1596 DOI 10.1002/mnfr.201200160 Mol. Nutr. Food Res. 2012, 56, 1596–1600 F
OOD
& F
UNCTION
Diet supplementation with donkey milk upregulates liver mitochondrial uncoupling, reduces energy efficiency and improves antioxidant and antiinflammatory defences in rats
Lill `a Lionetti1∗, Gina Cavaliere1∗, Paolo Bergamo2, Giovanna Trinchese1, Chiara De Filippo1, Giorgio Gifuni1, Marcello Gaita1, Angelica Pignalosa1, Immacolata Donizzetti1, Rosalba Putti1, Rossella Di Palo3, Antonio Barletta1 and Maria Pina Mollica1
1Dipartimento delle Scienze Biologiche, Universit `a degli Studi “Federico II”, Napoli, Italy 2Istituto di Scienze dell’Alimentazione, Consiglio Nazionale delle Ricerche (CNR-ISA), Avellino, Italy 3Dipartimento di Scienze Zootecniche e Ispezione degli Alimenti, Universit `a degli Studi “Federico II”, Napoli, Italy
Dietary PUFA, mainly those of the n-3 family, are known to play essential roles in the mainte- nance of energy balance and in the reduction of body fat deposition through the upregulation of mitochondrial uncoupling that is the main source of reactive oxygen species. We hypothe- sized that rat supplementation with raw donkey’s milk (DM), characterized by low-fat content and higher n3:n6 ratio, may affect energy balance, lipid metabolism, and prooxidant status as compared to animals treated with cow’s milk. In the present study, the effects of drinking raw DM (for 4 weeks) on energy balance, lipid metabolism, antiinflammatory, and antioxi- dant/detoxifying defences was compared to that produced by rat intake of an iso-energetic amount of raw cow’s milk. The hypolipidemic effect produced by DM paralleled with the en- hanced mitochondrial activity/proton leakage and with the increased activity or expression of mitochondrial markers namely, carnitine palmitoyl transferase and uncoupling protein 2. The association of decreased energy efficiency with reduced proinflammatory signs (TNF-α and LPS levels) with the significant increase antioxidant (total thiols) and detoxifying enzyme activities (glutathione-S-transferase NADH quinone oxidoreductase) in DM-treated animals, indicated that beneficial effects were attributable, at least in part, to the activation of nuclear factor 2 erythroid-related factor 2 pathway.
Keywords: Donkey milk / Energy expenditure / Inflammatory parameters / Mitochondrial efficiency / Redox status
Received: March 22, 2012 Revised: June 1, 2012 Accepted: July 13, 2012
association Milk and milk products contain a number of essential nutri-
of higher dietary intake of saturated fatty acids, ents but, in the western societies, their growing consumption
contained in milk and dairy foods, with the development of has raised some concerns. Indeed, cow milk (CM) was rec-
several human pathologies is still unclear [1]. During the past ognized the leading cause for allergy however the positive
years, donkey milk (DM) has been indicated as promising food for children affected by cow’s milk protein allergy or
Correspondence: Dr. Paolo Bergamo, Istituto di Scienze
multiple food intolerance [2, 3] and, although the presence of dell’Alimentazione (CNR-ISA), Avellino, Italy
higher n-3 PUFA content or n3:n6 ratio have been accounted E-mail: p.bergamo@isa.cnr.it
for healthy effects produced by DM intake [4], nevertheless Fax: +39-0825-299105
the underlying mechanisms have never been investigated.
Abbreviations: CM, cow milk; CPT, carnitine palmitoyl trans- ferase; DM, donkey milk; EE, energy expenditure; FFA, free fatty
In this context, as mono and poly-unsaturated have been re- ported as the better substrates for mitochondrial ß-oxidation
acids; GSHtot, total thiols; GST, glutathione-S-transferase; ME,
pathway [5], which is central to the provision of energy and metabolizable energy; NQO1, NADH quinone oxidoreductase; Nrf2, nuclear factor 2 erythroid-related factor 2; UCP2, uncou- pling protein 2
∗These authors contributed equally to this study.
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Mol. Nutr. Food Res. 2012, 56, 1596–1600 1597
the main source of reactive oxygen species (ROS) [6], thus we decided to use DM as a model of naturally PUFA-rich milk and to compare the effects produced by CM intake on rodents lipid metabolism, redox status, and inflammatory response.
Fat content, fatty acid composition of DM, and CM were preliminarily determined and the obtained values were com- parable with those reported in literature [4] (Supporting In- formation Table S1). Moreover, as both milk treatments were unable to alter cholesterol and alanine aminotransferase lev- els (marker of hepatic damage) (data not shown), thus their hepatotoxic effect can be excluded.
The energy intake of rats treated with different dietary sup- plement was first evaluated. Interestingly, despite the higher lipid content (27%) and the lesser carbohydrate content (10%) as provided by CM-supplemented diet, as compared to DM intake, a comparable energy density (isocaloric diet) was pro- vided by the different treatments. In particular, the ingestion of comparable chow and milk amount (87 and 13%, respec- tively) in supplemented animals resulted in similar energy intake (Supporting Information Table S2). Next, when the effect of milk supplementation on body composition (water, lipid, and protein) was evaluated, increased relative percent- age of lipids was observed in CM-treated animals (p < 0.05) whereas only unimportant alterations were found in DM- treated rats as compared to controls. Similarly, the signifi- cant reduction of serum triglycerids in DM-treated rats (as compared to controls), associated to decreased body weight, lipid gain, and liver lipids as compared to CM-treated animals (p < 0.01) (Table 1).
To investigate the mechanism underlying the hypolipi- demic effect produced by DM supplement, metabolic param- eters were evaluated. Interestingly, although CM and DM treatments resulted in similar increase of metabolizable en- ergy (ME), nevertheless DM had no effect on body weight gain but improved animal energy expenditure (EE) and de- creased energy efficiency, as compared to CM treated or to control rats. Moreover, CM supplement associated with higher protein gain, lipid gain, protein gain/ME intake, and lipid gain/ME intake, by contrary, these values were markedly reduced in DM-treated animals (p < 0.01). Interestingly, the enhanced energy efficiency found in CM-treated animals was consistent with increased lipid gain and body lipid content (Table 1), in addition, the milk-induced shift from glucose to fat as metabolic substrate milk was indicated by the low respiration quotient. Moreover the increased production of CO
2
and higher O
2
consumption in DM-treated animals were consistent with the increase of daily EE (Supporting Informa- tion Table S3). Taken together, these data demonstrated that DM intake improved the ability to utilize fat as metabolic fuel and suggested that, in these animals, almost all of the extra energy was dissipated through an increased metabolic activity.
As liver greatly contributes to whole-body EE and lipid utilization, thus protein mass, oxidative capacity, and en- ergy efficiency were evaluated in mitochondria isolated from the liver of differently treated rats. The increased citrate synthase activity (known mitochondrial marker enzyme) in DM-supplemented animals as compared with controls
Table 1. DM supplement reduces lipid gain, energy efficiency, and triglycerides levels and improves energy expenditure
Control CM DM
Body weight (g) Initial body weight 345 ± 5.0a 347 ± 4.0a 348 ± 6.0a Final body weight 476 ± 2.0a 500 ± 1.0b 472 ± 5.0a Body weight gain 131 ± 2.2a 153 ± 2.0b 125 ± 7.0a Body composition (%) Water 62.5 ± 0.36a 58.6 ± 1.6b 63.9 ± 0.14a Lipids 14.8 ± 0.17a 18.6 ± 1.1b 13.6 ± 0.13a Proteins 14.4 ± 0.67a 14.4 ± 2.60a 13.5 ± 0.30a Body energy (kJ/g) 9.2 ± 0.20a 10.7 ± 0.58b 8.5 ± 0.10a Energy balance (kJ) ME intake 10148 ± 261a 11508 ± 172b 11856 ± 231b Body weight gain (kcal intake /bw gain) 77.5 ± 5.5a 94.9 ± 6.3a 75.2 ± 4.2b Energy efficiency (%) 0.14 ± 0.01a 0.23 ± 0.01b 0.09 ± 0.01c Protein gain 245 ± 30a 552 ± 35b 115 ± 23c Lipid gain 1226 ± 39a 2113 ± 120b 978 ± 66c Protein gain/ME intake (%) 2.5 ± 0.3a 4.8 ± 0.5b 0.98 ± 0.1c Lipid gain/ME intake (%) 12.1 ± 0.3a 18.39 ± 2.0b 2.54 ± 0.5c Energy expenditure 8676 ± 293a 8843 ± 357a 10762 ± 141b Lipid metabolism Liver lipids (mg/g) 2.7 ± 0.1a 3.3 ± 0.1b 2.6 ± 0.1a TG (serum) (mg/dL) 117.6 ± 6.0a 130.2 ± 7.9a 89.0 ± 7.5b
Data are presented as mean ± SE from triplicate analyses on individual samples prepared (n = 8) for each experimental group. Differing superscript letters indicate statistically significant differences (p < 0.05). TG, triglycerides.
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1598 L. Lionetti et al. Mol. Nutr. Food Res. 2012, 56, 1596–1600
Figure 1. Dietary supplement with DM in- creases mitochondrial proton leakage. Res- piration rates of mitochondria, isolated from the liver of CM-, DM-treated, or control an- imals were examined by interpolation at 160 mV (A) and 150 mV (B) for basal and palmitate-induced proton leaks, respectively. ß-Oxidation rate (C), CPT activity (D) and UCP2 expression (E) in liver samples from milk sup- plemented and control animals were evalu- ated. Data are mean values ± SE from tripli- cate analysis on individual samples (n = 24). Differing superscript letters indicate statisti- cally significant differences (p < 0.05).
(10.88 ± 1.14 or 7.18 ± 0.67 μmoles/min/g wet liver, respec- tively) was indicative of an increased mitochondrial protein mass. Next, the significantly higher State 3 and State 4 res- piration rates, using succinate as a substrate, were found in CM-treated and DM-treated rats (p < 0.05). When glutamate was used as substrate, mitochondria from DM-treated rats showed higher State 3 and State 4 respiration rates (36 and 26%, respectively as compared to controls or to CM-treated rats (Supporting Information Table S4). As glutamate or suc- cinate oxidation involves NADH or FADH
2
basal proton leakage was evaluated in mitochondria prepared from milk-treated animals, DM-treated mitochondria showed the higher oxygen consumption to maintain the same mem- brane potential, as compared to untreated or CM-treated rats (Fig. 1A). By contrary, mitochondria from control animals (in FFA-acid-induced conditions) consumed less oxygen as compared to milk-supplemented animals that exhibited over- lapping kinetic curves (Fig. 1B). The activity of carnitine palmitoyl transferase (CPT), the rate-limiting enzymes in -linked pathways,
the ß-oxidation pathway [9] and the expression of uncou- respectively, presented data demonstrated for the first time
pling protein 2 (UCP2), which modulates the EE and FFA the ability of dietary DM to increase mitochondrial capacities
metabolism [10], were next determined. The significant in- in both NADH-linked and FADH
2
-linked respiration path-
crease of CPT and UCP2 levels further confirmed the DM ways. Of note, as improved ß-oxidation respiratory rate (State
ability to improve the FFA ß-oxidation pathway (Fig. 1C and 3, using palmitoyl-carnitine as a substrate) in CM-treated rats
D). Taken together these results showed that dietary sup- was further increased in rats supplemented with DM (Fig. 1)
plementation with DM associated with increased mitochon- thus we decided to investigate the effects produced by the
drial mass and activity (with both NADH-linked and FADH
2 different milk intake on the efficiency of free fatty acids (FFA) oxidation pathway.
Mitochondrial efficiency is mainly due to basal (i.e. in the absence of FFA) and to FFA-induced proton leakage. Basal proton leakage occurred in a variety of cells and tis- sues [7] and its key role in resting EE has been demon- strated [8]. To determine the effect of DM supplement on mitochondrial leakage, both basal and FFA-induced proton leakage was measured in differently treated animals. When
- linked substrates) and with reduced energy efficiency/higher proton leakage.
The protective role played by ß-oxidation against oxida- tive stress along with the effect produced by mitochondrial ROS on UCP2 activity has been reported [11]. Herein, the intracellular concentration of the major redox buffers (total thiols (GSHtot)) and activity of an intramitochondrial sensor of redox status (aconitase) [12], were measured to investigate the effects produced by DM-induced mitochondrial leakage
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Mol. Nutr. Food Res. 2012, 56, 1596–1600 1599
on animal antioxidant status. Interestingly, the beneficial ef- fects produced by DM supplementation was indicated by the higher GSHtot concentration or the improved aconitase ac- tivity (p = 0.0004 or p < 0.001, respectively) in liver (Fig. 2A and B). To further investigate the mechanism underly- ing the anti-oxidant effect elicited by DM intake, activity of NADH quinone oxidoreductase (NQO1), and glutathione-S- transferase (GST) were measured. In response to mild oxida- tive stress the basic leucine-zipper transcription factor nuclear factor 2 erythroid-related factor 2 (Nrf2) is released from its inhibitor (Keap1) and triggers the activation of distinct set of genes encoding detoxifying enzymes including NQO1 and GST [13]. Thus the significant increase of GST or NQO1 activ- ities in the liver of animals supplemented with DM (p = 0.001 or p = 0.02, respectively) indicated that the improved cytopro- tective defenses resulting from DM intake were attributable, at least in part, to activation of Nrf2–ARE pathway.
Finally, the reported association of enhanced antiinflam- matory defenses with Nrf2 signaling [14], prompted us to eval- uate the consequences of DM intake on the levels of several proinflammatory indicators. The beneficial effects produced by DM intake was indicated by lower TNF-α concentration in DM-treated rats (Fig. 2C) and the LPS reduction (marker of metabolic and inflammatory diseases) (Fig. 2D) [15]. The immune-modulatory effect produced by DM intake was fur- ther supported by immune-histological evaluation of rodent livers (Supporting Information Fig. S1), and the lower con- centration of serum TNF-α, as compared to liver, was indica- tive of an improvement of body antiinflammatory status.
Presented results demonstrate, for the first time, that di- etary supplementation with DM milk increases EE and de- creases body lipid accumulation via the mild augmentation of mitochondrial uncoupling pathway which associated to chemo-protective and antiinflammatory effects in rodents.
Figure 2. Effects of different milk supplement on anti-oxidant/detoxifying and on antiinflammatory status of rats. GSHtot content and aconitase activity (A), were measured in rat liver to examine the effects of the different treatments on animal redox status and their values were finally expressed as nmoles/mg/min or basal/total activity, respectively. GST and NQO1 activities (B) in liver were evaluated to determine the influence of the different milk supplement on detoxifying enzymes activity (expressed as nmoles chloro-dinitro benzene; CDNB/mg/min or nmoles NAD/mg/min, respectively). Hepatic expression of TNF-α, its concentration in blood or liver (C) and LPS levels in blood (D) were used to determine the effects of the different treatments on animal inflammatory status. TNF-α and LPS concentrations were expressed as ng/g protein or EU/mg protein, respectively. Representative Western immunoblot is shown (insert) and ß-actin was used as loading control. Data are presented as mean values ± SE from triplicate analyses on individual samples (n = 24). Differing superscript letters indicate statistically significant differences (p < 0.05).
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1600 L. Lionetti et al. Mol. Nutr. Food Res. 2012, 56, 1596–1600
In particular, metabolic shift associated to DM intake is not consistent with literature data produced by increased dietary fat/carbohydrate ratio [16, 17]. On the other hand as antiin- flammatory effects associated to DM supplement resembles those resulting fromn-3 PUFA intake [18], thus studies aimed at better understanding of n3 and n6 PUFA role, as well as Peroxisome proliferator-activated receptor (PPAR)α involve- ment, in the beneficial effects produced by DM consumption are in progress.
The authors have declared no conflict of interest.
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