Palmitic acid in chicken granulosa cell death-lipotoxic mechanisms mediate reproductive inefficacy of broiler breeder hens.

In vivo and in vitro approaches were used to elucidate mechanisms of palmitate-induced cytotoxicity of follicle granulosa cells in fuel-overloaded broiler hens. In contrast to their energy-restricted counterparts, broiler breeder hens fed ad libitum for 2 wk had dyslipidemia, atresia within hierarchical ovarian follicles, and a 34% reduction in egg production (P < 0.05). Based on vital staining of freshly isolated granulosa cells with annexin V/propidium iodide, there were increases in apoptosis consistent with suppressed Akt activation (P < 0.05). Supplementing primary granulosa cell cultures with 0.5 mM palmitate for 48 or 96 h increased apoptosis (P < 0.05). Palmitate-induced cell death was accompanied by increased acyl-CoA oxidase, carnitine palmitoyl transferase-1, serine palmitoyl transferase, and sphingomyelinase transcripts and increased concentrations of proinflammatory interleukin-1ß (P < 0.05). Triacsin-C inhibition of fatty acyl-CoA synthesis blunted interleukin-1ß production and rescued granulosa cultures from palmitate-induced cell death. That there was partial to complete prevention of cell death with addition of the free radical scavenger pyrrolidine dithiocarbamate, the sphingomyelinase inhibitor imipramine, or the de novo ceramide synthesis inhibitor fumonisin B1, supported the notion that palmitate-induced granulosa cell cytotoxicity operated through a palmitate-derived metabolite. Palmitoyl-CoA may be channeled into ß-oxidation and/or into bioactive metabolites that increase free radical generation, an inflammatory response, and ceramide production. In conclusion, palmitate-derived metabolites activated apoptotic machinery in avian granulosa cells, which caused ovarian follicular atresia and reduced egg production in fuel-overloaded broiler breeder hens.

Ceramide accumulation and up-regulation of proinflammatory interleukin-1ß exemplify lipotoxicity to mediate declines of reproductive efficacy of broiler hens.

The study was conducted to delineate fundamental mechanisms that initiate the deleterious effect of fuel overloading on reproductive efficacy of broiler breeder hens. Sixty hens at age 26 wk were fed recommended amounts of feed (160 g/d per hen) or allowed voluntary feeding (approximately 30% more than restriction). At age 35 and 50 wk, hens were sampled for further analyzes. Voluntary feeding resulted in poor egg production, high rate of mortality, and abnormal ovarian structure (mainly overt hierarchical follicle atresia at age 35 wk and ovarian involution at age 50 wk). In contrast to feed-restricted hens, voluntary feeding also induced metabolic dysregulations that comprised enhanced adiposity; hepatic triacylglycerol accumulation; and elevated concentrations of plasma glucose, NEFAs, very low density lipoprotein, triacylglycerol, phospholipids, and sphingomyelin (P < 0.05). Furthermore, hepatic and circulating ceramide and sphingomyelin accumulation, and up-regulation of proinflammatory IL-1ß expression in liver and adipose tissues (P < 0.05) systemically manifested the development of lipotoxicity in feed-satiated hens. Lipotoxicity leading to impaired ovarian dysfunctions, including follicle atresia, ovarian regression, and a decline of circulating estradiol levels (P < 0.05) in feed-satiated hens, was further exemplified by ceramide accumulation and up-regulation of IL-1ß, serine palmitoyltransferase, and sphingomyelinase transcript abundance, but suppressed protein kinase Akt activation (P < 0.1 to 0.05) within the hierarchical follicles. This study provides the first in vivo evidence of the actions of ceramide and IL-1ß in mediating overfeeding-induced follicle atresia and progression of ovarian involution in broiler hens.

Cancer-associated lactate dehydrogenase is a tyrosylphosphorylated form of human LDH-A, skeletal muscle isoenzyme.

Cancer-associated lactate dehydrogenase is a tyrosylphosphorylated form of human skeletal muscle isoenzyme, since the partial amino acid sequences of human liver LDH-K/A protein were found to be identical with the known primary structure of human LDH-A isoenzyme and the LDH-A isoenzymes from human placenta and bovine muscle were shown to be tyrosylphosphorylated. This tyrosylphosphorylated LDH-K/A protein was also found to be complexed with 21 kD, 30 kD, and 56 kD proteins.

Protein structure and gene organization of mouse lactate dehydrogenase-A isozyme.

The complete covalent structure of the 331 amino acids of mouse lactate dehydrogenase (LDH) A4 isozyme has been determined by sequence analyses of both the protein and the genomic DNA. The mouse LDH-A gene spans a length of at least 7000 bases from the translation initiation codon ATG to the end of the 3' untranslated region, and it contains six introns that interrupt the protein-coding sequence. The relationships between the exon-intron organization of LDH-A gene and the structural-functional domains of the protein are discussed.

Unique cholecystokinin peptides isolated from guinea pig intestine.

Fractionation on Sephadex G50 gel of methanol extracts of guinea pig intestine reveals two molecular forms of cholecystokinin (CCK) of about equal abundance. One elutes at the position of CCK8 while the other elutes at a position intermediate between CCK33 and CCK8. Purification and sequencing of these peptides identify them as CCK8 and CCK22, respectively. Guinea pig CCK8 differs from other mammalian CCK octapeptides isolated thus far in that there is a valine substituted for methionine at position 6 from the C-terminus. In addition to the substitution in CCK8, serine is substituted for asparagine in position 22, glycine for serine in position 19, and asparagine for serine in position 15 from the C-terminus compared to the pig sequence. HPLC separation on a C18 column yields two peaks each of CCK8 and of CCK22 in pig intestinal tissue obtained from a commercial supplier. The two CCK8 peptides have identical amino acid sequences as do the two CCK22 peptides. The CCK22 peptides are equally bioactive in the guinea pig pancreatic acinar cell assay but are about 10-fold less potent than synthetic CCK8(s). One of the guinea pig CCK8 peptides is fully bioactive whereas the other is about 50-fold less potent compared to synthetic CCK8(s).

Electron spin resonance for detecting polyadenylate tracts in RNA's.

Electron spin resonance is used to detect RNA's that contain polyadenylate tracts. The method depends on the ability of RNA's that contain polyadenylate sequences to associate with poly(2'-deoxy-2'-fluoro)uridylic acid, which has been spin-labeled with 4-(2-iodoacetamido)-2,2,6,6-tetramethylpiperidinooxyl. The formation of the hybridization product can be detected by monitoring the decrease in mobility of the spin probe.