Emmanuel Rogdakis

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 β-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.

Effects of caponization on growth performance, carcass composition and meat quality of males of a layer line

The present experiment was conducted in order to evaluate the effects of caponization on growth, carcass composition and meat quality of males of a layer line reared until the 34th week of age. Two hundred and fifty males of a layer line were purchased and randomly divided in two equal groups: intact males and capons. Caponization was conducted at 45 days of age. Three slaughters were performed at the ages of 26, 30 and 34 weeks of age. Caponization did not affect feed intake and final live weight. Capons had a heavier breast and lighter leg than intact males. Lipid accumulation was enhanced by the caponization and fat was stored mainly at the fat pad and the skin of the commercial parts excluding the drumstick. The Pectoralis major muscle of capons had higher intramuscular fat content, lightness (L) and yellowness (b*) values and lower redness values (a*). In conclusion, caponization could be applied to a layer genotype in order to produce commercial chicken meat.

Genetic modelling of test day records in dairy sheep using orthogonal Legendre polynomials

Test day milk yields of three lactations in Sfakia sheep were analyzed fitting a random regression (RR) model, regressing on orthogonal polynomials of the stage of the lactation period, i.e. days in milk. Univariate (UV) and multivariate (MV) analyses were also performed for four stages of the lactation period, represented by average days in milk, i.e. 15, 45, 70 and 105 days, to compare estimates obtained from RR models with estimates from UV and MV analyses. The total number of test day records were 790, 1314 and 1041 obtained from 214, 342 and 303 ewes in the first, second and third lactation, respectively. Error variances and covariances between regression coefficients were estimated by restricted maximum likelihood. Models were compared using likelihood ratio tests (LRTs). Log likelihoods were not significantly reduced when the rank of the orthogonal Legendre polynomials (LPs) of lactation stage was reduced from 4 to 2 and homogenous variances for lactation stages within lactations were considered. Mean weighted heritability estimates with RR models were 0.19, 0.09 and 0.08 for first, second and third lactation, respectively. The respective estimates obtained from UV analyses were 0.14, 0.12 and 0.08, respectively. Mean permanent environmental variance, as a proportion of the total, was high at all stages and lactations ranging from 0.54 to 0.71. Within lactations, genetic and permanent environmental correlations between lactation stages were in the range from 0.36 to 0.99 and 0.76 to 0.99, respectively. Genetic parameters for additive genetic and permanent environmental effects obtained from RR models were different from those obtained from UV and MV analyses.

Cellularity and enzymatic activity of adipose tissue in the Karagouniko dairy breed of sheep from birth to maturity

SUMMARY The cellular and enzymatic characteristics of dissected subcutaneous and perirenal adipose tissues were examined in a total of 102 male and female animals from commerical flocks of the Karagouniko dairy sheep. The animals were slaughtered in groups of three males and three females at 45-day intervals from birth to 720 days of age. The following determinations were made on each animal: (1) subcutaneous and perirenal chemical fat, (2) fat cell size and number and (3) the activities of NADP-isocitrate dehydrogenase and 6-phosphogluconate dehydrogenase in subcutaneous and perirenal adipose tissues. Subcutaneous adipose tissue expanded by a combination of fat cell hypertrophy and hyperplasia during the accelerating phase of the growth curve of chemical fat, after which the fat expansion was accomplished primarily by hyperplasia. The changes in perirenal chemical fat weight with increasing age were primarily due to hypertrophy of fat cells during the accelerating phase of the curve of chemical fat growth and due to a combination of hypertrophy and hyperplasia during the diminishing phase of the curve. The enzyme activities were higher in subcutaneous than in perirenal adipose tissue, with the NADP-isocitrate dehydrogenase being the most active enzyme in both tissues and sexes. The changes in enzyme activities in subcutaneous adipose tissue with increasing age were parallel to the changes in the rate of chemical fat weight during growth. ZUSAMMENFASSUNG: Zellularitaet und enzymatische Aktivitaet des Fettgewebes beim Karagouniko Milchschaf von der Geburt bis zur Reife. Es wurden die Anzahl und die Groesse der Fettzellen sowie die Aktivitaeten der NADP-Isocitrat-Dehydrogenase und der 6-Phosphogluconat Dehydrogenase in subcutanen und perirenalen Fettgewebe des Schafes waehrend des Wachstums undersucht. Das Tiermaterial bestand aus 51 maennlichen und 51 weiblichen Schafen der Karogouniko Milchrasse. Die Schafe wurden in Gruppen bestehend aus 3 maennlichen und 3 weiblichen Tieren in 45-taegigen Abstaenden von der Geburt bis zum Alter von 720 Tagen geschlachtet. Es wurden die folgenden Merkmale erfasst: Chemisches Fett, Anzahl and Groesse der Fettzellen, Aktivitaeten der gennanten Enzyme. Das subcutane Fettgewebe wuchs durch eine Kombination von Hypertrophie und Hyperplasia waehrend der beschleunigten Phase der Wachstumskurve fuer das chemische Fett. Nach dieser Wachstumsphase, das weitere Wachstum des subcutanen Fettgewebes kam vorwiegend durch Hyperplasia der Fettzellen zustande. Das perirenale Fettgewebe wuchs zunaechst durch Hypertrophie der Fettzellen und anschliessend durch eine Kombinationn von Hypertrophie und Hyperplasia. Die Aktivitaet der NADP-Isocitrat-Dehydrogenase war hoeher als die Aktivitaet der 6-Phosphogluconat-Dehydronase in beiden Geweben und Geschlechtern waehrend der gesamten Versuchsdauer. Die Aenderungen der Enzymaktivitaet waehrend des Wachstums stimmten gut ueberein mit den entsprechenden Aenderungen der taeglichen Wachstumstrate der subcutanen Fettgewebes.

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).

Effects of caponization on fat metabolism–related biochemical characteristics of broilers

A two-trial experiment was conducted to evaluate the effects of caponization on fat metabolism-related biochemical characteristics of broilers. Male Redbro broilers were purchased, caponized at 3 weeks and reared until either the 18th (Trial 1) or the 24th (Trial 2) week. In Trial 1, five slaughters were performed at 6, 9, 12, 15 and 18 weeks of age while in Trial 2 one slaughter at the end of the experiment (24 weeks). In each slaughter, the abdominal adipose tissue cellularity, the NADP dehydrogenase activity in the liver and some serum lipoproteins concentrations were assessed. Caponization had a marked effect on the adipocyte volume and NADP-malate dehydrogenase activity in the liver at 24 weeks but it did not affect adipocyte number or the activity of NADP-isocitrate dehydrogenase activity at any age (p < 0.05). Regarding the lipoproteins, cholesterol and HDL-cholesterol were elevated in capon serum at 18 weeks of age while no difference was detected in the triglyceride concentration at any age. In conclusion, a relationship between fat deposition at the phenotypic level and the level of lipogenic enzymes and lipoproteins capons was established but not as pronounced as expected as some parameters displayed a constant increasing pattern while others did not.

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 ruminants 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.

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.

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

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.