Tratamiento de desordenes metabólicos en ganado lechero con

un activador del receptor proliferador peroxisomal alfa (PPARa)

Dr. Roberto Farina- Fatro Group, Italy

Durante el periodo de transición, como una consecuencia del incremento de las

demandas fetales, los ácidos grasos almacenados se liberan del adiposito y

son tomados por el hígado para cubrir los requerimientos de energía materna

para la preñez, la lactación así como los cambios dramáticos endocrinos. En el

hígado ellos pueden ser reesterificados a triacilgliceroles y formar parte de una

lipoproteína de muy baja densidad (Very low density lipoproteins, VLDL) o

desnaturalizado por la beta oxidación. Cuando la cantidad de ácidos grasos

que se incorporan al hígado excede la cantidad secretada u oxidada, los

triacilgliceroles se acumulan en el adiposito provocando una condición

conocida como hígado graso o lipidosis hepática. El hígado graso es una

condición posparto que afecta a la mitad de las vacas lecheras en producción

en la lactación temprana. La acumulación de lípidos hepáticos tiene efectos

detrimentales en la salud, y los parámetros de la producción y la reproducción.

Las funciones hepáticas alteradas debido a la infiltración grasa está asociada

estrechamente con las fallas en el sistema inmune, la reducción en la fertilidad

y las enfermedades peri y posparto.

Recientemente se ha descubierto que los ácidos grasos regulan el

metabolismo a través de una interacción con los receptores activados por

proliferadores peroxisomales (peroxisome proliferators activated receptors

PPARs), los cuales han emergido como un de los reguladores centrales de las

interacciones nutriente-gene. Los PPARs son receptores nucleares que han

estado implicados como parte importante en el rol de la homeostasis de la

energía, el síndrome de hígado graso y las enfermedades metabólicas. Se ha

desarrollado un importante progreso en la exploración de la biología del PPAR,

la cual señala nuevas estrategias para el control de enfermedades metabólicas

en la vaca lechera. El receptor activado por proliferador peroxisomal alfa

(PPAR- alfa) ha sido identificado como el regulador de transcripción mas

importante de varios genes que participan en muchos y muy probablemente en

todos los aspectos catabólicos de las grasas, en el transporte de ácidos grasos

en la circulación, su incorporación hacia el hepatocito, la unión intracelular de

ácidos grasos por ligadura de proteínas, y la activación de la acil-CoA sintetasa

entre otras funciones, así como también el catabolismo por oxidación-beta en el

peroxisoma y en la mitocondria y la oxidación-omega en los microsomas. Se ha

demostrado también que el PPAR alfa regula la expresión de varios genes

involucrados en la gluconeogénesis. Por lo tanto el PPAR alfa actúa como un

regulador universal del metabolismo de la energía que detecta cambios en el

estatus energético y los traslada como ajustes metabólicos para poder

mantener la homeostasis. El PPAR alfa también influencia la expresión de

numerosos genes que están involucrados en diversos procesos en el

metabolismo de los aminoácidos e interfiere con diversos pasos en al

respuesta inflamatoria al modular la expresión de las citoquinas, el receptor de

las citoquinas, las moléculas de adhesión y la fase aguda de las proteínas.

El 2-metil-2 fenoxil-acido propiónico (Hepagen™-Fatro) es el único activador de

PPAR alfa en medicina veterinaria. El 2-metil-2 fenoxil-acido propiónico actúa

incrementando y uniéndose al PPAR alfa regulando la trascripción de varios

genes involucrados en el metabolismo lípido y de la glucosa. Como

consecuencia, promueve la activación del mitocondria y la beta-oxidación

peroxisomal y la gluconeogenesis hepática. El incremento en la oxidación

lipidica provoca una reducción en la acumulación de los triglicéridos en el

hepatocito, recuperando la función hepática e incrementando la producción de

energía.

La regulación de genes involucrados en la gluconeogenesis intensifica la salida

de glucosa hepática y restaura el balance de energía. Las propiedades anti-

inflamatorias de los PPAR ayudan a la vaca considerando las condiciones

inflamatorias que están en juego durante la patogénesis del hígado graso y

otras muchas enfermedades. El 2-metil-2 fenoxil-acido propiónico presenta

también un efecto colerético y colagogo que son extremadamente útiles para el

tratamiento del hígado graso.

El 2-metil-2 fenoxil-acido propiónico ha demostrado ser muy efectivo en el

tratamiento del hígado graso, cetosis, acidosis, problemas digestivos,

intoxicaciones y cuando hay desordenes hepáticos. Gracias a su amplio y

especifico efecto sobre el metabolismo de la energía, la coleresis y la

inflamación el 2-metil-2 fenoxil-acido propiónico representa la primera línea de

terapia para el tratamiento de los problemas metabólicos.

Keywords: PPAR-alpha, fatty liver, metabolic disorders, dairy cow.

Roberto Farina

Peroxisome Proliferator-Activated Receptor Alpha

A New Target for Metabolic Disorders of Dairy Cows

Introduction

During the transition period, in support of lacta-tion and fetal growth, cows must mobilise body fat tomeet the increased demand for energy. In almost allhigh-producing cows, large amounts of fatty acids arereleased into the blood and accumulated in the liveras triglycerides, leading to varying degrees of fattyliver. Hepatic lipid accumulation has detrimental ef-fects on health status, well-being, productivity andreproductive performance. Impaired liver functionsdue to fatty in�ltration are closely associated withimpairment of the immune system, reduced fertilityand peripartum diseases, including ketosis, left dis-placement of the abomasum, milk fever, downer cowsyndrome, retained placenta, metritis and mastitis.Peroxisome proliferator-activated receptors

(PPARs) are nuclear receptors that have beenimplicated to play an important role in energyhomeostasis, fatty liver and metabolic diseases.Recent advances in identifying selective ligandshave helped delineate the subtype-speci�c functionsand the therapeutic potential of these receptors.Peroxisome proliferator-activated receptor alpha(PPARα) has been identi�ed as a key transcriptionregulator of many genes involved in lipid transportand oxidation. This transcription factor plays a keyrole in lipid metabolism and energy homeostasis. Inthe liver, PPARα directly regulates genes involved infatty acid uptake and oxidation and gluconeogenesis.

2-methyl-2-phenoxy-propionic acid (HepagenTM -Fatro), a veterinary drug recently introduced in Mex-ico, is the only PPARα agonist available in veterinarymedicine. This substance, through the activation ofPPARα, regulate the transcription of a number ofgenes that facilitate lipid metabolism, reduce fattyin�ltration of the liver and promote gluconeogenesis,appearing as a unique tool for the treatment and pre-vention of the most important and widespread disor-ders a�ecting dairy cows.

Lipid Metabolism During the

Transition Period

When glucose availability is low during periods ofenergy de�cit, as we �nd in cows facing the transi-tion to lactation, fats stored in adipose tissue are

Figure 1. Lipid metabolism during the transition period

hydrolyzed to free fatty acids and mobilized intoplasma to reach the liver [1]. In the liver, thesenon-esteri�ed fatty acids (NEFA) are either con-verted into triacylglycerols and secreted as very lowdensity lipoproteins (VLDL) or oxidized by the mi-tochondrial and peroxisomal beta-oxidation systems.Partial oxidation of fatty acids leads to the produc-tion of acetyl coenzyme A (acetyl-CoA), which thencondenses with itself to form ketone bodies. Ketonebodies are then exported out of the liver to serveas fuels for other tissues (Fig. 1). The energy re-leased in the process of ÿ-oxidation is used by theliver to carry out gluconeogenesis from substratessuch as lactate, amino acids and glycerol. Thus, e�-cient hepatic fatty acid oxidation is obligatory to themetabolic response to energy de�cit. This is true inruminants more then in other animals, since they arein a state of continuous gluconeogenesis due to thedegradation of most of the dietary carbohydrate tovolatile fatty acids in the rumen fermentation. He-patic β-oxidation is necessary to maintain glucoseoutput by the ruminant liver and become vital duringfasting or periods of negative energy balance [31].

Control of Lipid Metabolism

The regulation of lipid metabolism is central toenergy homeostasis. It involves control systems thatare sensitive to stimuli and conditions such as theavailability of food, physical activity, temperature,production status, stress and other physiological or

1

pathological changes. The coordination of the re-sponses to signals triggered by these stimuli mustoccur on several levels to ensure a well adapted en-ergy balance, ranging from hypothalamic functionsin the brain to the direct control by lipids and carbo-hydrates of their own fate [3]. Fatty acids and carbo-hydrates participate together with several hormonesin the regulation of gene expression in response tofood intake and qualitative nutritional changes. Re-cently it has been found that fatty acids regulatemetabolism mainly through their interaction withperoxisome proliferator activated receptors (PPARs),which have emerged as one of the central regula-tors of the nutrient/gene interactions [2]. PPARsare lipid-activable transcription factors that regulategenes controlling lipid and glucose metabolism andadipogenesis. The PPARs control a variety of genesin several pathways of lipid metabolism, includingfatty acid transport, uptake by the cells, intracellularbinding and activation, as well as catabolism or stor-age. Rapid progress has been made in the explorationof PPAR biology, which indicates new strategies forthe control of important metabolic disorders of dairycows.

Peroxisome Proliferator Activated

Receptors

PPARs are members of the nuclear hormone recep-tor superfamily of ligand activated transcription fac-tors that are related to retinoid, steroid and thyroidhormone receptors [14]. Their name is derived fromthe fact that they were originally identi�ed as mole-cules that mediate the transcriptional e�ects of drugsthat induce peroxisome proliferation in hepatocytes[4]. The PPAR subfamily is composed of three mem-bers: PPARα, PPARβ (or δ), and PPARγ [18]. Theydisplay di�erential tissue distribution and each of thethree isotypes performs speci�c functions. PPARα isexpressed mainly in the liver, skeletal muscle, heartand kidney and is thought to regulate many genesinvolved in the catabolism of fatty acids. The γ iso-type is mainly expressed in adipose tissue, where itplays an important role in adipocyte di�erentiationan lipid storage [29] and in macrophages, where itmodulates di�erentiation and cytokine production.PPARδ is expressed in all tissues examined and hasbeen implicated in embryo implantation and woundhealing.Fatty acids and their derivatives are natural lig-

ands for PPARs. A number of unsaturated fattyacids and eicosanoids have been shown to activatePPARs [5]. PPARs stimulate target gene expressionvia the formation of heterodimeric transcription fac-tor complexes with the retinoid x receptor (RXR).RXR is also member of the nuclear hormone recep-tor superfamily and it is activated by 9-cis retinoic

acid. The activated PPAR/retinoid X receptor com-plex regulate gene expression by binding to DNAsequence elements in the promotor regions of spe-ci�c target genes, termed PPAR response elements(PPREs).

PPARα: a global regulator of energy

metabolism

PPARα regulates many genes involved in the con-trol of lipid and lipoprotein metabolism. In the liver,PPAR target genes form a comprehensive ensembleof genes which participates in many if not all aspectsof lipid catabolism. It includes transport of fattyacids in the circulation, their uptake by the hepato-cytes, intracellular binding by fatty acid binding pro-teins, activation by the acyl-CoA synthase, as well ascatabolism by β-oxidation in the peroxisomes and mi-tochondria, and ω-oxidation in the microsomes [16].Experiments with PPARα-null mice have been in-

valuable in elucidating the physiologic role of PPARαand have indicated that hepatic PPARα is partic-ularly important during fasting and in general insituation of energy de�cit [17, 30]. Mice de�cientin PPARα display hepatomegaly and severe liverfatty in�ltration following starvation (Fig. 2). Micelacking functional PPARα are incapable to induceexpression of a variety of genes required for themetabolism of fatty acids in peroxisomes, mitochon-dria, and other cellular compartments. PPARα-nullmice accumulate massive amounts of lipid in theirlivers when fasted (Fig. 3). Fasting also results insevere hypoglycemia and elevated plasma levels ofnonesteri�ed fatty acid, indicating a defect in fattyacid uptake and oxidation caused by dysregulationof these genes [17]. Furthermore, it is demonstratedthat to accommodate the increased requirement forhepatic fatty acid oxidation during fasting, PPARαmRNA is induced in wild-type mice. Reduction inhepatic phospholipid contents and unchanged car-nitine levels in PPARα-null mice following starva-tion show that this receptor plays also an impor-tant role in phospholipid homeostasis and carnitinemetabolism during energy deprivation [15, 17]. Thisdata indicate that PPARα is a key part of a complexnetwork of signaling pathways in the liver that op-erate during energy de�cit and stimulate fatty acidoxidation to produce energy and form substrates thatcan be metabolized by other tissues.PPARα does not function exclusively as a regula-

tor of lipid metabolism. Numerous lines of evidenceindicate that PPARα in�uences glucose homeostasisas well. First of all, PPARα participates to main-tain blood glucose during acute metabolic stress, asshown in PPARα-null mice, which develop severe hy-poglycemia when fasted [30, 19]. It has also beenshown that PPARα up-regulates the expression ofseveral genes involved in gluconeogenesis. Lipolysis

2

Figure 2. Livers from PPARα +/+ (Left) and PPARα-/- (Right) mice after a 24-h fast.

Leone, Teresa C. et al. (1999) Proc. Natl. Acad. Sci. USA 96,

7473-7478

Figure 3. Liver morphology in fed and fasted wild typemice and mice lacking PPARα (Oil Red O-stained frozen

sections of liver)

Fed wild type mouse

Fed PPARα null mouse

48-h fasted wild type mouse

48-h fasted PPARα null mouse

Adapted from Hashimoto, T. et al. J. Biol. Chem.

2000;275:28918-28928

in adipose tissue together with fatty acids releasesglycerol, which is carried to the liver for furthermetabolism. The metabolic fate of glycerol is alsounder the control of PPARα, which stimulates itsconversion to glucose in liver.Therefore PPAR acts as a global regulator of en-

ergy metabolism that senses changes in energy sta-tus and translates them into metabolic adjustmentsaimed at maintaining homeostasis [33].PPARα also in�uences the expression of numerous

genes implicated in major pathways of amino acidmetabolism [34] and possesses anti-in�ammatoryproperties. PPARα interferes with di�erent stepsof the in�ammatory response by modulating the ex-pression of cytokines, cytokine receptors, adhesionmolecules and acute phase proteins [36, 35].

Fatty Liver

During the transition period, as a consequenceof the increasing fetal demands, the maternal en-ergy requirements for pregnancy and lactation andthe dramatic endocrine changes, stored fatty acidsare released from the adipocyte and taken up byliver [20]. There they are either reesteri�ed to tri-acylglycerols and assembled into VLDL or brokendown through ÿ-oxidation. When the amount ofNEFA incorporated into the liver exceeds the amountsecreted or oxidized, triacylglycerols accumulate inthe adipocyte leading to a condition known as fattyliver or hepatic lipidosis [21, 25]. Overfeeding dur-ing the non-lactating stage, reduced feed intake andstress near parturition accelerate the release of NEFAfrom adipose tissues, resulting in an excess uptake ofNEFA by the liver.Fatty liver is a common postpartum condition af-

fecting up to 50% of dairy cows in early lactation(Table 1). Fatty liver is associated with reducedhealth status, well-being, productivity and reproduc-tive performance [22, 21]. High-yielding dairy cowsare susceptible to several diseases during the peripar-tum period, including ketosis, left displacement of theabomasum, retained placenta, milk fever and downercow syndrome. Several evidences suggest that thesediseases originate from fatty liver [7, 6, 8, 9, 26].Although the precise pathogenesis in each disease isunknown, decreased metabolic functions of the liverdue to fatty in�ltration are believed to be closely re-lated to the development of these disorders. Triglyc-erides accumulation in cultured hepatocytes has beenshown to decrease ureagenesis [24] and gluconeogen-esis [27]. Toxic substances such as bile constituentsaccumulate in the liver of cows with fatty liver dueto a reduction in bile �ow [25] and impaired detoxi�-cation processes. Synthesis of lipoproteins, which areimportant in lipid packaging, secretion and metabo-lization, also decreases [10, 12, 13, 11]. Fatty liveris associated with impairment of the immune system

3

Table 1. Incidence of di�erent categories of fatty liver in dairy cows.

Category of fatty liver Incidence Country Breed of cows Reference

Moderate 48% England Holstein Reid, 1980

Severe 15%

Moderate 33% England Guernsey Reid, 1980

Severe 5%

Severe 15% Finland Ayrshire Gröhn et al., 1987

Moderate 65% France not reported Mazur et al., 1988

Severe 5%

Moderate 53% Germany not reported Schäfer et al., 1991

Severe 20%

Moderate 33% Japan Holstein Acorda et al., 1995

Severe 11%

Moderate 45% Netherlands not reported Jorritsma et al., 2000

Moderate 40% Netherlands not reported Jorritsma et al., 2001

Severe 14%

Moderate 20% United States not reported Gerlo� et al., 1986a

Severe 15%

Severe 24% United States not reported Herdt, 1991

From Bobe G et al. J Dairy Sci. 2004 Oct;87(10):3105-24.

Figure 4. 2-methyl-2-phenoxy-propionic acid chemicalstructure

and increased risk and severity of infectious diseasessuch as mastitis and metritis [28]. Declining fertilityin dairy cows is also suggested to arise from increasedaccumulation of triacylglycerol in the liver [23].Fatty liver has detrimental e�ects on cow health

status and productivity and its presence even if notclinically apparent can predispose to or worsen manypathological conditions. Therefore, treatment andprevention of excessive hepatic lipid accumulation isextremely important to obtain complete ad rapid re-covery from numerous diseases a�ecting dairy cows.

2-Methyl-2-Phenoxy-Propionic Acid

2-methyl-2-phenoxy-propionic acid (HepagenTM

- Fatro) (Fig 4), is a speci�c PPARα ago-nist, acting in the same fashion of natural lig-ands and human hypolipidemic drugs [37, 40, 39].2-methyl-2-phenoxy-propionic acid binds to PPARα,activates the PPAR-RXR complex and ultimatelyup-regulates the transcription of several genes in-volved in lipid and glucose metabolism. As a resultit promotes the activation of mitochondrial and per-

Figure 5. Hepagen mechanism of action

oxisomal β-oxidation pathways and liver gluconeoge-nesis (Fig 5).Enhanced lipid oxidation brings to the reduction

of triglyceride accumulation in hepatocytes, recoveryof liver functions and increased energy availabilityfor glucose production. Direct up-regulation of genesinvolved in gluconeogenesis intensi�es hepatic glucoseoutput and restores energy balance.The anti-in�ammatory properties of this PPARα

agonist further help cows, considering that in�am-matory conditions play a role in the pathogenesis offatty liver and many other diseases [38].2-methyl-2-phenoxy-propionic acid has also a

choleretic and colagogue e�ect (Fig. 6) extremelyuseful for the treatment of fatty liver [41]. Bile �owis decreased in cows with fatty liver. Increased con-centrations of bile constituents are toxic and increasethe production of free radicals, which cause in�am-mation and tissue damage[25].

4

Figure 6. Choleresis increase induced by2-methyl-2-phenoxy-propionic acid in comparison

with menbutone

2-methyl-2-phenoxy-propionic acid has beenshown e�ective for the treatment of fatty liver,ketosis, acidosis, digestive disorder, intoxicationsand impaired hepatic activity, and thanks to its spe-ci�c and broad-scale action on energy metabolism,choleresis and in�ammation represents a �rst line oftherapy for the treatment of metabolic disorders.

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