Metabolic changes associated with sustained 48-Hr shivering thermogenesis in the newborn pig

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Comp. Biochem. Physiol. Vol. l14B, No. 4, pp. 327-335, 1996 Copyright © 1996 Elsevier Science Inc.

ELSEVIER

Metabolic Changes Associated with Sustained 48-Hr Shivering Thermogenesis in the Newborn Pig Delphine Berthon,* Patrick Herpin,* Roseline Bertin, 4 Frangoise De Marco* and Jean le Dividich* *INRA, STATIONDE RECnERCHESPORCINES, 35590 ST GILLES, FRANCE;AND tEPHE, 105 BD RASPAIL, 75006 PARIS, FRANCE ABSTRACT. Metabolic changes associated with sustained 48-hr shivering thermogenesis were studied in piglets maintained at 34 (thermoneutrality) or 25°C (cold) between 6 and 54 hr of life. Despite their high shivering activity and elevated heat production, cold-exposed piglets exhibited a slightly lower rectal temperature than thermoneutral animals (-1.1°C; P < 0.01) at the end of the treatment. The enhancement of heat production and shivering activity were associated with a decrease in muscle glycogen (-47%; P < 0.05) and total lipid content (-23%; P < 0.05), a reduction of blood lactate levels (P < 0.05) and an enhancement of muscle cytochrome oxidase activity (+ 20%; P < 0.05), which suggests that muscle oxidative potential was increased by cold exposure. Potential for capturing lipids (lipoprotein lipase activity) was also higher in the red rhomboideus muscle (+ 71%; P < 0.01) and lower in adipose tissue (-58%; P < 0.01 ) of the cold-exposed piglets. Measurements performed at the mitochondrial level show no changes in rhomboideus nmscle, but respiratory capacities (state IV and FCCP-stimulated respiration) and intermyofibrillar mitochondria oxidative and phosphorylative (creatine kinase activity) capacities were enhanced in longissimus dorsi muscle (P < 0.05). These changes may contribute to provide muscles with nonlimiting amount of readily oxidable substrates and ATP necessary for shivering thermogenesis. A rise in plasma norepinephrine levels was also observed during the second day of cold exposure (P < 0.05). come BIOCHEMPHYS1OL114B;4:327-335, 1996. KEY WORDS. Piglets, cold, shivering, mitochondria, cytochrome oxidase, creatine kinase, lipoprotein lipase, catecholainines

INTRODUCTION Newborn pigs are known to be very susceptible to cold because of their low energy reserves, lack of brown adipose tissue and poor insulation. Their main thermogenic mechanism is shivering (8,45), but biochemical mechanisms underlying the maintenance of high levels of shivering activity are not fully elucidated. However, it is likely that heat production during shivering involves mechanisms close to those associated with contraction of skeletal muscle myofibrils during exercise (30). This means that heat production during shivering would rely, at least in part, on substrate availability (i.e., energy stores, enzyme activities and muscle blood flow), efficiency of ATP synthesis and hydrolysis during contractions and active ion pumping, and muscle fiber type. Indeed, endurance training and long-term electrical stimulation of muscle are situations that can be compared, to some extent, with continuous shivering (21). They have been found to induce biochemical, morphological and physCorrespondence to: Dr. Patrick Herpin, Station de Recherches Porcines,

}5590 St-Gilles, France. Received 1 :} May 1995; revised 10 January 1996; accepted 30 January 1996.

iological adaptations, including changes in lipoprotein lipase (LPL) activity, oxidative capacity, muscle blood flow and myofiber type (review in 28,29,46). Further, in 2month-old pigs, prolonged cold exposure also induces marked changes in skeletal muscle oxidative and respiratory enzymes, morphology and fiber type distribution (13,15, 21,22). Whether some of these changes in muscle energy metabolism can be observed during continuous shivering thermogenesis in the neonatal period remains to be determined in pigs. The postnatal control of thermogenesis in pigs has been attributed essentially to thyroid hormones (7,52). However, the sympathoadrenal system also responds to acute cold exposure (33,36), and the thermogenic response to adrenaline is potentiated by previous administration of thyroxine (32). Considering the key role of catecholamines in vasomotor adjustments and stiraulation of glycogenolysis and lipolysis, they are probably inw~lved in the metabolic adjustments taking place during shivering. Therefore, the aims of the present study are first to determine the effects of 48-hr sustained shivering thermogenesis on some striking aspects of muscle energy metabolism, including provision of energy substrates, potential for captur-

328

ing and oxidation of lipids and mitochondrial respiration, and second to describe concomitant changes in circulating catecholamines, under conditions of controlled ambient temperature and milk intake, between 6 and 54 hr of life. MATERIALS AND METHODS

Animals Fifty-five large white newborn pigs were used to evaluate the metabolic changes associated with sustained shivering thermogenesis. Piglets were weighed soon after birth and allowed to suckle colostrum to acquire immune protection. At 4 hr of life, those exhibiting a positive weight gain were either maintained at thermoneutrality for 2 hr and killed at 6 hr of life (n = 6) for tissue analysis or catheterized in the jugular vein under sterile conditions and general anesthesia. At 6 hr of life, these piglets were placed in plasticcoated wire cages in groups of three to four animals and allotted at random to two environmental temperature treatments, 34°C (thermoneutral, TN) or 25°C (cold, C) for a 48-hr period. To take into account the changes in the lower critical temperature with age (7), the two temperatures were decreased by l°C every 12 hr to reach 30°C and 21°C at the end of the treatment (i.e., at 54 hr of age). Piglets from both groups were subjected to three different experimental protocols involving different types of measurements: circulating metabolites in experiment I (n = 2 × 10), plasma catecholamine levels in experiments I and II (n -- 2 × 9), shivering intensity in experiment III (n -- 5 and 6 in TNand C-piglets, respectively) and heat production in experiments I, II and III. Piglets from experiments I and II were killed at the end of the treatment, allowing collection of tissue samples for chemical and biochemical measurements.

Feeding Piglets were taught to drink an infant formula milk (Milumel, Miluma, Bagnolet, France) from an infant nursing bottle as described previously (9) every hour from 8 to 18 hr, every 2 hr from 18 to 29 hr and every 3 hr from 31 to 54 hr of life. The formula contained, on an as fed basis, 79% water, 7.3% lactose, 2.7% lipids, 4.7% crude protein and 4.3 kJ/g. The total intake over the 48 hr investigation averaged 415 g/kg body weight in both treatments. Measurements

Body weight and rectal temperature were recorded at 6 and 54 hr of age, heat production was determined by continuous indirect calorimetry (7) during 90 rain at the end of the treatment and shivering intensity was estimated by recording the electromyographic activity in longissimus dorsi muscle (8). Blood samples were taken via the catheter at 6, 18, 30 and 54 hr of age. One part of the collected blood was

Berthon et al.

directly analyzed for glucose and lactate concentrations, whereas the other part was immediately centrifuged at 13,000 g for 3 min at 4°C. Plasma was then stored at - 4 0 ° C until analyzed for free fatty acid (FFA) and epinephrine and norepinephrine concentrations. Glucose and lactate levels were determined by the glucose oxidase and the lactate oxidase methods, respectively, using an automatic analyser (YSI 27, YSI Corporated, Yellow Spring, OH, U.S.A.) Plasma FFA levels were determined by an enzymatic method using an NEFA-C kit (Wako Chemicals, Unipath, France). Plasma catecholamines were analyzed by highperformance liquid chromatography with electrochemical detection after purification and concentration of the amines on alumin as described by Bertin et al. (10).

Tissue Sampling and Analysis Piglets were anesthetized by halothane inhalation and then killed by exsanguination at the beginning (6 hr) or at the end (54 hr) of the treatments. Liver, heart, longissimus dorsi and rhomboideus muscles and subcutaneous adipose tissue were removed, immediately frozen in liquid nitrogen and stored at -80°C. Liver and muscles were analyzed for glycogen and total lipid content and lon~ssimus dorsi muscle for lactate content. Cytochrome oxidase (CO, EC 1.9.3.1) activity was measured in liver, muscles and adipose tissue, whereas LPL (EC 3.1.1.34) activity was determined in heart, muscles and adipose tissue. In muscles, the functional properties of isolated mitochondria were studied in experiment II. Glycogen content in tissues was measured by the glucose oxidase method after dissolution of the pulverized tissue in hot KOH, subsequent precipitation with cold ethanol and acid hydrolysis of the purified glycogen. Total lipid content was determined according to the method of Folch et al. (18) and lactate concentration in muscle homogenates with an enzymatic method using a commercial kit (Boehringer Mannheim, Meylan, France). LPL activity was assessed as previously described (23), and results were expressed in/./Eq of fatty acids released per hour per gram of tissue. CO activity was measure d polarographically according to the procedure of Barr~ et al. (3) and expressed in natoms O consumed per minute per mg of tissue. Moreover, to assess the functional properties of muscle subsarcolemmal and intermyofibrillar mitochondria, the two populations of mitochondria were isolated by differential centrifugation as described by Herpin et al. (25). Oxygen consumption was followed polarographically at 25°C, using succinate as substrate, to determine both basal and ADP-stimulated respiration. Similarly, the fully uncoupled respiratory rate was determined after addition of the mitochondrial uncoupler FCCP (carbonyl cyamide p-trifluoromethoxyphenylhydrazone). CO and creatine kinase (CK, EC 2.7.3.2.) activities were measured on isolated mitochondria. CO activity was measured

Shivering Thermogenesis in the Newborn Pig

as described for the tissues. CK activity, which is assumed to be an index ofphosphorylative capacity (4), was measured by spectrophotometry as described by Foster et al. (19) and expressed in mU/mg mitochondrial protein.

Statistical Analysis The results are expressed as means + SEM. Heat production, rectal temperature and circulating catechotamine levels were similar in the different trials, so the results were pooled across trials and were computed using the General Linear Model procedure (49), using analysis of variance to determine the effects of age and treatment on the different parameters. However, when plasma levels of epinephrine were below the sensitivity of the assay (0.3 ng/ml), this lower limit of detection was taken as an experimental result, and therefore the data were analyzed with a nonparametric test (Kruskall and Wallis, Wilcoxon test). Although means +SEM are given for epinephrine, they were not used for statistical analysis.

329

TABLE 1. Effect of sustained 48-hr shivering thermogenesis on circulating metabolites of newborn pigs 54 hr Treatment

6 hr

TN

C

Bloc,d glucose (rag/l) 791 -+ 47 (19) 865 +- 93 (7) 806-+ 84 (8) Plasma FFA (#tool/l) 234 -+ 25 (20)" 88-+ 17(7P 156 + 26 (8)' Blood lactate (mg/l) 166 -+ 10 (17)' 339 -+ 47 (5)~ 194 -+ 36 (4) ~' Values are means -+ SEM with n in parentheses.TN, piglets raised at thermoneutrality; C, piglets raised in the cold. *b'~Meanswithin a row with different superscript letters differ (P < 0.05). muscle glycogen content was 47% lower (P < 0.05) in C than in TN animals. In the liver, depletion of glycogen stores occurred in both groups but was only significant in the cold (P < 0.05). Concomitantly, tissue lipid content decreased by 13% (P < 0.01) in the liver and increased by 16 and 24% (P < 0.05) in rhomboideus and lon~ssimus dorsi muscles, respectively, at thermoneutrality. Cold exposure 14 a

RESULTS 12-

In Vivo Measurements The average body weight of piglets aged 6 hr was 1.50 _+ 0.30 kg. During the 48-hr treatment, body weight gain was slightly higher in T N than in C piglets (121.3 + 13.7 g vs 90.3 + 12.9 g, respectively). Rectal temperature of the piglets averaged 38.5 +- 0.1°C at 6 hr of life and was slightly lower (P < 0.001) in C than in T N piglets at the end of the treatment (38.2 + 0.1 vs 39.3 -+ 0.1°C). Heat production was 62% higher (P < 0.001) in C than in T N animals (32.1 + 0.9 vs 19.8 -+ 0.7 kJ/h/kg body weight, respectively). As expected, shivering intensity was minimal at thermoneutrality and elevated in the cold, averaging 4.6 + 1.4 and 121.3 + 25.4 mV/min (P < 0.001), respectively. During the 48-hr investigation, piglets huddled together and had a gathered posture in the cold, and a relaxed posture at thermoneutrality.

ttO 0 t'-" 01 0 O

Blood glucose levels were not affected by age and treatment and averaged 810 + 37 mg/l (Table 1). Plasma FFA levels decreased in both groups (P < 0.05) between 6 and 54 hr of age but were 77% (P < 0.05) higher in C than in T N piglets at the end of the treatment. Blood lactate levels doubled in 48 hr (P < 0.05) in T N piglets but remained unchanged in C piglets so that they were 43% (P < 0.05) lower in C than in T N piglets at 54 hr of age. Between 6 and 54 hr of age, glycogen content decreased by 62% (P < 0.001) inrhomboideus and by 27% (P < 0.001) in longissimus dorsi muscles of T N piglets (Fig. 1). In the cold, the drop was more accentuated in both muscles, and

b

10-

a b

864-

20 Liver

RH

a

4-

tO

Energy Substrate Concentrations in Plasma and Tissues

a

o~

LD

[ ] TO [ ] TN

_

3-

b

O

"o 2"EL ,m m

1O F-

0 Liver

RH

LD

FIG. 1. Effect of sustained 48.hr shivering thermogenesis on glycogen and lipid content of liver, rhornboideus (RH) and longisslmus dorsi (LD) muscles of newborn pigs. Values are means +- SEM (n = 5 - 1 0 ) and are expressed as percentage of wet tissue. TO, piglets aged 6 hr; TN, piglets aged 54 hr and raised at thermoneutrality; C, piglets aged 54 hr and raised in the cold. "b'cwithin a tissue, values with different superscript letters differ ( P < 0.05).

Berthon et al.

330

30

[] TO [] TN

m 25o~ 20.d

IIC

b

O"

15-

~

10-

a

° ~

m .J

5-

~

0

a

Hea~

RH

a

LD

a

~ c

Igl AT

FIG. 2. Effect of sustained 48-hr shivering thermogenesis on lipoprotein lipase activity (LPL) in the heart, rhomboideus (RH) and longissimus dorsi (LD) muscles and adipose tissue (AT) of newborn pigs. Values are means -+ SEM (n = 6 at 6 hr and 7-10 at 54 hr of age, except in AT, where n -- 4). TO, piglets aged 6 hr; TN, piglets aged 54 hr and raised at thermoneutrality; C, piglets aged 54 hr and raised in the cold. "'b'cWithin a tissue, values with different superscript let. ters differ ( P < 0.05). did not affect the lipid content in the liver but decreased it by 19 and 27% (P < 0.05) in rhomboideus and longissimus dorsi muscles, respectively. Lactate content in longissimus dorsi muscle was similar in both groups, averaging 604 -+ 26/2g/g tissue at 54 hr of age.

LPL and CO Activities At birth, the highest LPL activity was found in the heart, averaging 26.7 + 1.8/IEq FA/h/g tissue and being about 3.1-fold higher than in rhomboideus muscle and adipose tissue. Between 6 and 54 hr of age, LPL activity decreased by 62% (P < 0.001) in rhomboideus muscle and heart from TN piglets (Fig. 2). However, in both tissues, the activity was about 71% higher (P < 0.01) in C than in TN piglets at the end of the treatment. The reverse picture was found in adipose tissue with LPL activity increasing by 67% (P < 0.01) at thermoneutrality and decreasing by 58% (P < 0.01) in the cold between 6 and 54 hr of life. In longissimus dorsi muscle, no change was observed with age or treatment. CO activity (Fig. 3) was enhanced by 66% (P < 0.01) in the liver and by 84% (P < 0.01) in rhomboideus muscle, between 6 and 54 hr of age at thermoneutrality, whereas it remained constant in lon~ssimus dorsi muscle and adipose tissue. Cold exposure increased CO activity by 20% in the liver, rhomboideus and longissimus dorsi muscles (P < 0.05). On the contrary, the enzyme activity was 33% lower (P < 0.001) in adipose tissue from C than from TN piglets. Mitochondria Functional Properties Cold exposure had no effect on the amount of isolated mitochondrial proteins in both muscles (Table 2). The amount

of subsarcolemmal and intermyofibrillar mitochondria averaged 1.10 -+ 0.14 and 2.77 -+ 0.18 mg/g oflongissimus dorsi and 1.44 -+ 0.07 and 3.46 -+ 0.15 mg/g ofrhomboideus, respectively. In longissimus dorsi muscle, cold exposure increased basal (state IV) respiration by 50% (P < 0.05), the changes in the ADP-stimulated one (state III) being not significant. As a result, the respiratory control ratio (RCR) decreased (P < 0.05) in the cold in intermyofibrillar mitochondria (5.0 vs 3.7 for TN and C piglets, respectively). FCCP-stimulated respiration was increased by 56 and 115% (P < 0.01) in the cold, in intermyofibriUar and subsarcolemmal mitochondria, respectively. CO activity tended to increase in the cold (P < 0.1), the increase being significant in intermyofibrillar mitochondria (+36%, P < 0.05), and CK activity increased in both types of mitochondria (+ 57% in intermyofibrillar mitochondria, P < 0.05; +77% in subsarcolemmal mitochondria, P < 0.01), Therefore, the ratio of CK to CO activities rose (P < 0.05) in the cold. In rhomboideus muscle, cold exposure had no effect on mitochondrial respiration and CO and CK activities. However, as in longissimus dorsi muscle, FCCP-stimulated respiration was increased (P < 0.01) in the cold.

Epinephrine and Norepinephrine Plasma Levels Plasma epinephrine levels increased with age (Fig. 4) in both groups and were not significantly affected by the treatment. Plasma norepinephrine levels averaged 1.36 + 0.17 ng/ml during the first 24 hr of treatment and then increased (P < 0.01) by 61 and 127% between 30 and 54 hr of age in TN and C piglets, respectively. As a result, plasma nor-

g

40 [ ] TO

30 o

20

a

10 oo

0

[] TN IIC

c

~ Liver

aa~ RH

LD

aab AT

FIG. 3. Effect of sustained 48-hr shivering thermogenesis on cytochrome oxidase (CO) activity in the liver, rhomboideus (RH) and longissimus dorsi (LD) muscles and adipose tissue (AT) of newborn pigs. Values are means -4"-SEM (n = 6 at 6 hr, and 7-10 at 54 hr of age, except in AT, where n = 4). T0, piglets aged 6 hr; TIN, piglets aged 54 hr and raised at thermoneutrality; C, piglets aged 54 hr and raised in the cold. a'b'cWithin a tissue, values with different superscript let. ters differ ( P < 0.05).

Shivering Thermogenesis in the Newborn Pig

331

TABLE 2. Effect of sustained 48-hr shivering thermogenesis on the respiration and enzyme activities of subsarcolemmal and intermyofibrillar mitochondria from rhomboldeus and longissimus dorsi muscles of newborn pigs

Intermyofibrillar mitochondria Treatment

Longissimus &rrsi muscle Basal respiration ADP-stimulated respiration FCCP-stimulated respiration CO activity CK activity Rhomboideus muscle Basal respiration ADP-stimulated respiration FCCP-stimulated respiration CO activity CK activity

TN

C

Subsarcolemmal mitochondria TN

Statistical significance

C

M

T

20.7 102.1 88.6 261.0 266.7

-+ 3.7 _+ 8.9 -+ 20.2 _+ 22.7 + 37.9

30.9 110.6 138.2 354.0 418.8

_+ 4.2 -+ 10.9 -+ 23.5 -+ 42.0 + 35.0

12.2 30.6 22.8 244.3 196.8

_+ 2.3 + 5.8 -+ 10.8 -+ 46.8 _+ 42.6

18.6 41.6 49.0 283.9 349.0

-+ 3.2 +- 4.8 + 15.3 -+ 16.4 -+ 35.9

** *** *** NS NS

* NS *** NS **

32.8 125.2 99.2 367.8 548.7

_+ 3.1 _+ 5.6 + 30.1 -+ 18.7 -+ 101.3

34.0 121.9 151.0 423.5 520.2

-+ 1.8 _+ 5.9 _+ 16.1 + 29.5 -+ 45.1

27.0 49.0 43.9 418.9 426.3

-+ 4.5 +- 6.7 -+ 11.9 + 54.7 + 49.6

24.1 39.7 50.0 434.5 425.2

+ 3.8 -+ 2.9 -+ 18.6 -+ 60.5 _+ 44.9

NS *** *** NS NS

NS NS ** NS NS

Values are means -+ SEM (n = 5 - 8 for longissimus dorsi and n = 3 - 6 for rhomboideus muscle). TN, piglets raised at thermoneutrality; C, piglets raised in the cold. Basal (state IV), ADP-stimulated (state 111) and FCCP-stimulated respirations and CO activity are expressed in natoms O/rain/rag mitochondrial protein. CK activity was expressed in m U / m g mitochondrial protein. Respiration was measured in the presence of 1% bovine serum albumin, except when FCCP was added. Statistical significance: T, effect of treatment; M, difference between the two populations of mitochondria. There was no interactive effects. *P < 0.05; **P < 0.01; ***P < 0.001.

epinephrine level was 46% (P < 0.05) higher in C than in TN piglets at 54 hr of age.

DISCUSSION

Present results confirm the well-developed shivering capabilities of the newborn pig. Further, they suggest that the enhancement of heat production and shivering activity induced by 48-hr cold exposure are associated with noticeable changes in muscle energy metabolism, including an increased utilization of lipids and glycogen, an enhancement of oxidative potential and a rise in respiratory, oxidative and phosphorylative capacities of intermyofibrillar mitochondria isolated from lon~ssimus dorsi muscle. Whether these changes are part of an adaptative process or simply reflect the status quo biochemical capacity for shivering thermogenesis cannot be assessed from the present results.

Response of the Whole Animal to Cold Exposure As expected, a 48-hr cold exposure was associated with sustained shivering activity and elevated heat production, and one can calculate that the difference in heat production between C and TN piglets over 48 hr represents approximately 590 kJ/kg body weight. However, this value was probably slightly overestimated because measurements were made on animals kept singly in the respiratory chamber, whereas they lived in groups of three or four during the whole investigation and thus were able to huddle together to reduce heat loss. Despite this, a 10°C decrease in ambient temperature is known to be sufficient to induce significant

changes in the energy metabolism of neonatal pigs (34,41) and rats (10). Further, considering the well-known cold sensitivity of the newborn pig, a higher cold stress would have resulted in the progressive development of hypothermia that has dramatic effects on energy metabolism (14,40,53). During short-term cold exposure, shivering is the main thermogenic mechanism in newborn pigs (8), and it is unlikely that a 48-hr cold exposure could have induced the development of muscular nonshivering thermogenesis because, first, mitochondria isolated from the muscles of cold-exposed piglets were not loose-coupled (present results) and, second, several weeks of cold acclimatation are usually necessary for such a development in growing mammals (31). Therefore, we can assume that our experimental design is adequate to study the metabolic and biochemical mechanisms associated with shivering thermogenesis in the homeothermic piglet. Despite their vigorous response to cooling, cold piglets exhibited a slightly lower rectal temperature than thermoneutral animals as if they were unable to strictly maintain homeothermia, in agreement with previous results in pigs (24,34) and other species (27,43). This was neither due to a limited thermogenic capacity, because the peak metabolic rate was much higher and was obtained at a lower ambient temperature (7) nor to a lack of energy substrates because lipid and glycogen stores were far from being depleted at the end of the investigation and level of heat production is similar when cold-exposed piglets are supplied with an additional amount of dietary energy (26). Therefore, this inability to further increase metabolic rate probably reflect the inadequate development of the temperature

Berthon et al.

332

~lasmn epinephrine (nglml)

--c>- TN

I

I

6

18

I

30

I

54

Plasma noropinephrine (nglml) 6

piglets fed sow colostrum (22,35) and in spite of the low fat content of the milk substitute. Surprisingly, this was not associated with a rise in muscle LPL activity, which even decreased during this period. It suggests that LPL activity is not a limiting factor for lipid accretion in muscles of piglets raised at thermoneutrality. Alternatively, this is in agreement with the work of Planche et al. (48), which showed that although clearing of circulating chylomicrons during suckling largely depends on muscle LPL in rats, its activity is not clearly regulated by nutritional status during the first 2 weeks of life, as in older animals. Finally, the decrease in plasma FFA, which contrasts with the age-related rise usually observed in piglets fed sow colostrum (6) was probably due to the low fat content of the milk, because piglets given a low fat colostrum present a similar profile in plasma FFA (23,35).

Q _

--o-

4

Metabolic Responses to Sustain Shivering Thermogenesis

TN

-

3 2 1 0

I

I

8

18

I

30 Age (hours)

54

FIG. 4. Effect of sustained 48-hr shivering thermogenesis o n plasma epinephrine and norepinephrine levels in the newborn pig. Values are means -+ SEM (n = 6-13). TN, piglets raised at thermoneutrality (n); C, piglets raised in the cold (11). Effects o f t r e a t m e n t at 5 4 hr o f age: * P < 0.05.

control systems at birth. In other words, neonatal pigs can accept slight changes in body temperature without developping corresponding thermoregulatory mechanisms.

Utilization of Energy Substrates at Thermal Neutrality At 6 hr of age, glycogen content in liver and muscles was within the range of Elliot and Lodge's (17) results, whereas its postnatal decline in the liver was less pronounced probably because the synthetic milk contained more lactose (7.3%) than sow colostrum and milk (4-5%). In muscles, glycogen is the main source of energy at birth and its content decreases with age as previously reported (17), but the extent of the decrease was higher in rhomboideus than in longissimus dorsi muscle, probably in relation with the higher oxidative capacities of rhomboideua muscle (22, present results). Concomitantly, muscle lipid content increased, as in

Blood lactate results primarily from muscle glycogenolysis. Since muscle glycogen is known to be depleted at a higher rate in the cold (16,42; present results) and lactate production enhanced during cold-induced shivering (44), lactate production was likely increased in the cold group. However, blood lactate levels were reduced in cold-exposed piglets, which suggests that lactate utilization was also greatly enhanced. Lactate utilization could occur within the muscles since lactate represents a major fuel source during exercise (12) or in the liver, lactate being a main gluconeogenic precursor (38,47). Since muscle oxidative capacities were increased in the cold, it was also possible that the glycolytic pathway ended up with pyruvate and acetylCoA at the expense of lactate, which would favor a direct oxidative utilization of muscle glycogen and, thus, reduced lactate production. It is well known that total lipid content is not a good indicator of mobilizable lipid since it includes also structural fat and that variations in tissue lipid content reflect the balance between lipid accretion and utilization. However, lipid synthesis is negligible in the neonatal pig (37), and the reduction of muscle lipid content in the cold indicates either that lipid utilization was enhanced, in good agreement with the enhancement of CO activity (i.e. oxidative metabolism) in both muscles or that lipid deposition was reduced, which is very unlikely considering the unchanged LPL activity in longissimus dorsi muscle and its higher level in rhomboideus muscle. For the same reasons, the higher plasma FFA levels observed in the cold-exposed piglets at 54 hr of life are more likely due to a greater lipid mobilization than to a reduced FFA oxidation. Simultaneously, the potential for lipid capture and oxidative metabolism is reduced in adipose tissue. Overall, this would change the metabolic fate of blood triglycerides and enhance fatty acid availability in the

Shivering Thermogenesis in the Newborn Pig

thermogenic effectors (i.e., in the muscles). In addition, the differential response of LPL activity from white and red muscles during cold exposure has already been reported in rats during exercise (2), starvation (39) or cold exposure (5). Therefore, present changes are likely to have been induced by cold exposure and sustained muscle activity during shivering. These changes may contribute, at least in part, to provide the muscles with high amounts of readily oxidable substrates (i.e., carbohydrate and/or lactate and fatty acids). Overall, these cold-induced and age-induced changes occurred in a very short period of time (i.e. within only 48 hr), which confirms that the adaptative potential of the piglet is very high during the early neonatal period. Taken together, the decrease in muscle glycogen and total lipid content, the reduction of blood lactate levels and the enhancement of CO activity in the cold would suggest that muscle oxidative potential was increased during cold exposure. Measurements performed at the mitochondrial level clearly show that this rise was not related to an increase in mitochondrial mass, which is consistent with the slowness of the mitochondrial biogenesis process (20), but to the enhancement of mitochondrial respiratory capacities in lon~ssimus dorsi muscle. This is evidenced through the stimulation of state IV respiration rate and total respiratory chain capacity (i.e. FCCP-stimulated respiration). The oxidative potential was also enhanced in intermyofibrillar mitochondria. In this context, the absence of major changes in state III respiration rate and the associated slight reduction of the RCR in intermyofibrillar mitochondria could reflect a delay in the development of ATP-synthetase capacity, because the phosphorylative potential, estimated from CK activity and the CK:CO ratio, was also enhanced in the cold. In muscle, the function of mitochondrial CK is to synthetize creatine phosphate from creatine and ATP generated de novo and at the same time to return ADP to the respiratory system, thereby stimulating oxidative phosphorylation ( 11 ). Because of this functional relationship between mitochondrial CK activity and oxidative phosphorylation in muscle, the enzyme activity is a good indicator of the coupling state of muscle mitochondria. Present coldinduced enhancement of phosphorylative capacities is likely to have been induced by sustained muscle activity during shivering, in agreement with previously reported increase in CK activity in chronically stimulated fast-twitch muscle from rabbits (50) and human gastrocnemius muscle from long distance runners (1). Therefore, taken together, these results suggest that capacities to synthetize ATP are increasing in the cold in intermyofibrillar mitochondria from longissimus dursi muscle. This will contribute to supply the muscle with adequate amounts of ATP during shivering activity, as previously observed in muscle of animals during endurance exercise (28). By contrast, in rhomboideus muscle, 48 hr cold exposure induced only minor and inconsistent changes in mitochondria properties. This would suggest

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that this muscle, which plays a key role in thermoregulation during long-term cold acclimatation (22), plays only a minor role during short-term cold exposure (i.e., during shivering thermogenesis).

Age. and Cold.Related Changes in Circulating Catecholamines Blood samples were taken via the jugular catheter in nonstressful conditions while the piglet rested quietly in its cage. Plasma epinephrine and norepinephrine levels clearly increased with age in both groups, which could reflect the functional development of both the sympathetic nervous system and the adrenal glands. This is in agreement with the fact that the adrenal medullary neural innervation is not fully developed at birth (51) and with the increase in basal levels of plasma catecholamines between 2 and 24 hr of life (36). During the first part of the experiment (i.e., between 6 and 30 hr of life), plasma levels of both hormones were not modified by the cold treatment, which would suggest that the contribution of the adrenomedullary and sympathetic systems to the regulation of body temperature was weak during this period. This result contrasts with the higher plasma levels of catecholamines reported by Mayfield et al. (41) and Le Dividich et al. (36) during a cold challenge. However, their observations were only made during the initial period of cold exposure (first hour), and no informations were given concerning the persistence of the response over the following hours. Further, Mayfield et al. (41 ) pointed out that this hormonal response was significant only in the hypothermic piglets, a situation that was also encountered in the work of Le Dividich et al. (36). Our piglets were not hypothermic during the first day of cold exposure, and therefore our experimental protocol (i.e. prolonged exposure to a moderate cold with blood sampling every 6 hr) was probably not appropriate to detect such changes. However, after 2 days of cold exposure, coldexposed piglets had a slightly lower body temperature than control piglets, and the significant increase in plasma noradrenaline levels observed in the cold could be related to the difference in body temperature between groups, as suggested by Mayfield et al. (41). Interestingly, we have shown recently (26) that this hormonal response was modulated by energy intake, cold-exposed pigs given an additional amount of milk to compensate for the extra energy demand in the cold exhibiting no changes in plasma catecholamine levels. Therefore, the actual role of catecholamines in early neonatal thermoregulation certainly needs further investigation in pigs, with careful attention to degree of hypothermia, age, nutritional state and degree and duration of cold exposure. We gratefully acknowledge the technical assistance of J. Chevalier, M. Fillaut, J. Gauthier, J.C. Hulin and F. Strullu.

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