Douglas et al. (1991)
showed a 12% reduction in
whole-body net protein loss when young lambs (16 kg, 3 to 5 mo of
age), deprived of food for 48 h, were infused systemically with
0.17 nmol/kg · h for 300 min (i.e., systemic insulin was raised
substantially, from 0.03 to 0.13 nmol/L). There were no changes,
however, in protein fractional synthesis rate for skeletal muscle,
heart or liver. Similarly, in a study by Oddy et al. (1987)
using food-deprived, preruminant young lambs, an
increase in insulin from 0.06 to 2.0 nmol/L resulted in a 30% decrease
in hind limb protein degradation, and a further increase in insulin to
11.3 nmol/L decreased degradation by another 25%. When these same
lambs consumed feed ad libitum, infusion of insulin (to raise
concentration 5-fold, i.e., from 2.1 to 12.2 nmol/L) had no effect on
hind limb protein metabolism. This compares with the present study
where older (6 mo vs. 10 to 60 d), maintenance-fed lambs,
showed a 12-fold increase in net Phe uptake in response to an elevation
of plasma insulin from 0.15 to 0.64 nmol/L. Only one other ruminant
study involving fed lambs has reported a protein anabolic effect, where
chronic infusion (45 d) of insulin increased hind limb muscle gain 5%,
compared with the contra-lateral control leg (Wolff et al. 1989
).
In the present study, infusion of BCAA increased net Phe uptake across
the hind limb. Elevation of BCAA was not confined to the vascular
supply of the leg, however, and much recirculation occurred because of
relatively low rates of clearance compared with rates of infusion. One
consequence of elevated BCAA was a 25% decrease in systemic Phe
concentration. Studies in overnight-fasted humans which examined
the effects of oral administration of BCAA also found systemic effects.
Whole-body Phe flux (representing degradation) was decreased by
20%, despite no change in net Phe balance across the leg
(Ferrando et al. 1995
). In contrast, intravenous
infusion of amino acids to post-absorptive humans increased protein
synthesis and decreased protein degradation across muscles of both the
forearm and leg, despite no change in plasma insulin concentration
(Svanberg et al. 1996a
). These authors contended that
specific amino acids triggered the changes in protein balance, but an
alternative explanation is that sensitivity of skeletal muscle to
insulin was increased by the presence of BCAA. Garlick and Grant (1988)
observed that in overnight food-deprived young rats
infused with BCAA, maximal stimulation of skeletal muscle protein
synthesis could be achieved with a dose of insulin only 13% of that
required when BCAA were not infused. This, they considered, provided an
explanation of the acute response in muscle metabolism to feeding.
That there was no further enhancement of muscle protein anabolism
through co-infusion of BCAA and low insulin in the present study
may be because maximal responsiveness was already achieved at that
hormone dose. This is supported by the lack of further response in net
Phe uptake when the insulin dose was doubled. Wolff et al. (1989)
also reported that the response in skeletal muscle
protein gain was already maximized at chronic insulin infusions of 36.3
pmol/min, the lowest dose they examined, which was between the infusion
rates in the present study of 10.5 and 52 pmol/min. The authors also
concluded that the lambs differed in sensitivity to insulin with both
age and body composition. In the present study, enhancement of insulin
action may have occurred with BCAA infusion alone sensitizing muscle to
endogenous hormone concentration. The response to insulin was thus
maximized, similar to Garlick and Grant (1988)
, at a
lower concentration of hormone.
In conclusion, short-term, close-arterial infusion of insulin
across the hind limb of fed lambs resulted in increased net Phe uptake
and, presumably, net protein gain. BCAA alone also increased Phe
uptake. This may be a direct stimulatory effect or may involve
enhancement of the action of endogenous insulin, similar to that
observed in rats (Garlick and Grant 1988
). Maximum
stimulation seemed to have been achieved with infusion of the lower
insulin dose because there was no increase in net Phe uptake with the
higher hormone dose or from co-infusion with BCAA. The lack of
change in peripheral metabolite and hormonal concentrations argue for
local (direct) effects rather than systemic mechanisms.
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FOOTNOTES |
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3 Abbreviations used: BCAA, branched-chain amino acids; BSA, bovine serum albumin; BW, body weight; Ile, isoleucine; Leu, leucine; Phe, Phenylalanine; Val, valine.
Manuscript received May 6, 1999. Revision accepted November 29, 1999.
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REFERENCES |
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1. Cherrick G. R., Stein S. W., Leevy C. M., Davidson C. S. Indocyanine green: observations on its physical properties, plasma decay, and hepatic extraction. J. Clin. Invest. 1960;39:592-600
2. Cole N. A., Purdy C. W., Hallford D. M. Influence of fasting and postfast diet energy level on feed intake, feeding pattern and blood variables of lambs. J. Anim. Sci. 1988;66:798-805
3. Douglas R. D., Gluckman P. D., Ball K., Breier B., Shaw J. H. F. The effects of infusion of insulin-like growth factor (IGF) I, IGF-II, and insulin in glucose and protein metabolism in fasted lambs. J. Clin. Invest. 1991;88:614-622
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Ferrando A. A., Williams B. D., Stuart C. A., Lane H. W., Wolfe R. R. Oral branched-chain amino acids decrease whole-body proteolysis. J. Paren. Enter. Nutr. 1995;19:47-54
5. Fryburg D. A., Jahn L. A., Hill S. A., Oliveras D. M., Barrett E. J. Insulin and insulin-like growth factor-I enhance human skeletal muscle protein anabolism during hyperaminoacidemia by different mechanisms. J. Clin. Invest. 1995;96:1722-1729
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