Increase of energy balance significantly alters major lipogenic gene expression in lactation ewes.

The objective of the present study was to examine changes observed in the expression of cytosolic NADP isocitrate dehydrogenase (ICDH) and glucose 6-phosphate dehydrogenase (G6PD) genes, the major implicated genes in ruminant lipogenesis in terms of produce NADPH, during the early post-weaning period in dairy ewes in respect to energy intake, and to further correlate the noted changes with their respective enzymatic activities. A total of 21 subcutaneous adipose tissue samples were obtained from seven lactating (2nd lactation period) dairy ewes of the Chios breed. Adipose tissue samples were taken from the tail head region at weeks 1, 2, and 4 after weaning (45 days after parturition). Dairy ewes were in negative energy balance during weeks 1 and 2 after weaning and they moved into a strong positive energy balance at week 4 after weaning. Expression of ICDH and G6PD genes and their respective enzymatic activity was determined. Results showed that both genes' expression and enzymatic activities were significantly minimal at week 1 after weaning, reaching a maximum level at week 4 after weaning (P < 0.05). A 3.5-fold and a 5-fold increase of G6PD and ICDH mRNA levels were observed, respectively. Concerning their respective enzymatic activities, a 5.5-fold and 2-fold increase was noted, respectively. A positive correlation was found between ICDH and G6PD gene expression (P < 0.001) indicating a synchronized response to energy intake changes. Almost similar changes were observed for enzymatic activities, rendering these enzymes as potential biochemical markers of ovine lipogenesis.

The resurgence of Hormone-Sensitive Lipase (HSL) in mammalian lipolysis.

The ability to store energy in the form of energy-dense triacylglycerol and to mobilize these stores rapidly during periods of low carbohydrate availability or throughout the strong metabolic demand is a highly conserved process, absolutely essential for survival. In the industrialized world the regulation of this pathway is viewed as an important therapeutic target for disease prevention. Adipose tissue lipolysis is a catabolic process leading to the breakdown of triacylglycerols stored in fat cells, and release of fatty acids and glycerol. Mobilization of adipose tissue fat is mediated by the MGL, HSL and ATGL, similarly functioning enzymes. ATGL initiates lipolysis followed by the actions of HSL on diacylglycerol, and MGL on monoacylglycerol. HSL is regulated by reversible phosphorylation on five critical residues. Phosphorylation alone, however, is not enough to activate HSL. Probably, conformational alterations and a translocation from the cytoplasm to lipid droplets are also involved. In accordance, Perilipin functions as a master regulator of lipolysis, protecting or exposing the triacylglycerol core of a lipid droplet to lipases. The prototype processes of hormonal lipolytic control are the ß-adrenergic stimulation and suppression by insulin, both of which affect cytoplasmic cyclic AMP levels. Lipolysis in adipocytes is an important process in the management of body energy reserves. Its deregulation may contribute to the symptoms of type 2 diabetes mellitus and other pathological situations. We, herein, discuss the metabolic regulation and function of lipases mediating mammalian lipolysis with a focus on HSL, quoting newly identified members of the lipolytic proteome.

Enzymatic and mRNA Transcript Response of Ovine 6-Phosphogluconate Dehydrogenase (6PGD) in Respect to Different Milk Yield.

Ovine 6-phosphogluconate dehydrogenase (6PGD) is an enzyme of the pentose phosphate pathway, providing the necessary compounds of NADPH for the synthesis of fatty acids. Much of research has been conducted both on enzymatic level and on molecular level. However, to our knowledge, any correlation between enzymatic activity and 6PGD gene expression pattern related to different physiological stages has not been yet reported. With this report, we tried to highlight if any correlation between enzymatic activity and expression of ovine 6PGD gene exists, in respect to different milk yield. According to the determined enzymatic activities and adipocytes characteristics, ewes with low milk production possessed a greater (P ≤ .001) 6PGD activity and larger adipocytes than the highly productive ewes. Although 6PGD expression pattern was higher in low milk yield ewes than in ewes with high milk production, this difference was not found statistically significant. Thus, 6PGD gene expression pattern was not followed by so rapid and great/sizeable changes as it was observed for its respective enzymatic activity, suggesting that other mechanisms such as post translation regulation may be involved in the regulation of the respective gene.

Enzymatic and mRNA transcript response of ovine 6-phosphogluconate dehydrogenase (6PGD) in respect to different weights from weaning to four months of age.

Ovine 6-phosphogluconate dehydrogenase (6PGD), an enzyme of the pentose phosphate pathway, provides the necessary compounds of NADPH for the synthesis of fatty acids. Much of research has been conducted not only on the enzymatic level, but also on molecular level elucidating its cDNA sequence. Herein, we tried to elucidate if any correlation between enzymatic activity and expression of ovine 6PGD gene exists, in respect to two different weights from weaning to 4 months old. 18 male and 16 female lambs of Chios breed were randomly selected after weaning and assigned to two groups based on sex in a different experimental open-plan shed. Two subgroups were defined in each sex and they were slaughtered at 25 kg and 30 kg, respectively. Samples of adipose tissue (tail, perirenal and shoulder site) were collected and 6PGD enzymatic activity, gene expression, and characteristics of adipocytes were determined. According to the determined data, tail subcutaneous adipose tissue matures later than the others examined tissues and has a diminished lipogenic activity. A 6PGD gene expression pattern was not followed by analogous changes of its enzymatic activity, suggesting that other mechanisms such as post transcription or/and post translation regulation may be involved.

Molecular study of ovine glucose 6-phosphate dehydrogenase gene expression in respect to different energy intake.

Glucose 6-phosphate dehydrogenase (G6PD) plays an important role in a ruminant's metabolism catalyzing the first committed reaction in the pentose phosphate pathway as it provides necessary compounds of NADPH for the synthesis of fatty acids. The cloning of ovine (Ovis aries) G6PD gene revealed the presence of two cDNA transcripts (oG6PD(A) and oG6PD(B)), with oG6PD(B) being a product of alternative splicing and with no similarity to any other previously reported G6PD transcript. Here, we attempt to study the effect of energy balance in ovine G6PD transcript expression, trying simultaneously to find out any potential physiological role of the oG6PD(B) transcript. Changes of energy balance that lead to synergistic changes in the expression of both transcripts, but in opposite directions and not in a proportional way. Negative energy balance favours the presence of the oG6PD(B) transcript leading to a significant increase of its expression, compared to oG6PD(A) expression (P<0.05). In contrast, positive energy balance leads to a significant increase of oG6PD(A) compared to oG6PD(B) expression (P<0.05). In either condition oG6PD(B) expression is unchanged. Regression analysis showed that there is an energy balance threshold where the expression of both transcripts shows no change.

The ovine plasminogen activator inhibitors type 1 and type 2 cDNAs: molecular cloning, characterization and expression in various tissues.

Two serine proteases, urokinase and tissue type, control the activation of plasminogen to plasmin. These proteases are in turn specifically inhibited by plasminogen activator inhibitors type 1 and 2 (PAI-1 and PAI-2), both of which belong to the serine protease inhibitor (serpin) superfamily. Very little information is available on the role of PAI-1 and PAI-2 in ruminants, in mammary gland involution and in the adipose tissue. In this paper we describe the isolation of the full-length cDNAs of ovine PAI-1 and PAI-2 using a polymerase chain reaction based strategy. The ovine PAI-1 cDNA comprised of 1460bp and it is characterized by a coding region of 1209bp, and 5'- and 3'-UTR regions of 147 and 104bp, respectively. The deduced amino acid sequence consists of 402 amino acids. The ovine PAI-2 cDNA is comprised of 2128bp and it is characterized by a coding region of 957bp and 5'- and 3'-UTR regions of 58 and 819bp respectively. The deduced amino acid sequence consists of 416 amino acids. Three-dimensional models of the putative protein products of both cDNAs showed that the proteins bear a high similarity with their human counterparts. Real-time PCR revealed that the two inhibitors were predominantly expressed in the ovine mammary gland and adipose tissue. Furthermore, PAI-1 and PAI-2 mRNA levels were higher in the involuting mammary tissue and the adipose tissue obtained from non-lactating ewes compared to the corresponding values in tissues obtained from lactating ewes. These data are consistent with the notion that the plasminogen activation cascade plays a key role in involution of the mammary gland. The upregulation of expression of both inhibitors in the adipose tissue during the non-lactating period is a rather enigmatic observation. However, the expression of both inhibitors (PAI-1 and PAI-2) together with that of urokinase type plasminogen activator and its receptor previously reported by our group, strengthen the suggestion that the adipose tissue functions as an endocrine besides an energy storage organ.

The ovine urokinase plasminogen activator and its receptor cDNAs: molecular cloning, characterization and expression in various tissues.

The activation of plasminogen plays a crucial role in a variety of extracellular proteolytic events such as, fibrinolysis, cell migration, ovulation, involution of the mammary gland and the activation of other protease classes and growth factors. In this paper we describe the isolation of the full-length cDNAs of ovine urokinase plasminogen activator (u-PA) and its receptor (u-PAR) using a polymerase chain reaction based strategy. The ovine u-PA cDNA comprised of 2350 bp and it is characterized by a coding region of 1302 bp, and 5'- and 3'-UTR regions of 129 and 919 bp, respectively. The deduced amino acid sequence consists of 433 amino acids. The ovine u-PAR cDNA is comprised of 1247 bp and it is characterized by a coding region of 957 bp and 5'- and 3'-UTR regions of 44 and 246 bp respectively. The deduced amino acid sequence consists of 318 amino acids. Three-dimensional models of the putative protein products of both cDNAs showed that the proteins bear a high similarity with their human counterparts. Real-time PCR revealed high levels of u-PA expression in the adipose tissue, followed by that in mammary gland and kidney. Lower levels of expression were detected in the adrenal glands, heart, ovaries, spleen, liver and cerebellum. A similar pattern was observed in u-PAR expression with noticeably lower levels of expression in heart, liver and cerebellum. To the best of our knowledge, this is the first paper reporting expression of u-PA and u-PAR in the adipose tissue. These data strengthen the suggestion that adipose tissue functions as an endocrine organ besides an energy storage organ. Furthermore, u-PA and u-PAR mRNA levels were 7 and 8.5 fold higher respectively in involuting mammary tissue obtained from non-lactating ewes compared to that detected in mammary tissue obtained from lactating ewes. These data are consistent with the notion that upregulation of u-PA and u-PAR expression may play a key role in the process of involution of the mammary gland.

Cloning and functional characterization of the ovine malic enzyme promoter.

While in human and rodents lipogenesis occurs predominantly in the liver, adipose tissue is the major site in ruminants. Here we report the nucleotide sequence of the 5'-flanking region of the ovine malic enzyme gene (ME1). The ME1 promoter is located within a GC-rich region fulfilling the criteria of CpG islands and lacks a TATA-box. Deletion analysis identified a region (-231/-170) that suppressed promoter activity in luciferase assays in HepG2 hepatoma cells but not in 3T3-L1 adipocytes. This region contains a putative triiodothyronine response element (T3RE) that differs from the human ME1 T3RE by two nucleotides. When the human ME1 T3RE was introduced into the ovine ME1 promoter context, transcriptional activity was increased in the hepatic cell lines HepG2 and H4IIE but not in differentiated 3T3-L1 cells. Our results suggest that the sequence of the T3RE in the ME1 promoter determines differences in the tissue/species activity of malic enzyme in ruminants and human.

Cloning and functional characterization of the 5' regulatory region of ovine Hormone Sensitive Lipase (HSL) gene.

Hormone Sensitive Lipase (HSL) catalyzes the rate-limiting step in the mobilization of fatty acids from adipose tissue, thus determining the supply of energy substrates in the body. HSL enzymatic activity is increased by adrenergic agonists, such as catecholamines and glucagons, which induce cyclic AMP (cAMP) intracellular production, subsequently followed by the activation of Protein Kinase A (PKA) and its downstream signaling cascade reactions. HSL constitutes the critical enzyme in the modulation of lipid stores and the only component being subjected to hormonal control in terms of the recently identified Adipose Triglyceride Lipase (ATGL). In order to acquire detailed knowledge with regard to the mechanisms regulating ovine HSL (ovHSL) gene transcription activity, we initially isolated and cloned the 5' proximal and distal promoter regions through a genome walking approach, with the utilization of the already characterized ovHSL cDNAs. As evinced by BLAST analysis and a multiple alignment procedure, the isolated genomic fragment of 2.744 kb appeared to contain the already specified 5'-untranslated region (5'-UTR), which was interrupted by a relatively large intron of 1.448 kb. Regarding the upstream remaining part of 1.224 kb, it was demonstrated to represent a TATA-less promoter area, harboring several cis-regulatory elements that could be putatively recognized by relatively more general transcription factors, mainly including Stimulating protein 1 (Sp1), CCAAT-box Binding Factors (CBFs), Activator Protein 2 (AP2) and Glucocorticoid Receptor (GR), as well as other cis-acting regions denominated as Insulin Response Element (IRE), Glucose Response Element (GRE), Fat Specific Element (FSE) and cAMP Response Element (CRE), which could likely function in a nourishment (i.e. glucose)-/hormone-dependent fashion. When different genomic fragments were directionally (5' to 3') cloned into a suitable reporter vector upstream of a promoter-less luciferase gene and transiently transfected into 3T3-L1 (mouse fibroblasts) as well as T24 (human bladder cancer) cell lines, strong promoter activities were unambiguously detected, with the -140/+18 nucleotide sequence bearing the highest transcriptional response, thus indicating that the 1.224 kb 5' flanking region, isolated by genome walking, veritably contains the ovHSL gene promoter. Of particular significance are the observations that the functional promoter fragments could trigger the transcriptional activity of luciferase gene only under high concentration of glucose conditions in both cell lines.

Molecular cloning and characterization of the sheep malic enzyme cDNA.

Malic enzyme catalyzes decarboxylation of malate to pyruvate and CO(2), providing de novo biosynthesis of fatty acids with NADPH. Since lipogenesis in ruminants, contrarily to some monogastric species like human and rodents, occurs predominantly in adipose tissue, the activity of many lipogenic enzymes is higher in adipose tissue compared to liver. Expression of malic enzyme is regulated by nutrition; refeeding after a period of starvation results to an induction of the enzyme. Here we present the nucleotide sequence of two transcripts of the ovine cytosolic malic enzyme gene that differ at the length of the 3' UTR. These are the first published cDNA sequences for ruminant species and share high similarity with the corresponding sequences of other species. Malic enzyme mRNA was present in every ovine tissue that was examined. In agreement with the fact that adipose tissue is the major lipogenic site for ruminants, mRNA levels in adipose tissue were higher than in liver. Refeeding after two weeks of caloric restriction resulted in a two-fold increase of the mRNA level of malic enzyme in adipose tissue.

Cloning and functional characterization of the ovine Hormone Sensitive Lipase (HSL) full-length cDNAs: an integrated approach.

Hormone Sensitive Lipase (HSL) is a highly regulated enzyme that mediates lipolysis in adipocytes. HSL enzymatic activity is increased by adrenergic agonists, such as catecholamines and glucagons, which induce cyclic AMP (cAMP) intracellular production, subsequently followed by the activation of Protein Kinase A (PKA) and its downstream signalling cascade reactions. Since HSL constitutes the key enzyme in the regulation of lipid stores and the only enzyme being subjected to hormonal regulation [in terms of the recently identified Adipose Triglyceride Lipase (ATGL)], the ovine Hormone Sensitive Lipase (ovHSL) full-length cDNA clones were isolated, using a Polymerase Chain Reaction-based (PCR) strategy. The two isolated isoforms ovHSL-A and ovHSL-B contain two highly homologous Open Reading Frame (ORF) regions of 2.089 Kb and 2.086 Kb, respectively, the latter having been missed the 688th triplet coding for glutamine (DeltaQ(688)). The putative 695 and 694 amino acid respective sequences bear strong homologies with other HSL protein family members. Southern blotting analysis revealed that HSL is represented as a single copy gene in the ovine genome, while Reverse Transcription-PCR (RT-PCR) approaches unambiguously dictated its variable transcriptional expression profile in the different tissues examined. Interestingly, as undoubtedly corroborated by both RT-PCR and Western blotting analysis, ovHSL gene expression is notably enhanced in the adipose tissue during the fasting period, when lipolysis is highly increased in ruminant species. Based on the crystal structure of an Archaeoglobus fulgidus enzyme, a three-dimensional (3D) molecular model of the ovHSL putative catalytic domain was constructed, thus providing an inchoative insight into understanding the enzymatic activity and functional regulation mechanisms of the ruminant HSL gene product(s).

Cloning, characterization and computational analysis of the 5' regulatory region of ovine glucose 6-phosphate dehydrogenase gene.

To better understand the structure and the function of ovine glucose 6-phosphate dehydrogenase (G6PD) promoter region, a genome-walking procedure was followed to isolate and sequence a 1628 bp fragment, containing the 5' regulatory region of the G6PD gene. In silico analysis of the sequence showed many conserved blocks and features with other known mammalian G6PD promoter regions. The analysis also revealed the presence of one TATA box, three GC boxes, two E-boxes and several binding sites for Stimulating Protein 1 (Sp1) and Activator Protein 2 (AP2). Moreover, elements involved in the regulation of lipogenesis like USF (Upstream stimulating factor), HSF (Heat Shock Factor), F2F (Prolactin receptor), RAR (Retinoid Acid Receptor), STRE (STress Response Element), RORa (Retinoid related Orphan Receptor alpha), GATA (GATA binding factor), RFX (Regulatory Factor X), SREBP (Sterol Regulatory Element Binding Protein), MEP (Metal Element Protein), CREB (insulin receptor), PRE (Progesterone receptor), and HNF4 (Hepatic Nuclear Factor 4) were detected. The most important regulatory motifs were found to be conserved as compared to those in human and mouse counterparts. However, some differences were noted, likely indicating differences in the transcription regulation of G6PD gene between ruminant and non-ruminant species.

Cloning and characterization of an alternative transcript of ovine glucose 6-phosphate dehydrogenase gene: comparative approach between ruminant and non-ruminant species.

Glucose 6-phosphate dehydrogenase (G6PD) plays an important role in ruminant's lipogenesis, as it provides necessary compounds of NADPH for the synthesis of fatty acids catalyzing the first committed reaction in the pentose phosphate pathway. In this work the full length ovine glucose 6-phosphate dehydrogenase cDNA was isolated using a polymerase chain reaction based strategy. Two isoforms (OG6PDA and OG6PDB) were detected encoding a protein of 515 and 524 amino acids, respectively. Both deduced amino acid sequences reveal a well conserved protein containing all the important residues for its catalytic role. The extra nine amino acids encoded by OG6PDB cause a frameshift in the polypeptide chain resulting in changes around the area of the potential substrate binding site. A three-dimensional model of ovine G6PD protein shows that this frameshift cause structural changes in the catalytic binding "pocket" of the molecule. Southern blot and RT analysis revealed that ovine G6PD appears as a single copy gene while it is expressed, with slight variability, in all tissues analyzed. Moreover, expression analysis of the ovine G6PD isoforms showed that OG6PDB is expressed only in tissues where lipogenesis is high in ruminants. Thus, we hypothesize that in ruminants G6PD may be regulated by the ratio of the two transcripts, according to the existence stimulus.