Caffeine stimulated lipolysis protects pigs against catecholamine-induced dark cutting

Meat Science 8 (1983) 53-64

Caffeine Stimulated Lipolysis Protects Pigs Against Catecholamine-induced Dark Cutting G. S. G. Spencer, L. J. Wilkins & D. Lister ARC Meat Research Institute. Langford, Bristol BSI8 7DY, Great Britain

(Received: 21 April, 1982)

SUMMARY High ultimate pH dark cutting meat was obtained in Pietrain pigs following ocernight intracenous infusion oJ" the ~-adrenergic agonist, isoproterenol. This effect was enhanced by simultaneous administration of the antilipolytic agent nicotinic acid and completely reeersed by pretreatment of the animals with caffeine (which stimulates lipolysis). These results suggest that the arailability of energy substrates from adipose tissue (free ./'atO' acids and glycerol) is an important factor in the production of dark cutting meat. Stimulation of lipolysis in animals prior to pre-slaughter stresses (such as transport and lairing) could possibly reduce the incidence of dark cutting meat.

INTRODUCTION Ante-mortem depletion of muscle glycogen reduces the amount of postmortem glycolysis possible and, thereby, the extent of acidification of the meat. Most meat attains an ultimate pH in the range 5.5 to 6.0. However, at higher ultimate pH (6-2-6-3) the meat retains a darker colour. Such meat is called 'dark cutting' and presents as dark cutting beef or dark, firm and dry (DFD), pork. The main energy substrates in the living animal are glucose and free fatty acids (FFA). These are released from liver glycogen, muscle glycogen and adipose tissue. A complex combination of nervous and hormonal control of metabolism usually ensures that there is a relatively equitable contribution from each of these stores to the total energy 53

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G. S. G. Spencer. L. J. ~t,~lkins, D. Lister

54

requirement of the animal. However, the large stores of adipose tissues supplying FFA are practically inexhaustible and it has been suggested that reliance on muscle glycogen results when F F A are not so readily available from fat which may be the case during periods of nervous and metabolic stimulation (Lister, 1979). This view has been supported by recently reported experiments using sheep and bulls. When the availability o f FFA in these animals was reduced by administration of antilipolytic agents, there was severe ante-mortem depletion of muscle glycogen which resulted in dark cutting meat (Lister & Spencer, 1981, 1983). The sympathetic nervous system appears to play a central role in mediating the stress-induced metabolic changes (Lister et al., 1976) and both lipolysis and muscle glycolysis are controlled by fl-adrenergic mechanisms (Himms-Hagen, 1967; Bowman & Nott, 1969). We have used an infusion of the fl-adrenergic agonist, isoproterenol, to stimulate prolonged stress in pigs, and have examined the effects of both reducing and increasing the availability of FFA on the occurrence of dark cutting in the meat from these pigs. To counteract the lipolytic effect of isoproterenol, nicotinic acid was administered as an antilipolytic agent, while additional stimulation of lipolysis was effected by administration of caffeine.

MATERIALS A N D M E T H O D S Twenty-one Pietrain pigs of approximately 55 kg bodyweight were fitted bilaterally with indwelling jugular vein catheters while under thiopentone-induced anaesthesia. After the operation the pigs were placed in individual metabolism crates and allowed at least 7 days to recover and to become accustomed to the handling and sampling procedures. Throughout this period they had access to food and water ad libitum and all the pigs were eating a normal a m o u n t by 3 days after the operation. On the day of the experiment the pigs were offered food between 08.00 and 18.00 h--all food was then removed; water remained freely available throughout the experiment. The pigs were allocated one of the following treatments: 1.

lsoproterenol (fl-adrenergic) stimulation: the eight animals in this group received a continuous infusion of 0"1/.lg/kg/min of iso-

Lipolysis protects pigs against dark cutting

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proterenol (Sigma) in sterile saline (Steriflex) to which was added 10 U/ml of heparin (Pularin) and 0.3 g/1 of ascorbic acid (British Drug Houses) as an antioxidant. The infusion began at 03.00 h and continued until slaughter at approximately 09.30h the following morning. lsoproterenol stimulation plus nicotinic acid." the seven pigs receiving this treatment were infused with 20 g of nicotinic acid (Niacin, Sigma). The nicotinic acid was dissolved in 50ml 3s NaOH, the pH adjusted to about 7-4 with 2N HCI, and added to sterile saline containing 10 U/ml of heparin. The infusion started at 23.30h and lasted for l h. Immediately following this the animals received the isoproterenol infusion as described in point 1 above. A bolus boost of nicotinic acid (5g in 16ml 3N NaOH and adjusted to pH 7-4) was administered through the catheter every 2 h and the isoproterenol infusion continued. The animals were slaughtered at 09.30 h as above. lsoproterenol stimulation plus caffeine: 2-5 g of caffeine (Sigma) and 2"5g of sodium benzoate (Hopkin and Williams) were dissolved in 25 ml of water and administered through the catheter to each of six pigs, over a 3-4 min period, at 22.30 h. At 00.30 h an isoproterenol infusion began and the subsequent protocol was as in point 1 above. Controlpigs: fourteen Pietrain pigs of a similar weight were used as control animals. These pigs were completely untreated but were starved overnight before being slaughtered as were the treated animals.

Blood samples were taken prior to treatment (22.30h), prior to isoproterenol infusion (00.30 h) and at 2-h intervals thereafter (before the nicotinic acid booster administration in those pigs receiving this treatment). The plasma was immediately separated by centrifugation and stored for later assay of glucose, lactate, free fatty acids and glycerol, as described elsewhere (Lister & Spencer, 1983). Slaughter was by electrical stunning and exsanguination by vena cava puncture. Samples of M. longissimus dorsi were taken at 5 min, 45 min and 24 h post-mortem for the measurement of lactate, glycogen and pH (Lister & Spencer, 1983). Samples of liver were taken within 15min of slaughter for glycogen estimation. Forty-eight hours after slaughter colour measurements were made on

56

G. S. G. Spencer, L. J. Wilkins, D. Lister

the M . longissimus dorsi, in the carcass, using a fibre-optic probe (MacDougall & Jones, 1975) and drip loss was estimated by the method of Taylor & Dant (1971).

RESULTS

lsoproterenol treatment In agreement with other studies, isoproterenol treatment produced marked lipolysis and muscle glycolysis. These changes in metabolism were reflected in increased levels of plasma free fatty acids (FFA) and lactate (Fig. 1). Throughout the period of infusion glucose levels did not change significantly in the isoproterenol-treated pigs. At slaughter, muscle glycogen levels were severely depleted compared with the levels in 14 untreated control Pietrains of similar age (Table 1). It should be noted, however, that since the control pigs were untreated in any way this may have some bearing on the results. The low level of muscle glycogen at slaughter in the isoproterenol-treated pigs did not allow adequate acidification of the muscle post-mortem as indicated by

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the change in muscle pH and lactate levels between 5 min after slaughter and 24 h post-mortem (Table 2). This incomplete acidification of the muscle resulted in low fibre-optic values and little drip loss. In short, the meat was dark cutting or D F D . Despite the absence of a significant rise in plasma glucose levels (presumably because of a slow, prolonged, glycogenolysis over 10 h), liver glycogen was also low at the time of death.

Isoproterenol and nicotinic acid Nicotinic acid is a potent inhibitor of lipolysis and successfully counteracted the isoproterenol stimulation of fatty acid release, at least until the final stages of the infusion (Fig. 2). As in pigs treated with isoproterenol alone plasma glucose levels did not change significantly, but the absence of complementary energy from fat breakdown caused a greater reliance on muscle glycogen. This could, at least partially, account for the high levels of plasma lactate in these animals. The increased catabolism of muscle energy stores was shown in the meat post-mortem. Although the differences were not statistically significant, muscle glycogen at 5 min post-mortem was lower than in the

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pigs treated with isoproterenol alone and the colour, drip, and liver glycogen levels were also lower (Table 1). lsoproterenol and caffeine Preliminary experiments in similar pigs showed that intravenous administration of caffeine alone could stimulate FFA release (Fig. 3). In the present experiments, the contrast between the nicotinic acid treatment and the lipolytic effect of caffeine could be seen in the elevated levels of plasma FFA in the animals treated with caffeine (Fig. 4). The lipolytic effect of caffeine, in addition to the lipolytic effect of isoproterenol alone, is more clearly seen in the change in FFA levels during infusion (Fig. 5A) and in the increased FFA: glycerol ratio (Fig. 5B). The changes in plasma lactate and the unchanged plasma glucose levels were similar to those in the animals treated with isoproterenol alone. There was a significant difference between the isoproterenol- and the caffeine-treated pigs in the post-mortem changes. Table 1 shows that muscle glycogen was significantly higher (p < 0-01) at 5 min post-mortem and pH was lower at 45 rain (p < 0.01) and 24 h post-mortem (p < 0-001); 30.

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G. S. G. Spencer, L. J. Wilkins. D. Lister

liver glycogen was much higher (p < 0-001) as were the colour (p < 0.01) and drip-loss levels (p<0-01). The increase in post-mortem muscle lactate content was also elevated (Table 2).

DISCUSSION It has been proposed (Lister, 1979) that excessive glycogen utilisation is exacerbated by reduced availability of energy from fat sources. Experimental evidence supporting this hypothesis has been provided by experiments in which catecholamine-induced muscle glycolysis in sheep resulted in dark cutting meat only when fl-adrenergic stimulation was accompanied by blockage oflipolysis (Lister & Spencer, 1983). Metabolic events in sheep are, however, different from those in pigs. In contrast to ruminants, which use considerable amounts of volatile fatty acids as an energy source, the main fuel for oxidation in monogastric animals is glucose. It might therefore be expected that the monogastric pig would be more susceptible to catecholamine-stimulated glycogen depletion. This would appear to be the case since, in contrast to the effects of similar infusions in sheep, isoproterenol alone caused marked muscle glycogen depletion and resulted in dark cutting meat in these pigs. Concurrent treatment with nicotinic acid inhibited lipolysis, reducing the availability of energy from FFA and producing even greater depletion of muscle (and liver) glycogen and consequently even darker, drier meat. A corollary of this finding is that treatment with a lipolytic agent could protect the animal against excessive reliance on muscle glycogen and, thereby, prevent the production of D F D pork. The results presented here show that treatment with caffeine has a significant effect in stimulating liver gluconeogenesis and in sparing liver and muscle glycogen, thus permitting the development of normal pH and meat quality characteristics post-mortem. There are two points of methodology which require some comment. First, although the drip values recorded are somewhat high compared with those obtained by other methods of measurement, the mean drip value from stress-resistant Gloucester Old Spot pigs obtained by this method (Taylor & Dant, 1971) in our laboratory is 8.1 ~,o- The second point relates to glycogen levels. There is an apparent discrepancy between glycogen change and the increase in lactate post-mortem in the isoproterenol- and the isoproterenol + nicotinic acid-treated pigs. This is

Lipolysis protects pigs against dark cutting

63

probably explained by the limit of precision of the glycogen assay (about + 1 mg/g). The low values for the means of these groups can only, therefore, serve as an indication of very low levels. The mechanism of action of caffeine in causing its glycogen sparing effect on muscle is not clear. Apart from being a potent lipolytic agent, caffeine also is known to have direct effects on muscle (such as rendering it less susceptible to fatigue) and on the cardiovascular system (where it increases cardiac output and decreases peripheral capillary resistance) (Ritchie, 1975). It might, therefore, favour oxidative rather than anaerobic metabolism. However, there was no indication of any major change in muscle metabolism, as indicated by the levels of metabolites in the plasma. After calculation of the areas underneath the curves for FFA, glucose, lactate and glycerol, and the FFA:glycerol ratio (an indicator of the degree of re-esterification taking place) no significant difference was found between the animals treated with isoproterenol alone and those treated with isoproterenol and caffeine for any of these indicators. It remains to be established whether treatment of animals with caffeine prior to endogenous secretion of catecholamines pre-slaughter will prevent the occurrence of dark cutting meat.

ACKNOWLEDGEMENTS We are grateful to Sheila Jones for the colour measurements and to Kathy Buckley for laboratory assistance and preparation of the manuscript and figures.

REFERENCES Bowman, W. C. & Nott, M. W. (1969). Pharmac. Rev., 21, 27. Himms-Hagen, J. (1967). Pharmac. Rev., 19, 367. Lister, D. (1979). Acta Agric. Stand., Supplementum 21, 281. Lister, D., Lucke, J. N. & Hall, G. M. (1976). In: Production diseases i, farm aninlals, p. 141. Pudoc, Wageningen. Lister, D. & Spencer, G. S. G. (1981). In: The problem of dark cutting beeJ[ (Hood, D. & Tarrant, P. V. (Eds)), Martinus Nijhoff, The Hague, p. 129.

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Lister, D. & Spencer, G. S. G. (1983). Meat Sci., 8, 41. MacDougall, D. B. & Jones, S. J. (1975). Proc. 21st Eur. Meeting Meat Res. Wkrs., Bern, Switzerland, p. 113. Ritchie, J. M. (1975). In: The pharmacological basis of therapeutics, 5th Edn. (Goodman, L. S. & Gilman, A. (Eds)), p. 367. Taylor, A. A. & Dant, S. J. (1971). J. Fd. Technol., 6, 131.