The ability of adenosine, insulin and human acylation-stimulating protein to modify fat synthesis was determined using cultures of fat tissue from steers. Adenosine did not influence fat synthesis. However, acylation stimulating protein and insulin promoted fat synthesis. These observations, coupled with knowledge of how fat synthesis is regulated in other species, justify investigation of whether cattle synthesize acylation-stimulating protein, and how this synthesis is regulated. An understanding of how acylation-stimulating protein production and action is regulated should expose potential places for intervention to manipulate fat synthesis in cattle

Jacobi, Sheila

Miner, Jess L.

English

DigitalCommons@University of Nebraska

University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln Nebraska Beef Cattle Reports Animal Science Department January 1999 Hormonal Influence on Fat Synthesis in Cattle Sheila Jacobi University of Nebraska-Lincoln Jess L. Miner University of Nebraska-Lincoln, jminer1@unl.edu Follow this and additional works at: https://digitalcommons.unl.edu/animalscinbcr  Part of the Animal Sciences Commons Jacobi, Sheila and Miner, Jess L., "Hormonal Influence on Fat Synthesis in Cattle" (1999). Nebraska Beef Cattle Reports. 408. https://digitalcommons.unl.edu/animalscinbcr/408 This Article is brought to you for free and open access by the Animal Science Department at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Nebraska Beef Cattle Reports by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. Page 69 — 1999 Nebraska Beef Report13) gave closer estimates to the other two techniques. Endogenous N values were estimated using reference values which vary. Endogenous N was assumed to be 2.2 g for each kg DM intake. The in situ technique is not an in vivo technique. Restriction of microbial access to the protein, bag size, bag porosity, sample quantity, sample particle size, incubation time, diet and animals are all concerns. Passage rate is required to calculate escape protein in the in situ technique. External markers are used to calculate passage rate, but they do not estimate lag prior to passage.NDIP can estimate EP values simply using the ratio of internal particulate marker in the diet and the omasal contents with ND insoluble protein content of omasal contents. Therefore, NDIP can estimate EP contents of brome hay and smooth bromegrass more precisely, simply and faster than OSPT, and provide reasonable EP digestibility values. NDIP is superior to the in situ technique because it is an in vivo tech-nique and better correlated with OSPT. Use of NDIP to measure the EP content of forages will enhance our understanding of the protein components of forages.1Abdullah Can, graduate student; Terry Klop-fenstein, professor Animal Science, Lincoln; Ryan Mass, research technician, Lincoln.IntroductionMarbling is a major reason behind feeding grain to cattle. This intramuscu-lar fat improves the eating qualities of beef. A problem associated with feeding cattle to increase marbling, however, is the parallel fat deposition in other parts of the carcass. The synthesis of fat in feed energy, although this fat is of little value to beef customers. Beef producers -uct quality by using a management tool which shifts fat synthesis to intramuscu-lar depots. Development of this tool will require a better understanding of how fat synthesis is regulated in cattle.Although the hormones controlling fat synthesis in cattle have not been and mouse cells. Adenosine, a molecule secreted by adipocytes, can stimulate fat synthesis in mice. Another hormone stimulating fat synthesis in both humans and mice is acylation-stimulating protein (ASP). ASP is also secreted by adipo-cytes. The objective of these experiments was to determine if adenosine and ASP can stimulate fat synthesis in cattle.Procedure-plantsCultures of fat tissue obtained from steers were used to test effects of adenosine and ASP. This technique provides a rapid (less than six hours) analysis of fat synthesis and does not require injecting animals with radio-active materials. For each experiment, fresh tissue obtained by biopsy was incubated at body temperature in a solution which provided all essential nutrients, was pH buffered and oxygen-ated. Synthesis of fat was measured by monitoring the incorporation of 14C-labeled substrates into triacylglycerol. In cattle, fat is synthesized mainly from acetate, but also from fatty acids like palmitate or oleate. Both acetate and palmitate were used in these experi-ments.The hypothesis was that adenos-ine would increase synthesis of fat in cultured tissue. Since adipose tissue contains some adenosine, an enzyme (adenosine deaminase) was used to Sheila JacobiJess Miner1Fat synthesis was enhanced by acylation-stimulating protein in cultured bovine tissue. This supports the idea that external fat or marbling this hormone in cattle.Summaryhuman acylation-stimulating protein to modify fat synthesis was determined using cultures of fat tissue from steers. However, acylation stimulating protein observations, coupled with knowledge of how fat synthesis is regulated in other species, justify investigation of whether cattle synthesize acylation-stimulat-ing protein, and how this synthesis is regulated. An understanding of how acylation-stimulating protein production and action is regulated should expose potential places for intervention to ma-nipulate fat synthesis in cattle.(Continued on next page)1999 Nebraska Beef Report — Page 70destroy the pre-existing adenosine. This allowed comparison between untreated and treated cultures. Two additional treat-ments provided a test of added adenosine. Additional cultures were treated with low and high doses of the adenosine analog, PIA (N6-phenylisopropyl adenosine), which resists degradation by adenosine deaminase.Experiment 2 was similar to experi-ment 1 except the dose range for PIA was greater. The treatments were: 1) no hormone; 2) 0.75 U/ml adenosine deaminase; 3) 0.75 U/ml adenosine de-aminase plus 10 nM PIA; 4) 0.75 U/ml adenosine deaminase plus 100 nM PIA; and 5) 0.75 U/ml adenosine deaminase plus 1,000 nM PIA.-lating protein (ASP)The second hormone suspected to increase synthesis of fat in cattle is ASP. To test the effect of ASP on fat synthesis in cattle, cultures were designed with in-creasing concentrations of ASP and some cultures with added insulin, a glucose uptake-promoting hormone. It is possible that ASP only increases fat synthesis if insulin is present. On the other hand, an effect of ASP may be more pronounced if insulin is absent. The treatments were: 1) no hormone; 2) 0.5 M ASP; 3) 5 MASP; 4) 10 nM insulin; 5) 0.5 M ASP plus 10 nM insulin; and 6) 5 M ASP plus 10 nM insulin.protein dose curveIn experiment 4, a wider range of ASP doses was evaluated: 1) no hormone; 2) 0.01 M ASP; 3) 0.1 M ASP; 4) 1 M ASP; 5) 5 M ASP; and 6) 10 M ASP.protein dose curve with and without insulinTreatments were: 1) no hormone; 2) 0.01 M ASP; 3) 0.1 M ASP; 4) 1 MASP; 5) 5 M ASP; 6) 10 M ASP; 7) 1 nM Insulin; 8) 0.01 M ASP plus 1 nM insulin; 9) 1 M ASP plus 1 nM Insulin; and 10) 10 M ASP plus 1 nM insulin.triacylglycerol synthesis.Following incubation, tissues were extracted in 2 ml of chloroform:metha-nol (2:1, v/v), and washed with both 1 M KCl-0.15N HCl, and 0.38 M sodium carbonate. Incorporation of radioactivity was determined in a beta counter.Triacylglycerol was distinguished from total fat by use of thin layer chromatography. Fat synthesis was calculated from radioactivity incor-porated and expressed as nmol acetate (or palmitate)/ mg tissue / six hours. Data were analyzed by ANOVA with main effects of animal and treatment. The animal x treatment interaction -cance.ResultsFat synthesis in cattle occurs in two general steps. First, fatty acids are syn-thesized. Second, fatty acids are linked to glycerol phosphate in a process called high-fat diets, such as humans or mice, fatty acid synthesis is less than in cattle. Incorporation of 14C-acetate estimates while incorporation of 14C-palmitate fatty acid synthesis.synthesis in bovine adipose tissue.Neither fatty acid synthesis nor fatty -dition of PIA (an adenosine analog) or adenosine deaminase (an enzyme which removal of adenosine (deaminase) and addition of an adenosine analog (PIA). No effect of treatment was detected.9876543210Acetate9876543210PalmitateFigure 2. (Experiment 2). Effect of adenosine on synthesis of fat from acetate (left) and palmitate (right). No effect of treatment was detected.6543210Acetate Palmitate6543210Page 71 — 1999 Nebraska Beef Reportdestroys adenosine) to cultures of adi-pose tissue (Figures 1 and 2). Adenosine apparently does not have an effect on fatty acids in cattle.-tein on triacylglycerol synthesis in bovine adipose tissue.The second hormone evaluated was ASP. Initially, it appeared ASP stimulates fat synthesis (Figure 3). In this experi-ment, however, the degree of variation in fat synthesis was large and treatment shown in Figure 4, ASP did increase the use of acetate for fatty acid and triacylg-lycerol synthesis. However the synthesis of fat from palmitate (a fatty acid) was not affected by ASP. This indicates ASP may have a greater effect on enzymes of enzymes. This would be logical, as most fat synthesis in cattle is from acetate and dietary fat intake is low. Finally, the interaction of insulin and ASP in regard to fat synthesis was evaluated (Figure 5). Fat synthesis was enhanced by increasing doses of ASP only when cultures also contained insulin. Our interpretation is that while both insulin and ASP promote different parts of the pathway.In conclusion, human ASP appears capable of promoting fat synthesis in bovine tissue. Our prediction is that bovine ASP is at least as potent. Based on information from a cDNA clone of bovine ASP, we know this molecule dif-fers from human at several amino acid positions. We are developing a method future we hope to determine whether intramuscular fat cells utilize ASP dif-ferently than fat cells in other fat depots. Perhaps this can lead to a method for promoting marbling while reducing total carcass fat.1Sheila Jacobi, graduate student. Jess Miner, assistant professor, Animal Science, Lincoln.Figure 3. (Experiment 3). Effects of ASP and insulin on fat synthesis from acetate (left) or palmi-9876543210Acetate Palmitate9876543210Figure 4. (Experiment 4). Effect of varying concentration of ASP on fat synthesis in absence of insulin. Acetate incorporation (left graph) was increased (P<.05) by ASP but all doses 876543210876543210Acetate PalmitateFigure 5. (Experiment 5). Interaction of ASP with insulin in stimulation of fat synthesis from acetate (left) and palmitate (right). Hatched bars represent cultures treated with 10 nM insulin. ASP treatment ( M ) is indicated below bars. Incorporation of both acetate interaction indicates that insulin is required for ASP to stimulate fat synthesis. *P<.10.1312111098765432101312111098765432100 .01 .1 1 5 10 0 .1 1 10 0 .01 .1 1 5 10 0 .1 1 10

Hormonal Influence on Fat Synthesis in Cattle

UNL | Libraries

University of Nebraska - Lincoln 
DigitalCommons@University of Nebraska - Lincoln 
Nebraska Beef Cattle Reports Animal Science Department 
January 1999 
Hormonal Influence on Fat Synthesis in Cattle 
Sheila Jacobi 
University of Nebraska-Lincoln 
Jess L. Miner 
University of Nebraska-Lincoln, jminer1@unl.edu 
Follow this and additional works at: https://digitalcommons.unl.edu/animalscinbcr 
 Part of the Animal Sciences Commons 
Jacobi, Sheila and Miner, Jess L., "Hormonal Influence on Fat Synthesis in Cattle" (1999). Nebraska Beef 
Cattle Reports. 408. 
https://digitalcommons.unl.edu/animalscinbcr/408 
This Article is brought to you for free and open access by the Animal Science Department at 
DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Nebraska Beef Cattle 
Reports by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. 
Page 69 — 1999 Nebraska Beef Report
13) gave closer estimates to the other two 
techniques. Endogenous N values were 
estimated using reference values which 
vary. Endogenous N was assumed to be 
2.2 g for each kg DM intake. The in situ 
technique is not an in vivo technique. 
Restriction of microbial access to the 
protein, bag size, bag porosity, sample 
quantity, sample particle size, incubation 
time, diet and animals are all concerns. 
Passage rate is required to calculate 
escape protein in the in situ technique. 
External markers are used to calculate 
passage rate, but they do not estimate 
lag prior to passage.
NDIP can estimate EP values simply 
using the ratio of internal particulate 
marker in the diet and the omasal 
contents with ND insoluble protein 
content of omasal contents. Therefore, 
NDIP can estimate EP contents of 
brome hay and smooth bromegrass more 
precisely, simply and faster than OSPT, 
and provide reasonable EP digestibility 
values. NDIP is superior to the in situ 
technique because it is an in vivo tech-
nique and better correlated with OSPT. 
Use of NDIP to measure the EP content of 
forages will enhance our understanding 
of the protein components of forages.
1Abdullah Can, graduate student; Terry Klop-
fenstein, professor Animal Science, Lincoln; Ryan 
Mass, research technician, Lincoln.
Introduction
Marbling is a major reason behind 
feeding grain to cattle. This intramuscu-
lar fat improves the eating qualities of 
beef. A problem associated with feeding 
cattle to increase marbling, however, is 
the parallel fat deposition in other parts 
of the carcass. The synthesis of fat in 
feed energy, although this fat is of little 
value to beef customers. Beef producers 
-
uct quality by using a management tool 
which shifts fat synthesis to intramuscu-
lar depots. Development of this tool will 
require a better understanding of how fat 
synthesis is regulated in cattle.
Although the hormones controlling 
fat synthesis in cattle have not been 
and mouse cells. Adenosine, a molecule 
secreted by adipocytes, can stimulate 
fat synthesis in mice. Another hormone 
stimulating fat synthesis in both humans 
and mice is acylation-stimulating protein 
(ASP). ASP is also secreted by adipo-
cytes. The objective of these experiments 
was to determine if adenosine and ASP 
can stimulate fat synthesis in cattle.
Procedure
-
plants
Cultures of fat tissue obtained 
from steers were used to test effects 
of adenosine and ASP. This technique 
provides a rapid (less than six hours) 
analysis of fat synthesis and does not 
require injecting animals with radio-
active materials. For each experiment, 
fresh tissue obtained by biopsy was 
incubated at body temperature in a 
solution which provided all essential 
nutrients, was pH buffered and oxygen-
ated. Synthesis of fat was measured by 
monitoring the incorporation of 14C-
labeled substrates into triacylglycerol. 
In cattle, fat is synthesized mainly from 
acetate, but also from fatty acids like 
palmitate or oleate. Both acetate and 
palmitate were used in these experi-
ments.
The hypothesis was that adenos-
ine would increase synthesis of fat in 
cultured tissue. Since adipose tissue 
contains some adenosine, an enzyme 
(adenosine deaminase) was used to 
Sheila Jacobi
Jess Miner1
Fat synthesis was enhanced by 
acylation-stimulating protein in 
cultured bovine tissue. This supports 
the idea that external fat or marbling 
this hormone in cattle.
Summary
human acylation-stimulating protein to 
modify fat synthesis was determined 
using cultures of fat tissue from steers. 
However, acylation stimulating protein 
observations, coupled with knowledge of 
how fat synthesis is regulated in other 
species, justify investigation of whether 
cattle synthesize acylation-stimulat-
ing protein, and how this synthesis is 
regulated. An understanding of how 
acylation-stimulating protein production 
and action is regulated should expose 
potential places for intervention to ma-
nipulate fat synthesis in cattle.
(Continued on next page)
1999 Nebraska Beef Report — Page 70
destroy the pre-existing adenosine. This 
allowed comparison between untreated 
and treated cultures. Two additional treat-
ments provided a test of added adenosine. 
Additional cultures were treated with low 
and high doses of the adenosine analog, 
PIA (N6-phenylisopropyl adenosine), 
which resists degradation by adenosine 
deaminase.
Experiment 2 was similar to experi-
ment 1 except the dose range for PIA 
was greater. The treatments were: 1) 
no hormone; 2) 0.75 U/ml adenosine 
deaminase; 3) 0.75 U/ml adenosine de-
aminase plus 10 nM PIA; 4) 0.75 U/ml 
adenosine deaminase plus 100 nM PIA; 
and 5) 0.75 U/ml adenosine deaminase 
plus 1,000 nM PIA.
-
lating protein (ASP)
The second hormone suspected to 
increase synthesis of fat in cattle is ASP. 
To test the effect of ASP on fat synthesis 
in cattle, cultures were designed with in-
creasing concentrations of ASP and some 
cultures with added insulin, a glucose 
uptake-promoting hormone. It is possible 
that ASP only increases fat synthesis if 
insulin is present. On the other hand, an 
effect of ASP may be more pronounced 
if insulin is absent. The treatments were: 
1) no hormone; 2) 0.5 M ASP; 3) 5 M
ASP; 4) 10 nM insulin; 5) 0.5 M ASP 
plus 10 nM insulin; and 6) 5 M ASP 
plus 10 nM insulin.
protein dose curve
In experiment 4, a wider range of ASP 
doses was evaluated: 1) no hormone; 
2) 0.01 M ASP; 3) 0.1 M ASP; 4) 
1 M ASP; 5) 5 M ASP; and 6) 10 
M ASP.
protein dose curve with and without 
insulin
Treatments were: 1) no hormone; 2) 
0.01 M ASP; 3) 0.1 M ASP; 4) 1 M
ASP; 5) 5 M ASP; 6) 10 M ASP; 7) 1 
nM Insulin; 8) 0.01 M ASP plus 1 nM 
insulin; 9) 1 M ASP plus 1 nM Insulin; 
and 10) 10 M ASP plus 1 nM insulin.
triacylglycerol synthesis.
Following incubation, tissues were 
extracted in 2 ml of chloroform:metha-
nol (2:1, v/v), and washed with both 1 
M KCl-0.15N HCl, and 0.38 M sodium 
carbonate. Incorporation of radioactivity 
was determined in a beta counter.
Triacylglycerol was distinguished 
from total fat by use of thin layer 
chromatography. Fat synthesis was 
calculated from radioactivity incor-
porated and expressed as nmol acetate 
(or palmitate)/ mg tissue / six hours. 
Data were analyzed by ANOVA with 
main effects of animal and treatment. 
The animal x treatment interaction 
-
cance.
Results
Fat synthesis in cattle occurs in two 
general steps. First, fatty acids are syn-
thesized. Second, fatty acids are linked 
to glycerol phosphate in a process called 
high-fat diets, such as humans or mice, 
fatty acid synthesis is less than in cattle. 
Incorporation of 14C-acetate estimates 
while incorporation of 14C-palmitate 
fatty acid synthesis.
synthesis in bovine adipose tissue.
Neither fatty acid synthesis nor fatty 
-
dition of PIA (an adenosine analog) or 
adenosine deaminase (an enzyme which 
removal of adenosine (deaminase) and addition of an adenosine analog (PIA). No effect 
of treatment was detected.
9
8
7
6
5
4
3
2
1
0
Acetate
9
8
7
6
5
4
3
2
1
0
Palmitate
Figure 2. (Experiment 2). Effect of adenosine on synthesis of fat from acetate (left) and palmitate 
(right). No effect of treatment was detected.
6
5
4
3
2
1
0
Acetate Palmitate
6
5
4
3
2
1
0
Page 71 — 1999 Nebraska Beef Report
destroys adenosine) to cultures of adi-
pose tissue (Figures 1 and 2). Adenosine 
apparently does not have an effect on 
fatty acids in cattle.
-
tein on triacylglycerol synthesis in bovine 
adipose tissue.
The second hormone evaluated was 
ASP. Initially, it appeared ASP stimulates 
fat synthesis (Figure 3). In this experi-
ment, however, the degree of variation 
in fat synthesis was large and treatment 
shown in Figure 4, ASP did increase the 
use of acetate for fatty acid and triacylg-
lycerol synthesis. However the synthesis 
of fat from palmitate (a fatty acid) was 
not affected by ASP. This indicates ASP 
may have a greater effect on enzymes of 
enzymes. This would be logical, as most 
fat synthesis in cattle is from acetate 
and dietary fat intake is low. Finally, the 
interaction of insulin and ASP in regard 
to fat synthesis was evaluated (Figure 5). 
Fat synthesis was enhanced by increasing 
doses of ASP only when cultures also 
contained insulin. Our interpretation is 
that while both insulin and ASP promote 
different parts of the pathway.
In conclusion, human ASP appears 
capable of promoting fat synthesis in 
bovine tissue. Our prediction is that 
bovine ASP is at least as potent. Based 
on information from a cDNA clone of 
bovine ASP, we know this molecule dif-
fers from human at several amino acid 
positions. We are developing a method 
future we hope to determine whether 
intramuscular fat cells utilize ASP dif-
ferently than fat cells in other fat depots. 
Perhaps this can lead to a method for 
promoting marbling while reducing total 
carcass fat.
1Sheila Jacobi, graduate student. Jess Miner, 
assistant professor, Animal Science, Lincoln.
Figure 3. (Experiment 3). Effects of ASP and insulin on fat synthesis from acetate (left) or palmi-
9
8
7
6
5
4
3
2
1
0
Acetate Palmitate
9
8
7
6
5
4
3
2
1
0
Figure 4. (Experiment 4). Effect of varying concentration of ASP on fat synthesis in absence of 
insulin. Acetate incorporation (left graph) was increased (P<.05) by ASP but all doses 
8
7
6
5
4
3
2
1
0
8
7
6
5
4
3
2
1
0
Acetate Palmitate
Figure 5. (Experiment 5). Interaction of ASP with insulin in stimulation of fat synthesis from 
acetate (left) and palmitate (right). Hatched bars represent cultures treated with 10 
nM insulin. ASP treatment ( M ) is indicated below bars. Incorporation of both acetate 
interaction indicates that insulin is required for ASP to stimulate fat synthesis. *P<.10.
13
12
11
10
9
8
7
6
5
4
3
2
1
0
13
12
11
10
9
8
7
6
5
4
3
2
1
0
0 .01 .1 1 5 10 0 .1 1 10 0 .01 .1 1 5 10 0 .1 1 10


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Hormonal Influence on Fat Synthesis in Cattle

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