Structure and regulation of acetyl-CoA carboxylase genes of metazoa.

Acetyl-CoA carboxylase (ACC) plays a fundamental role in fatty acid metabolism. The reaction product, malonyl-CoA, is both an intermediate in the de novo synthesis of long-chain fatty acids and also a substrate for distinct fatty acyl-CoA elongation enzymes. In metazoans, which have evolved energy storage tissues to fuel locomotion and to survive periods of starvation, energy charge sensing at the level of the individual cell plays a role in fuel selection and metabolic orchestration between tissues. In mammals, and probably other metazoans, ACC forms a component of an energy sensor with malonyl-CoA, acting as a signal to reciprocally control the mitochondrial transport step of long-chain fatty acid oxidation through the inhibition of carnitine palmitoyltransferase I (CPT I). To reflect this pivotal role in cell function, ACC is subject to complex regulation. Higher metazoan evolution is associated with the duplication of an ancestral ACC gene, and with organismal complexity, there is an increasing diversity of transcripts from the ACC paraloges with the potential for the existence of several isozymes. This review focuses on the structure of ACC genes and the putative individual roles of their gene products in fatty acid metabolism, taking an evolutionary viewpoint provided by data in genome databases.

Characterisation of an N-terminal variant of acetyl-CoA carboxylase-alpha: expression in human tissues and evolutionary aspects.

mRNA encoding a variant acetyl-CoA carboxylase (ACC)-alpha isozyme, transcribed from a downstream promoter, PIII, was detected in human tissues. Such exon 5A-containing transcripts (E5A-mRNA) encode ACC-alpha with a distinct N-terminus, with 15/17 residues identical to those encoded by the ovine mRNA. In the current study we used antisera directed against the E5A N-terminus to verify that ovine E5A translates are present in tissues consistent with the distribution of E5A-mRNA. The presence of E5A alters the context of adjacent regulatory phosphorylation sites in E6, which may indicate altered regulation of activity for this isozyme. Sequences with high identity to the proximal promoter of PIII and E5A are present in the mouse and rat ACC-alpha genes, however, the coding region of E5A is not conserved, and E5A transcripts are not detected in tissues. Thus E5A must have been present in a common ancestor of rodents, primates, and ruminants, and has become nonfunctional in the former. A minor human PIII-derived mRNA containing an additional 111-bp sequence encoded by a downstream exon, E5B, was also detected. E5B encodes an in-frame stop-codon such that the E5A open-reading frame is terminated, however, ACC-alpha translation may be re-initiated from a downstream AUG in E6, potentially generating an isozyme lacking the N-terminal phosphorylation sites. Transcription of human ACC-alpha from at least three promoters and the potential to generate ACC-alpha isozymes with differential susceptibilities to phosphorylation indicate that the regulation of fatty acid synthesis in human tissues is likely to be complex.

Induction of transcripts derived from promoter III of the acetyl-CoA carboxylase-alpha gene in mammary gland is associated with recruitment of SREBP-1 to a region of the proximal promoter defined by a DNase I hypersensitive site.

ACC-alpha (acetyl-CoA carboxylase-alpha), a key regulator of fatty-acid metabolism, is encoded by mRNAs transcribed from three promoters, PI, PII and PIII, in the ovine genome. Enhanced expression of transcripts encoded by PIII in mammary gland during lactation is associated with alterations in chromatin structure that result in the detection of two DNase I hypersensitive sites, upstream of the start site. The most proximal site, located between -190 and -10, is characterized by the presence of an inverted-CCAAT box, C2 at -167, and E-boxes, E1 and E2, at -151 and -46. Deletion of these motifs, which bind nuclear factor-Y and upstream stimulatory factors respectively in gel-shift assays, attenuates the activity of luciferase reporter constructs in transfected cells. Chromatin immunoprecipitation demonstrated that these transcription factors were associated with PIII in vivo in both lactating and non-lactating mammary tissues. The basic helix-loop-helix-leucine zipper transcription factor, SREBP-1 (sterol-regulated-element-binding protein-1), transactivated PIII reporter constructs in transfected HC11 mammary cells, and this was dependent on the presence of E1, but not on C2 or E2. SREBP-1 was only associated with PIII in chromatin from lactating animals, which was coincident with a 4-fold increase in the precursor (125 kDa) form of SREBP-1 in microsomes and the appearance of the mature form (68 kDa) in the nucleus. SREBP-1 motifs are also present in the proximal region of PII, which is also induced in lactation. This indicates that SREBP-1 is a major developmental regulator of the programme of lipid synthesis de novo in the lactating mammary gland.

Growth hormone, acting in part through the insulin-like growth factor axis, rescues developmental, but not metabolic, activity in the mammary gland of mice expressing a single allele of the prolactin receptor.

The heterozygous prolactin (PRL) receptor (PRLR(+/-)) mouse fails to develop a fully functional mammary gland at the end of the first pregnancy and shows markedly impaired lobuloalveolar development and milk secretion in young females. PRL and GH, acting through the IGF system, have interactive effects to enhance epithelial cell survival. Thus, we propose that a reduction in the expression of the PRLR may lead to increased IGFBP-5 expression (proapoptotic) and that GH may rescue mammary development by increasing IGF-I, an important mitogen and survival factor for the mammary epithelium. Mammary IGF-binding protein-5 (IGFBP-5) concentrations and plasmin activity in PRLR(+/-) mice were increased on d 2 postpartum, indicative of increased cell death and extracellular matrix remodeling. After GH treatment, a restoration of mammary alveolar development and a reduction in the activities of IGFBP-5 and plasmin were observed. Despite the severely impaired mammary development in PRLR(+/-) mice, both mRNA and protein expression for caseins and acetyl-coenzyme A (acetyl-CoA) carboxylase and acetyl-CoA caboxylase-alpha mRNA increased at parturition, although not to the extent in wild-type animals. Surprisingly, GH treatment actually led to a further decrease in milk protein and acetyl-CoA carboxylase-alphaexpression when expressed per cell. This was confirmed by the smaller alveolar size, the relative paucity of milk in the mammary glands of GH-treated animals, and the inability of their pups to gain weight. In a subsequent study IGFBP-5 was administered to wild-type mice and produced a 45% decrease in mammary DNA content, a 30% decrease in parenchymal tissue, and impaired lactation. These results suggest that GH can improve mammary development in PRLR(+/-) mice, but that it fails to enhance metabolic activity. This may be due to the maintenance by GH/IGF-I of a proliferative, rather than a differentiative, phenotype.

Diet-induced obesity impairs mammary development and lactogenesis in murine mammary gland.

We have developed a mouse model of diet-induced obesity that shows numerous abnormalities relating to mammary gland function. Animals ate approximately 40% more calories when offered a high-fat diet and gained weight at three times the rate of controls. They exhibited reduced conception rates, increased peripartum pup mortality, and impaired lactogenesis. The impairment of lactogenesis involved lipid accumulation in the secretory epithelial cells indicative of an absence of copius milk secretion. Expression of mRNAs for beta-casein, whey acid protein, and alpha-lactalbumin were all decreased immediately postpartum but recovered as lactation was established over 2-3 days. Expression of acetyl-CoA carboxylase (ACC)-alpha mRNA was also decreased at parturition as was the total enzyme activity, although there was a compensatory increase in the proportion in the active state. By day 10 of lactation, the proportion of ACC in the active state was also decreased in obese animals, indicative of suppression of de novo fatty acid synthesis resulting from the supply of preformed fatty acids in the diet. Although obese animals consumed more calories in the nonpregnant and early pregnant states, they showed a marked depression in fat intake around day 9 of pregnancy before food intake recovered in later pregnancy. Food intake increased dramatically in both lean and obese animals during lactation although total calories consumed were identical in both groups. Thus, despite access to high-energy diets, the obese animals mobilized even more adipose tissue during lactation than their lean counterparts. Obese animals also exhibited marked abnormalities in alveolar development of the mammary gland, which may partially explain the delay in differentiation evident during lactogenesis.

Asymmetric expression of transcripts derived from the shared promoter between the divergently oriented ACACA and TADA2L genes.

The mammalian gene (ACACA) encoding acetyl-CoA carboxylase-alpha, a key regulatory enzyme of fatty acid synthesis, is transcribed from multiple promoters. We have delineated the 5' boundary of ACACA in four species (human, mouse, rat, and ovine). The 5' end of ACACA is located within a 600- to 700-bp CpG island encompassing a bidirectional promoter shared with the divergently oriented TADA2L, which encodes a component of chromatin-modifying complexes. In mouse and rat, this promoter, now referred to as Acaca PI, is located 43 kb upstream of the previously known regulatory regions. The shared promoter coregulates transcripts for TADA2L and ACACA in an asymmetric fashion in human and mouse tissues. A higher concentration of RNA polymerase II (Pol II) within the intergenic region in brain compared to liver of mouse reflects the greater abundance of the two transcripts in brain. The concentration of Pol II tracking downstream, which is lower than at the promoter, is not significantly different in either gene in the two tissues and does not reflect the 10- and >200-fold greater abundance of Tada2l and Acaca PI transcripts, respectively, in brain. Thus, regulation of clearance of Pol II from the promoter and the rate of elongation may therefore be determinants of the asymmetric expression of these transcripts.

Localization of messenger RNAs encoding enzymes associated with malonyl-CoA metabolism in mouse brain.

Malonyl-CoA acts a fuel sensor in the pancreas, liver and muscle. Similarly, malonyl-CoA is implicated in satiety regulation in the brain. Expression of genes encoding enzymes implicated in regulation of malonyl-CoA levels was examined in murine brain. Acetyl-CoA carboxylase (ACC) alpha-isoform, fatty acid synthase and malonyl-CoA decarboxylase are highly expressed in the hippocampus, habenula nucleus, cerebral cortex and areas of the hypothalamus, whereas the ACC-beta isoform and liver-type carnitine palmitoyltransferase I (CPTI-L) are principally expressed in the choroid plexus. Thus different brain regions appear to be functionally configured primarily for either fatty acid synthesis or beta-oxidation. Localization of transcripts encoding enzymes involved in fatty acid synthesis and beta-oxidation in distinct nuclei of the hypothalamus supports a role for malonyl-CoA as a potential effector of satiety.

Insulin-like growth factor binding protein-5 (IGFBP-5) induces premature cell death in the mammary glands of transgenic mice.

We have previously demonstrated that IGFBP-5 production by mammary epithelial cells increases dramatically during involution of the mammary gland. To demonstrate a causal relationship between IGFBP-5 and cell death we created transgenic mice expressing IGFBP-5 in the mammary gland using a mammary-specific promoter, beta-lactoglobulin. DNA content in the mammary glands of transgenic mice was decreased as early as day 10 of pregnancy. Histological analysis indicated reduced numbers of alveolar end buds, with decreased ductal branching. Transgenic dams produced IGFBP-5 in their milk at concentrations similar to those achieved at the end of normal lactation. Mammary cell number and milk synthesis were both decreased by approximately 50% during the first 10 days of lactation. BrdU labelling was decreased, whereas DNA ladders were increased in transgenic animals on day 1 of lactation. On day 2 postpartum, the epithelial invasion of the mammary fat pad was clearly impaired in transgenic animals. The concentrations of the pro-apoptotic molecule caspase-3 and of plasmin were both increased in transgenic animals whilst the concentrations of 2 prosurvival molecules Bcl-2 and Bcl-x(L)were both decreased. In order to examine whether IGFBP-5 acts by inhibiting the survival effect of IGF-I we examined IGF receptor phosphorylation and Akt phosphorylation and showed that both were inhibited. We attempted to "rescue" the transgenic phenotype by using growth hormone to increase endogenous IGF-I concentrations or by implanting minipumps delivering an IGF-1 analogue, R(3)-IGF-1, which binds weakly to IGFBP-5. Growth hormone treatment failed to affect mammary development suggesting that increased concentrations of endogenous IGF-1 are insufficient to overcome the high concentrations of IGFBP-5 produced by these transgenic animals. In contrast mammary development (gland weight and DNA content) was normalised by R3-IGF-I although milk production was only partially restored. This is the first demonstration that over-expression of IGFBP-5 can lead to; impaired mammary development, increased expression of the pro-apoptotic molecule caspase-3, increased plasmin generation and decreased expression of pro-survival molecules of the Bcl-2 family. It clearly demonstrates that IGF-I is an important developmental/survival factor for the mammary gland and, furthermore, this cell death programme may be utilised in a wide variety of tissues.