Temperature, but not excess of glycogen, regulates "in vitro" AMPK activity in muscle samples of steer carcasses

Abstract

Postmortem muscle temperature affects the rate of pH decline in a linear manner from 37.5°C to 0-2°C. The pH decline is correlated with the enzymatic degradation of glycogen to lactate and this process includes the metabolic coupling between glycogenolysis and glycolysis, and that are strongly upregulated by the AMPK. In this study, we used 12 samples previously characterized by have different muscle glycogen concentration, lactate and AMPK activity, selected from 38 steers that produced high final pH (>5.9) and normal final pH (<5.8) carcasses at 24 h postmortem. Moreover, we evaluated changes in the AMPK activity in samples from both categories incubated at 37, 25, 17 and 5°C and supplemented with exogenous glycogen. Finally, we analysed if there were structural differences between polymers from both categories. Our results showed that "in vitro" enzymatic AMPK activity evaluated at both 0.5 or 24 h was greater in samples from normal then high pH categories (p <0.01), and in all temperature of incubation analysed (17, 25 and 37°C). For other hand, a greater AMPK activity were obtained in samples incubated at 17 that 25 or 37°C, in normal carcasses at both 0.5 or 24 h (p < 0.01), as also in samples from carcasses categorized as high pH, but at 24 h (p < 0.05). Interestingly, AMPK activity was totally abolished at 5°C, independent of final pH category of carcasses, and was confirmed that the incubation temperature at which the maximum activity was obtained (p < 0.01), at least in carcasses with a normal pH is at 17°C. The enzymatic AMPK activity did not change in relation to excess glycogen (p > 0.05) and we did not detect structural differences in the polymers present in samples from both categories (p > 0.05), suggesting that postmortem AMPK activity may be highly sensitive to temperature and not to in vitro changes in glycogen concentration (p > 0.05). Our results allow concluding that normal concentrations of muscle glycogen immediately at the time of slaughter (0.5 h) and an adequate cooling managing of carcasses are relevant to let an efficient glycogenolytic/glycolytic flow required for lactate accumulation and pH decline, through the postmortem AMPK signalling pathway.

Fig 1. Temperature decline postmortem in steer carcasses M. longissimus thoracis (both categories, n = 12).

In the figure, slopes are shown (S1 and S2) corresponding to two components founded by non-linear function analysis.

Fig 2. Average values ±SEM of AMPK enzyme activity levels in samples of in M. longissimus thoracis categorized with normal (< 5.8) v/s high (> 5.9) final pH, evaluated at 0.5 h and 24 h and in function of temperature.

A: measure at 17, 25 and 37°C at the 0.5 h; B: measure at 17, 25 and 37°C at the 24 h. Two-way ANOVA and Bonferroni post-tests. Differences between categories (#, p < 0.0001) and between 17°C and 25°C or 37°C, within categories (*, p < 0.0001), n = 6.

Fig 3. Average values ±SEM of AMPK enzyme activity levels in samples of in M. longissimus thoracis categorized with normal (< 5.8) v/s high (> 5.9) final pH evaluated at different temperatures in the cooling phase (5°, 17°, 25° and 37°C at the 0.5 h).

Two-way ANOVA and Bonferroni post-tests. Differences between categories (#, p < 0.0001); between 17°C and 25°C or 37°C, within categories (*, p < 0.0001); between 17°C and 5°C, within categories (@. p < 0.0001 and p < 0.05, for normal and high final pH, respectively), n = 6.

Fig 4. Average values ±SEM of AMPK enzyme activity levels in samples of M. longissimus thoracis categorized with normal (< 5.8) v/s high (> 5.9) final pH, evaluated at 0.5 h and to 17°C, and in function of endogen and exogenous glycogen content.

Two-way ANOVA and Bonferroni post-tests. Differences between categories (#, p < 0.0001), n = 6.

Fig 5. Structural glycogen analysis.

A: Absorption spectrum of glycogen in samples of M. longissimus thoracis categorized with normal (< 5.8) v/s high (> 5.9) final pH, in presence of potassium iodide. B: maxima wavelength (nm). (n = 6).