Am J Physiol Regul Integr Comp Physiol 285: R1107R1115, 2003.

First published July 24, 2003; 10.1152/ajpregu.00320.2003.

Effect of insulin and growth hormone on plasma leptin

in periparturient dairy cows
Brian J. Leury,1 Lance H. Baumgard,2 Stephanie S. Block,2
Nthabisheng Segoale,2 Richard A. Ehrhardt,2 Robert P. Rhoads,2
Dale E. Bauman,2 Alan W. Bell,2 and Yves R. Boisclair2
1

School of Agriculture and Food Systems, Institute of Land and Food Resources,
University of Melbourne, Victoria, Australia, 3010; and 2Department
of Animal Science, Cornell University, Ithaca, New York 14853-4801
Submitted 11 June 2003; accepted in final form 22 July 2003

Leury, Brian J., Lance H. Baumgard, Stephanie S.

Block, Nthabisheng Segoale, Richard A. Ehrhardt,
Robert P. Rhoads, Dale E. Bauman, Alan W. Bell, and
Yves R. Boisclair. Effect of insulin and growth hormone on
plasma leptin in periparturient dairy cows. Am J Physiol
Regul Integr Comp Physiol 285: R1107R1115, 2003. First
published July 24, 2003; 10.1152/ajpregu.00320.2003.After
parturition, dairy cows suffer from an intense energy deficit
caused by the onset of copious milk secretion and an inadequate increase in voluntary food intake. We previously
showed that this energy deficit contributes to a decline in
plasma leptin. This decline mirrors that of plasma insulin
but is reciprocal to the profile of plasma growth hormone
(GH), suggesting that both hormones may regulate plasma
leptin in periparturient dairy cows. To study the role of
insulin, hyperinsulinemic-euglycemic clamps were performed on six dairy cows in late pregnancy (LP, 31 days
prepartum) and early lactation (EL, 7 days postpartum).
Infusion of insulin (1 g kg body wt1 h1) caused a progressive rise in the plasma concentration of leptin that
reached maximum levels at 24 h during both physiological
states. At steady states, the absolute increase in plasma
leptin was greater in LP than in EL cows (2.4 vs. 0.4 ng/ml).
Insulin infusion increased leptin mRNA in adipose tissue
during LP but not during EL. During lactation, mammary
epithelial cells expressed leptin mRNA but insulin did not
increase milk leptin output. In contrast, a 3-day period of GH
administration had no effect on plasma leptin during LP or
EL. Therefore, insulin increases plasma leptin in LP by
stimulating adipose tissue synthesis but has only marginal
effects in EL, when cows are in negative energy balance.
Other factors, such as increased response of adipose tissue to
-adrenergic signals, probably contribute to the reduction of
plasma leptin in early lactating dairy cows.
pregnancy; lactation; hyperinsulinemic-euglycemic clamp

IN HIGH-YIELDING DAIRY COWS,

the onset of lactation increases the total energy requirements by approximately fourfold, reflecting mostly the oxidative and
milk precursor needs of the mammary gland (5, 6).
Because the hyperphagia required to meet these additional demands develops slowly, shortfalls are met by
mobilization of endogenous reserves and by shifting

Address for reprint requests and other correspondence: Y. R.

Boisclair, 259 Morrison Hall, Dept. of Animal Science, Cornell Univ.,
Ithaca, NY 14853-4801 (E-mail: YRB1@Cornell.edu).
http://www.ajpregu.org

the pattern of nutrients used by nonmammary tissues

(5, 6). Peripheral mechanisms orchestrating these adaptations have been studied extensively in ruminants,
and they involve changes in the concentration and
actions of hormones (51). For example, hypoinsulinemia and decreased insulin responsiveness of skeletal
muscle and adipose tissue occur simultaneously in
early lactation (7, 51). The sum effect of these adaptations is increased availability of glucose for the mammary gland where uptake is independent of insulin (5,
7). Some of these adaptations, such as hypoinsulinemia, also occur in lactating rodents and humans
(40, 49).
The central nervous system (CNS) also plays important regulatory roles during this period and is receiving growing attention (45, 47, 49). This renewed interest reflects, in part, the recent discovery of leptin, a
protein hormone synthesized almost exclusively by
white adipose tissue (AT) (1, 29). In contrast to most
other metabolic hormones, leptin acts predominantly
on regions of the brain involved in the regulation of
energy metabolism, such as the arcuate, ventromedial,
and dorsomedial nuclei of the hypothalamus (1, 43, 45).
Ahima and Flier (1) proposed that a fall in plasma
leptin signals to the CNS that a state of energy insufficiency prevails in the periphery. Consistent with such
a role during the transition period, plasma leptin is
reduced around parturition in dairy cows (8, 31, 33).
Moreover, early lactation and other conditions in which
plasma leptin is low or absent share many adaptations
such as depressed reproductive and immune function
and higher metabolic efficiency (1, 5, 45, 49).
Factors responsible for the decline of plasma leptin
in periparturient dairy cows have not been characterized. Adiposity cannot be the primary cause because
plasma leptin falls before significant depletion of lipid
reserves occurs (8). The decline, however, matches almost exactly the profile of plasma insulin at that time
(6, 8). Insulin stimulates plasma leptin in simplestomached species such as rodents and humans (1, 12),

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LEPTIN REGULATION BY INSULIN IN PERIPARTURIENT DAIRY COWS

but its role in ruminants remains unknown (29). Moreover, growth hormone (GH), a hormone that attenuates
many actions of insulin in bovine AT (19, 51), is also
increased at parturition (6, 8). Therefore, the objectives
of this study were to determine whether insulin and
GH could regulate plasma leptin in periparturient
dairy cows. Our studies show that insulin increases
plasma leptin to a greater extent during pregnancy
than during lactation and that GH is unlikely to play a
major role.
MATERIALS AND METHODS

Animals and design. The Cornell University Institutional

Animal Care and Use Committee approved all experimental
procedures. Only multiparous Holstein dairy cows were used.
Effect of insulin. Six cows were studied in late pregnancy
(LP) and in early lactation (EL). At the start of these periods,
cows averaged 31 1.5 days prepartum (mean SE) and 7
1.6 days postpartum, respectively, and weighed 626 18 and
582 15 kg, respectively. Cows were housed in a controlled
environment (20C, lights on 05002100) and fed a total
mixed ration (TMR). Cows were fed according to recommended feeding standards (39). The TMR contained 1.56
Mcal of net energy for lactation (NEL) and 140 g crude
protein (CP)/kg dry matter (DM) during LP and 1.58 Mcal
NEL and 198 g CP/kg DM during EL. The TMR was offered
ad libitum in 12 bihourly meals with water available at all
times. During EL, cows also consumed a daily allowance of
long-stem hay (3.6 kg containing 1.23 Mcal NEL and 144 g
CP/kg DM). After parturition, cows were milked twice daily
at 0600 and 1800.
Hyperinsulinemic-euglycemic clamps were performed by
previously described procedures (22, 35). Briefly, cows were
fitted with chronic indwelling catheters in each external
jugular vein. Starting the next day (31 days prepartum
during LP and 7 days postpartum during EL), basal conditions were characterized by taking blood samples over 66 h,
followed by biopsies of subcutaneous AT from the tailhead
region (8). Biopsied tissue was snap frozen in liquid nitrogen
and stored at 80C. Immediately after biopsy, insulin was
infused at the rate of 1 g kg body wt1 h1 for 96 h during
LP and for 48 h during EL. Infusates were prepared by
diluting bovine pancreatic insulin (lot 615-70N-80, 26.6 IU/
mg; Lilly Research Laboratories, Indianapolis, IN) in sterile
saline with individual cow plasma as a carrier (2.5% vol/vol).
The concentration of blood glucose was determined at frequent intervals (515 min) for the first 23 h and hourly
thereafter with a Surestep glucometer (Lifescan, Milpitas,
CA). Plasma glucose was maintained to the concentration
observed during the basal period by varying the rate of
intrajugular infusion of a glucose solution (50% wt/vol dextrose solution; Abbott Laboratories, Morgan Hills, CA). Blood
samples were taken during the basal period and every 4 h
during insulin infusion. After addition of heparin (10 IU/ml),
plasma was obtained by centrifugation and stored at 20C
until being analyzed for hormones and metabolites. A second
AT biopsy was obtained just before the insulin infusion was
discontinued.
Source of mammary tissue and cells. Mammary gland and
subcutaneous AT were obtained at death from lactating dairy
cows and immediately frozen at 80C. For isolation of
mammary epithelial cells, parenchymal tissue was dissected
from the mammary glands of additional EL dairy cows.
Mammary epithelial cells were isolated by digestion with a
mixture of collagenase, hyaluronidase, and elastase, followed
AJP-Regul Integr Comp Physiol VOL

by gradient centrifugation (34). This procedure yields primarily mammary epithelial cells (95% of isolated cells), as
shown by morphology and expression of milk protein genes
and epithelial cell markers when cultured in vitro (34). Isolated cells were frozen immediately in liquid nitrogen until
total RNA was prepared.
Effect of GH. Twelve cows were used in the period from 5
wk prepartum to 5 wk postpartum (weeks 5 to 5 relative
to parturition). They were housed in individual tie stalls and
fed a TMR ad libitum once daily. Nutrient composition of the
TMR varied according to physiological state, with respective
NEL and CP content of 1.36 Mcal and 130 g/kg DM during
week 5, 1.56 Mcal and 141 g/kg DM between week 4 and
parturition, and 1.72 Mcal and 179 g/kg DM after parturition. Cows were randomly allocated to a control or a GH
group. The GH group received a daily injection of recombinant bovine GH bovine somatotropin (bST)], 45 mg/injection
im; lot 96J-B5128-002, Monsanto, St. Louis, MO] on three
consecutive days during weeks 5, 2, 1, and 5. Injections were initiated on the second day of each week, corresponding to days 30 4, 13 2, 2, and 30; variation
during pregnancy reflects difference between predicted and
actual time of parturition. By this mode of administration,
GH-induced changes in insulin secretion and AT occur within
1 day of injection and persist for over 24 h after last administration (26, 52). Blood samples were obtained immediately
before GH administration (day 0) and the day after the last
injection (day 4). Sampling was by coccygeal venipuncture
between 1000 and 1130. Control cows were sampled on the
same day of each week. Plasma was prepared immediately
and frozen at 20C until analyzed. Cows were weighed at
weekly intervals throughout the experiment and milked
twice daily at 1130 and 2330 during lactation.
Whole body energetics. Individual feed intakes were recorded on a daily basis. Feed samples were collected weekly
and analyzed for nutrient and chemical composition (Dairy
One Cooperative, Ithaca, NY). Milk was weighed and sampled at each milking. Individual milk samples (insulin experiment) or weekly composites (GH experiment) were analyzed
for protein, fat, and lactose content by infrared analysis
(Dairy One Cooperative). Chemical composition of feeds and
milk was used to estimate their energy content according to
the National Research Council (NRC) (39). These data and
body weights were used to calculate individual estimates of
net energy balance (EB) on a daily basis (8, 26). To estimate
changes in body fatness, two individuals independently assigned a body condition (BC) score (thin 1, fat 5) to each
cow at the start and end of each period (8).
Analysis of gene expression. Total RNA was extracted from
tissues and mammary epithelial cells by a modification of the
guanidinium thiocyanate-phenol-chloroform method (8, 41).
The concentration of total RNA was determined by absorbance at 260 nm, and its quality was verified by staining
formaldehyde agarose gel with Syber Green II (Molecular
Probes, Eugene, OR). Leptin mRNA was quantified by ribonuclease protection assay (RPA) with an RPA III kit [Ambion, Austin, TX] (8). The leptin probe corresponded to nt
64 to 316 (ATG, 1) of the bovine leptin cDNA and
contained a sequence derived from the last two exons of the
gene. The RPA also included a 10-fold molar excess of a
low-specificity 18S probe generated from a DNA template
(Ambion). Protected bands (253 bp for leptin, 80 bp for 18S)
were resolved on 6% polyacrylamide, 7 M urea gels. Signals
were quantified by phosphorimaging with a Fujix-Bio-Imaging Analyzer BAS 1000 (Fuji Medical Systems, Stanford,
CT). When comparing leptin expression between mammary
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LEPTIN REGULATION BY INSULIN IN PERIPARTURIENT DAIRY COWS

and adipose tissues, leptin signals were normalized to the

mass of input RNA determined by A260.
Analysis of metabolites and hormones. Plasma glucose was
measured by the glucose oxidase method and nonesterified
fatty acids (NEFA) by the acyl-CoA synthetase/oxidase
method (8). Plasma concentration of -hydroxybutyrate was
assayed with the -hydroxybutyrate dehydrogenase method
(kit 310-UV, Sigma, St. Louis, MO). Plasma concentrations of
insulin and GH were determined by established RIAs (8).
The concentration of leptin in plasma and milk was measured by a double-antibody bovine RIA developed in our
laboratory (16). Milk leptin was assayed exactly as plasma,
except that it was extensively mixed and sonicated before
being diluted in assay buffer (27). RIA performed adequately
with milk samples, as shown by parallel displacement curves
of 125I-labeled bovine leptin, by serial dilution of bovine milk
and standard, and by quantitative recovery of recombinant
bovine leptin added to whole milk (85%). The RIA for leptin
has a sensitivity of 0.5 ng/ml and a range of 0.520 ng/ml.
Inter- and intra-assay coefficients of variation for all metabolic and hormone assays averaged 8% and 9%, respectively.
Statistical methods. For the insulin study, energy-related
variables during LP and EL were averaged over the last 48 h
of the basal period and over the first 48 h of the clamp period.
For hormones and metabolites, averages were calculated at
steady state (entire basal period and the 3648 h clamp
period). Data were analyzed by a general linear model accounting for physiological state (STATE, LP vs. EL), insulin
(INS, basal vs. hyperinsulinemia), and their interaction
(STATE INS) as fixed effects and animal as the random
effect. Linear regression was used to assess the relationship
of leptin concentration between plasma and milk. A repeated-measure model was used to analyze the effect of time
and insulin on milk leptin concentration and output. For the
GH study, the response was calculated for each variable as
the difference between day 4 and day 0. Responses were
analyzed by a model accounting for GH (GH, control vs. GH),
time (TIME, weeks 5, 2, 1, and 5), and their interaction (GH TIME) as fixed effects and animal as the random
effect.
RESULTS

Insulin is a positive regulator of plasma leptin. Basal

concentrations of plasma insulin and glucose were 60%

and 20% lower in EL than in LP, respectively (Table 1;

P 0.001). Insulin infusion produced an almost immediate increase in plasma insulin concentration of 2
ng/ml, irrespective of physiological state (Fig. 1 and
Table 1; P 0.001). When expressed relative to basal
concentration, steady-state plasma insulin doubled
during LP and nearly quadrupled during EL. During
the clamps, plasma glucose concentration remained
within 10% of basal concentration for both physiological states (Fig. 1), although slightly lower than basal
state during the 3648 h period of both clamps (Table
1; INS, P 0.05). Despite lower plasma concentrations
of glucose and insulin during EL, maintenance of euglycemia required significantly higher rates of glucose
infusion (Fig. 1 and Table 2; P 0.001). These data
indicate that clamp conditions were rapidly achieved
and maintained during both physiological states
(Fig. 1).
Under basal conditions, EL cows consumed 42%
more dry matter (Table 2; P 0.05) and tended to
consume 33% more energy than LP cows (P 0.07).
This increase was still insufficient to meet the increased energy expenditures associated with milk production, as shown by the negative net EB during EL
(P 0.001). During EL, cows had lower BC scores and
higher plasma NEFA concentrations (Tables 1 and 2;
P 0.05), indicating an increased use of body fat
reserves. Despite this accelerated lipid mobilization,
there was no evidence of subclinical or clinical ketogenesis, because plasma concentrations of -hydroxybutyrate did not differ between states (Table 1).
Hyperinsulinemia had no effect on voluntary feed
and energy intake during LP but caused a 33% reduction in both variables during EL (Table 2; STATE
INS, P 0.05). Despite this negative effect, the energy
deficit of EL was not exacerbated (STATE INS, P
0.15). This is because the decreased feed intake during
EL was also associated with a 15% reduction in milk
energy output (INS, P 0.05), reflecting combined
reductions in yield as well as in fat and protein content
of milk. The negative EB during the EL clamp per-

Table 1. Response in plasma concentrations of hormones and metabolites to hyperinsulinemic-euglycemic

clamp during late pregnancy and early lactation
Late Pregnancy
Plasma Variable

Hormones
Insulin, ng/ml
Leptin, ng/ml
Growth hormone, ng/ml
Metabolites
Glucose, mg/dl
NEFA, M
-Hydroxybutyrate, mg/dl

Basal

Insulin

Early Lactation
Basal

Insulin

Significance
SE

STATE

INS

STATE INS

1.8
3.2
3.5

4.0
5.6
2.6

0.7
2.7
4.6

2.5
3.1
7.3

0.2
0.3
1.0

0.001
0.001
0.05

0.001
0.01
NS

NS
0.05
NS

49
95
7.7

45
62
5.1

39
533
10.7

38
155
4.3

1
60
1.2

0.001
0.001
NS

0.05
0.01
0.01

NS
0.05
NS

Multiparous cows (n 6) were studied during a 66-h basal period (basal) and a 48-h hyperinsulinemic-euglycemic clamp (insulin) in late
pregnancy (commencing at 31 1.5 days prepartum) and again in early lactation (7 1.6 days postpartum). Data represent the average of
the entire 66-h basal period and the average of the 3648 h clamp period. Significance level (P) for the effect of physiological state (STATE,
late pregnancy vs. early lactation), insulin (INS, basal vs. insulin), and their interaction (STATE INS) is shown; NS nonsignificant (P
0.05). NEFA, nonesterified fatty acids.
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LEPTIN REGULATION BY INSULIN IN PERIPARTURIENT DAIRY COWS

Fig. 2. Profiles of leptin and nonesterified fatty acids (NEFA) during

hyperinsulinemic-euglycemic clamps. Six dairy cows in late pregnancy (31 1.5 days prepartum) and early lactation (7 1.6 days
postpartum) were studied for 66 h under basal condition and for 48 h
during hyperinsulinemia-euglycemia. Pooled standard errors were
0.1 ng/ml for leptin and 8 M for NEFA.

sisted even after estimates were corrected for the caloric value of infused glucose (Table 2).
During both physiological states, plasma leptin
started to rise within 48 h of insulin infusion and was
maximally elevated after 24 h (Fig. 2); no further
changes were detected over the next 24 h of hyperinsulinemia during EL or the next 72 h during LP (Fig. 2
and results not shown). During the 3648 h interval of
insulin infusion, plasma leptin was increased nearly
75% during LP but only 13% during EL (Table 1;
STATE INS, P 0.05). This modest effect of insulin

Fig. 1. Profiles of glucose-related variables during hyperinsulinemiceuglycemic clamps. Six dairy cows in late pregnancy (31 1.5 days
prepartum) and early lactation (7 1.6 days postpartum) were
studied for 66 h under basal conditions and for 48 h during hyperinsulinemia-euglycemia. Dashed lines on blood glucose graph represent 10% of baseline blood glucose. Pooled standard errors were 0.1
ng/ml for insulin, 1 mg/dl for plasma glucose, and 1.2 g/h for glucose
infusion rate.

Table 2. Response in energy-related variables to hyperinsulinemic-euglycemic clamp during late

pregnancy and early lactation
Late Pregnancy
Variable

Intake
Dry matter, kg/day
Energy, Mcal/day
Milk
Yield, kg/day
Energy, Mcal/day
Glucose infused, g/h
Energy balance, Mcal/day
Corrected energy balance, Mcal/day
Body condition score

Basal

Insulin

13.2
20.7

12.5
19.6

9.0
9.0
3.4

73.4
7.6
14.1

Early Lactation

Significance

Basal

Insulin

SE

STATE

18.7
27.7

12.6
18.2

1.0
1.4

0.05
NS

37.5
31.3

34.6
27.1
121.5
21.9
11.5

1.0
1.1
4.3
2.0
1.9
0.1

14.6
14.6
3.1

0.001
0.001
0.01

INS

STATE INS

0.01
0.01

0.05
0.05

NS
0.05
0.001
0.05
0.05

NS
NS

Multiparous cows (n 6) were studied during a 66-h basal period (basal) and a 48-h hyperinsulinemic-euglycemic clamp (insulin) in late
pregnancy (commencing at 31 1.5 days prepartum) and again in early lactation (7 1.6 days postpartum). Data represent the average of
the entire 66-h basal period and the average of the 3648 h clamp period. Significance level for the effect of physiological state (STATE, late
pregnancy vs. early lactation), insulin (INS, basal vs. insulin), and their interaction (STATE INS); NS nonsignificant (P 0.05). Glucose
infused, average amount of glucose infused during the 36 h to 48 h clamp period. Net energy balance excluding (energy balance) or including
(corrected energy balance) the caloric value of infused glucose is shown. Body condition scores were estimated at the start of the basal period
during late pregnancy and early lactation.
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R1111

Fig. 3. Effects of insulin on adipose tissue expression of the leptin gene during pregnancy. Six dairy cows in late
pregnancy (31 1.5 days prepartum) were studied for 66 h under basal condition and for 96 h during hyperinsulinemia-euglycemia. Biopsies of adipose tissue were obtained at the end of the periods of basal measurement ()
and euglycemic hyperinsulinemia () when the plasma concentration of leptin averaged 3.2 and 5.3 ng/ml,
respectively. Total RNA (2 g) was analyzed simultaneously by a ribonuclease protection assay for the abundance
of leptin and 18S ribosomal RNA. Bars represent the means SE of leptin signal normalized to the 18S signal.
Bars with different letters differ at P 0.05.

on plasma leptin during EL was in contrast to its

equally effective inhibition of plasma -hydroxybutyrate during both states (P 0.01) and to its exaggerated inhibition of plasma NEFA during EL (Table 1
and Fig. 2; STATE INS, P 0.05). We conclude
that insulin is capable of increasing plasma leptin in
dairy cows but this effect is attenuated considerably
during EL.
To examine the mechanism underlying the effects of
insulin on plasma leptin, AT was obtained immediately
at the end of the basal and clamp periods and assayed
for abundance of leptin mRNA. Hyperinsulinemia increased the abundance of leptin mRNA during LP (Fig.
3; P 0.05) but had no detectable effect during EL
(results not shown).
Secretion of leptin by bovine mammary gland is not
increased by hyperinsulinemia in EL. In humans, the
concentration of leptin is higher in milk than in plasma
(27, 46). Moreover, epithelial cells derived from human
breast tissue express the leptin gene and contain immunoreactive leptin (46). To determine whether the

mammary gland is a site of synthesis in cattle, mammary tissue and isolated mammary epithelial cells
were assayed for the presence of leptin mRNA. As
shown in Fig. 4, leptin gene expression was readily
detected in epithelial cells isolated from lactating
mammary glands. A longer exposure was needed for
detection of the leptin signal in mammary tissue, possibly reflecting the difficulty of obtaining representative samples of the whole gland in large animals. We
conclude that, in the mammary gland of lactating dairy
cows, epithelial cells are the major site of leptin synthesis. Leptin mRNA abundance in mammary epithelial cells, however, is considerably lower than that of
AT (Fig. 4).
We also determined milk leptin content by RIA. On
average, the concentration of leptin was approximately
three times higher in milk than in plasma (Fig. 5).
Plasma and milk leptin were not correlated during
either the basal or the clamp periods. The milk leptin
concentration appeared to increase over time during
the clamp (INS TIME, P 0.08), but milk leptin

Fig. 4. Expression of the leptin gene in the mammary gland during lactation. Tissues were obtained at death from
2 (adipose tissue) or 3 (mammary gland) lactating dairy cows. Epithelial cells were obtained from 2 independent
isolations by digestion of the parenchymal mammary compartment of 2 dairy cows. A: total RNA (either 3 or 30 g
as indicated) was analyzed by ribonuclease assay for the abundance of leptin mRNA. The arrow indicates the
position of the single leptin signal of 253 bp (top). Before the ribonuclease assay was performed, 2 g of each sample
was evaluated for quality by denaturing agarose gel electrophoresis and Syber Green II staining (bottom). Lanes
16 are from a single autoradiogram exposed for 18 h. B: prolonged exposure (72 h) of autoradiogram corresponding
to mammary tissue and epithelial cells (lanes 14 from A). The arrow indicates the position of the leptin signal. C:
leptin signals obtained in A were quantified by phosphorimaging and corrected for the mass of RNA analyzed.
Signals were expressed relative to the mean signal obtained in subcutaneous adipose tissue. Means SE with
different letters differ at P 0.05.
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Fig. 5. Mammary gland secretion of leptin during lactation. Six dairy cows in early lactation (7 1.6 days
postpartum) were studied for 66 h under basal conditions and for 48 h during hyperinsulinemic-euglycemic clamp.
Cows were milked twice daily at 0600 and 1800. Aliquots of milk obtained at each milking (every 12 h) during basal
and clamp periods were analyzed for the concentration of milk leptin by RIA. Left: concentrations of leptin in milk
secreted over the last 24 h of the basal and clamp periods plotted against the mean plasma concentration of leptin
during the corresponding time interval. Right: milk leptin concentration and yield during the 48-h period preceding
(basal) and following the initiation of the hyperinsulinemic-euglycemic clamp (Clamp). The only effect approaching
significance was the interaction between treatment and time (INS TIME). Pooled SE for milk leptin concentration and yield were 0.4 ng/ml and 11.2 g/12 h, respectively.

output during the basal and clamp periods were identical (Fig. 5). We conclude that in EL the bovine mammary gland synthesizes leptin but that mammary secretion is not stimulated by insulin.
GH has no effect on plasma leptin in EL and LP. The
reduction of plasma leptin observed during the transition from LP to EL is also associated with increased
plasma GH (Table 1, P 0.05; Ref. 8). We examined
the possibility that GH might decrease plasma leptin
in cows at 5 and 2 wk prepartum and again at 1 and 5
wk postpartum. At each time, cows receiving GH were
sampled immediately before (day 0) and the day after a
3-day period of GH treatment (day 4). Control cows
were sampled at identical times but were not treated.
The transition from pregnancy to lactation caused
expected and identical changes in both groups of cows
(i.e., onset of negative net EB and decreased plasma
concentrations of insulin, glucose, and leptin; Fig. 6
and results not shown). As observed by others (52) , GH
treatment caused a significant increase in plasma insulin during pregnancy but not during lactation (Fig. 6;
GH TIME, P 0.05). In contrast, GH had no effect
on plasma leptin during either pregnancy or lactation.
Therefore, the periparturient increase in plasma GH is
unlikely to contribute to the postpartum reduction in
plasma leptin.
DISCUSSION

In dairy cattle, the transition from pregnancy to

lactation is associated with a reduction in plasma leptin (8, 31, 33). In other species, placental synthesis of
leptin (primates) or a soluble form of the leptin receptor (mice) contributes to elevated plasma leptin during
pregnancy (21, 25). However, the ruminant placenta,
including that of cattle, does not synthesize leptin (15).
Moreover, leptin binding activity is negligible when
plasma from pregnant cows is incubated with 125IAJP-Regul Integr Comp Physiol VOL

leptin and assayed by native PAGE or by gel filtration

chromatography (R. A. Ehrhardt and Y. R. Boisclair,
unpublished results). Instead, we have attributed the
periparturient decline in plasma leptin to inhibition of
AT leptin synthesis associated with the negative EB of
EL (8). This reasoning is supported by three lines of
evidence. First, the periparturient decline in plasma
leptin is correlated with the onset of negative EB (8,
33). Second, AT leptin gene expression is reduced in EL
(8, 47). Finally, when not milked after parturition,
dairy cows remain in positive EB and their plasma
leptin concentration is identical to that of dairy cows in
LP (8).
To understand the regulation of leptin in periparturient dairy cows, we focused on insulin. Insulin stimulates leptin synthesis in rodent and human AT (1, 12)
and, unlike other positive regulatory hormones (e.g.,
prolactin and glucocorticoids), its plasma concentration is decreased markedly in early lactating dairy
cows (6, 8). In support of a role for insulin, we show
that physiological elevations in plasma concentration
increased circulating leptin in both late pregnant and
early lactating cows. In both states, plasma leptin
responses to insulin took nearly 24 h to plateau. Similar sluggish responses have been observed in humans
when euglycemic clamps were performed at modest
hyperinsulinemia (42). The slow kinetics of this response may explain why others failed to detect positive
effects of insulin in ruminants after acute or shortterm elevations in plasma insulin (20, 32). Despite this
slow response, we believe the effects of insulin to be
direct because insulin alone stimulates indexes of leptin synthesis in ruminant AT explants (28) and because in vivo leptin responses are accelerated considerably when clamps are performed at supraphysiological concentrations of insulin (4, 48).
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LEPTIN REGULATION BY INSULIN IN PERIPARTURIENT DAIRY COWS

Fig. 6. Effect of growth hormone (GH) on plasma leptin in late

pregnancy and in early lactation. Twelve multiparous cows were
studied in the period between 5 wk before and 5 wk after parturition
(weeks 5 to 5 relative to parturition). Cows were randomly allocated to remain untreated (control) or to receive 3 consecutive daily
injections of GH (45 mg/injection) in each of weeks 5, 2, 1, and
5 (GH). During each week, blood samples were obtained immediately before (day 0) and the day after a 3-day period of GH injection
(day 4). Blood samples were obtained at similar times from the
control group. The plasma leptin concentration was analyzed for
each sample by RIA. Top: profile of plasma leptin under basal
conditions (day 0). Bottom: response in plasma leptin (leptin) and
insulin (insulin). For each group, the response was calculated as
the plasma concentration difference between day 4 and day 1. The
only significant effect was an interaction between treatment and
time (GH TIME) for insulin. This was the result of significant
increases in plasma insulin in the GH-treated cows during weeks 5
and 2. During these 2 weeks, basal concentrations (day 0) for
plasma insulin were 1.0 0.2 and 1.5 0.2 ng/ml, respectively.

At steady state, the plasma leptin response to insulin

was six times greater in LP than in EL (2.4 vs. 0.4
ng/ml). Insulin infusion produced similar absolute increases in plasma insulin in LP and EL cows, indicating a similar half-life in both states. However, the
degree of hyperinsulinemia during lactation was
slightly lower, reflecting lower endogenous insulin secretion. This factor alone is unlikely to account for the
AJP-Regul Integr Comp Physiol VOL

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muted leptin response during EL. First, an eightfold

increase in plasma insulin in EL cows produced only a
marginally larger response (0.6 ng/ml; W. R. Butler
and Y. R. Boisclair, unpublished results). Similarly
small leptin responses were observed in two 96-h
clamps performed in later lactating dairy cows (0.4 and
0.8 ng/ml) even though their hyperinsulinemia exceeded that achieved in LP in the present study (6.8
and 11.5 vs. 4.0 ng/ml; Ref. 9). Second, insulin-dependent glucose uptake is impaired in early lactating ruminants (7, 50), and recent evidence in other species
indicates that glucose uptake is a primary determinant
of AT leptin synthesis (36, 38). In humans, the plasma
leptin response during hypo- and euglycemic clamp
experiments is more closely related to glucose infusion
rates than either glucose or insulin concentration (53).
Glucose exerts these effects by increasing the production of metabolites such as UDP-N-acetylglucosamine,
which sense the energy status of the adipocyte, and
stimulates leptin synthesis accordingly (12, 13, 54).
Whether the reduced plasma leptin response in EL
relates to decreased AT glucose uptake and production
of UDP-N-acetylglucosamine remains to be demonstrated in ruminants.
GH inhibits insulin-mediated glucose uptake by AT
(19), and the impaired insulin stimulation of glucose
disposal in EL coincides with increased GH secretion
(7, 8, 51). Moreover, elevated plasma leptin levels seen
in GH-deficient humans are corrected by GH therapy,
even before a reduction in adiposity occurs (2, 17).
These observations prompted us to ask whether GH is
a negative regulator of plasma leptin in dairy cows
during the periparturient period. We found that exogenous GH treatment did not reduce plasma leptin,
either in EL when the plasma concentration of insulin
was unaltered or in LP when its concentration was
increased by one- to twofold. These results suggest that
GH has no independent effect but inhibits insulinmediated leptin synthesis, an interpretation supported
by identical observations in bovine AT explants (28).
Our results in LP, however, are in contrast to those
obtained in GH-treated castrate male cattle, which
experience simultaneous increases in plasma insulin
and AT leptin gene expression (28). The reason for this
discrepancy is unclear, but it could relate to the pregnant state of our nonlactating cows. Finally, -adrenergic signaling inhibits both basal and insulin-mediated leptin secretion in rodent adipocytes (11, 23). In
EL, ruminant AT displays increased -adrenergic responsiveness (6, 51), suggesting that this system could
also contribute to the reduction of plasma leptin after
parturition.
As reported for other species, we found significant
concentrations of immunoreactive leptin in bovine milk
(3, 18, 27, 46). Consistent with the possibility that most
of the leptin in milk is synthesized by the mammary
gland, milk leptin concentration is substantially
greater than, and not correlated with, the plasma leptin concentration. Bonnet et al. (10) recently showed
that, in sheep, mammary leptin gene expression is
highest during the first 80 days of gestation, before
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LEPTIN REGULATION BY INSULIN IN PERIPARTURIENT DAIRY COWS

declining during the remainder of gestation and during

EL. By immunohistochemistry, they mapped leptin
expression to differentiating epithelial cells during late
gestation and to myoepithelial cells during lactation, a
surprising finding given the high leptin content of
milk. In contrast, we show that leptin gene expression
is significant in mammary epithelial cells, suggesting
that they synthesize a significant fraction of milk leptin in lactating dairy cows. In agreement with our
findings, mammary epithelial cells from mice and humans have been shown to synthesize leptin (3, 46).
Smith and Sheffield (44) reported that insulin and
IGF-I stimulated leptin mRNA in a bovine mammary
epithelial cell line (MAC-T). However, we did not detect
increased leptin secretion in milk during the clamp,
despite chronic increases in both insulin (Fig. 1) and
IGF-I (R. P. Rhoads and Y. R. Boisclair, unpublished
results). These data indicate that, if they exist, the
positive effects of both insulin and IGF-I on mammary
leptin synthesis must be small.
Exogenous leptin administration reduces voluntary
feed intake in most animals, and this effect usually
requires 24 days of continuous treatment in ruminants (24, 37). Chronic hyperinsulinemic-euglycemic
clamps often reduce feed intake in lactating dairy cows
(22, 35), but our data do not support a role for leptin in
this effect. In the present study, feed intake was unaffected in LP, when the insulin-dependent response in
plasma leptin was the highest, and depressed in EL,
when the leptin response was small. We have noted a
similar lack of association between the depression in
feed intake and increased plasma leptin in other clamp
studies performed in early and in later lactation [Ref. 9
and Y. R. Boisclair, unpublished data]. This lack of
association is also inconsistent with the idea that a
decrease in plasma leptin around parturition drives
the hyperphagia of EL. This notion has been experimentally dispelled in rodents by showing that feed
intake varies in proportion to litter size but independently of serum leptin concentration (14, 49). Moreover, the transition from pregnancy to lactation is not
associated with increased leptin resistance in rodents
(30, 49). As we (8) and others (49) have suggested, the
hypoleptinemia of EL may serve to increase energy
conservation by suppressing functions that are dispensable in the short term (e.g., reproduction and immunity) and by promoting increased metabolic efficiency. The lactating dairy cow, with its substantial
energy deficit of EL, is an ideal model to determine the
role that leptin plays in these adaptations.
The authors thank Debra Dwyer and Ramona Ehrhardt for analysis of hormones and metabolites and Dr. Elvina Matitashvili for the
isolation of mammary epithelial cells.
DISCLOSURES
This work was supported by the U.S. Department of Agriculture
National Research Initiative Competitive Grant Program (Award
00-35206-9352 to Y. R. Boisclair) and by the Cornell University
Agricultural Experiment Station.
AJP-Regul Integr Comp Physiol VOL

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