The roles of DGAT1 and DGAT2 in human myotubes are dependent on donor patho-physiological background.

The roles of DGAT1 and DGAT2 in lipid metabolism and insulin responsiveness of human skeletal muscle were studied using cryosections and myotubes prepared from muscle biopsies from control, athlete, and impaired glucose regulation (IGR) cohorts of men. The previously observed increases in intramuscular triacylglycerol (IMTG) in athletes and IGR were shown to be related to an increase in lipid droplet (LD) area in type I fibers in athletes but, conversely, in type II fibers in IGR subjects. Specific inhibition of both diacylglycerol acyltransferase (DGAT) 1 and 2 decreased fatty acid (FA) uptake by myotubes, whereas only DGAT2 inhibition also decreased fatty acid oxidation. Fatty acid uptake in myotubes was negatively correlated with the lactate thresholds of the respective donors. DGAT2 inhibition lowered acetate uptake and oxidation in myotubes from all cohorts whereas DGAT1 inhibition had no effect. A positive correlation between acetate oxidation in myotubes and resting metabolic rate (RMR) from fatty acid oxidation in vivo was observed. Myotubes from athletes and IGR had higher rates of de novo lipogenesis from acetate that were normalized by DGAT2 inhibition. Moreover, DGAT2 inhibition in myotubes also resulted in increased insulin-induced Akt phosphorylation. The differential effects of DGAT1 and DGAT2 inhibition suggest that the specialized role of DGAT2 in esterifying nascent diacylglycerols and de novo synthesized FA is associated with synthesis of a pool of triacylglycerol, which upon hydrolysis results in effectors that promote mitochondrial fatty acid oxidation but decrease insulin signaling in skeletal muscle cells.

Molecular Mechanism for Hepatic Glycerolipid Partitioning of n-6/n-3 Fatty Acid Ratio in an Obese Animal Biomodels.

The n-6/n-3 metabolic pathway associated with hepatic glycerolipid portioning plays a key role in preventing obesity. In this nutrition metabolism study, we used in vivo monitoring techniques with 40 obese male Sprague-Dawley strain rats attached with jugular-vein cannula after obesity was induced by a high-fat diet to determine the molecular mechanism associated with hepatic glycerolipid partitioning involving the n-6/n-3 metabolic pathway. Rats were randomly assigned to four groups (10 animals per group), including one control group (CON, n-6/n-3 of 71:1) and three treatment groups (n-6/n-3 of 4:1, 15:1 and 30:1). They were fed with experimental diets for 60 days. Incorporation rates of [14C]-labeling lipid into glycerolipid in the liver were 28.87−37.03% in treatment groups fed with diets containing an n-6/n-3 ratio of 4:1, 15:1 and 30:1, which were significantly (p < 0.05) lower than that in the CON (40.01%). However, 14CO2 emission % of absorbed dose showed the opposite trend. It was significantly (p < 0.05) higher in a treatment groups (n-6/n-3 of 4:1, 15:1 and 30:1, 30.35−45.08%) than in CON (27.71%). Regarding the metabolic distribution of glycerolipid to blood from livers, phospholipid/total glycerolipid (%) was significantly (p < 0.05) lower in CON at 11.04% than in treatment groups at 18.15% to 25.15%. Moreover, 14CO2/[14C]-total glycerolipid (%) was significantly (p < 0.05) higher in treatment groups at 44.16−78.50% than in CON at 39.50%. Metabolic distribution of fatty acyl moieties flux for oxidation and glycerolipid synthesis in the liver were significantly (p < 0.05) better in order of 4:1 > 15:1 > 30:1 than in the CON. Our data demonstrate that n-6/n-3 of 4:1 could help prevent obesity by controlling the mechanism of hepatic partitioning through oxidation and esterification of glycerolipid in an obese animal biomodel.

Vitamin B12 Induces Hepatic Fatty Infiltration through Altered Fatty Acid Metabolism.

BACKGROUND/AIMS: Rise in global incidence of obesity impacts metabolic health. Evidence from human and animal models show association of vitamin B12 (B12) deficiency with elevated BMI and lipids. Human adipocytes demonstrated dysregulation of lipogenesis by low B12 via hypomethylation and altered microRNAs. It is known de novo hepatic lipogenesis plays a key role towards dyslipidaemia, however, whether low B12 affects hepatic metabolism of lipids is not explored. METHODS: HepG2 was cultured in B12-deficient EMEM medium and seeded in different B12 media: 500nM(control), 1000pM(1nM), 100pM and 25pM(low) B12. Lipid droplets were examined by Oil Red O (ORO) staining using microscopy and then quantified by elution assay. Gene expression were assessed with real-time quantitative polymerase chain reaction (qRT-PCR) and intracellular triglycerides were quantified using commercial kit (Abcam, UK) and radiochemical assay. Fatty acid composition was measured by gas chromatography and mitochondrial function by seahorse XF24 flux assay. RESULTS: HepG2 cells in low B12 had more lipid droplets that were intensely stained with ORO compared with control. The total intracellular triglyceride and incorporation of radio-labelled-fatty acid in triglyceride synthesis were increased. Expression of genes regulating fatty acid, triglyceride and cholesterol biosynthesis were upregulated. Absolute concentrations of total fatty acids, saturated fatty acids (SFAs), monounsaturated fatty acids (MUFAs), trans-fatty acids and individual even-chain and odd-chain fatty acids were significantly increased. Also, low B12 impaired fatty acid oxidation and mitochondrial functional integrity in HepG2 compared with control. CONCLUSION: Our data provide novel evidence that low B12 increases fatty acid synthesis and levels of individual fatty acids, and decreases fatty acid oxidation and mitochondrial respiration, thus resulting in dysregulation of lipid metabolism in HepG2. This highlights the potential significance of de novo lipogenesis and warrants possible epigenetic mechanisms of low B12.

How to Improve the Biocompatibility of Peritoneal Dialysis Solutions (without Jeopardizing the Patient's Health).

Peritoneal dialysis (PD) is an important, if underprescribed, modality for the treatment of patients with end-stage kidney disease. Among the barriers to its wider use are the deleterious effects of currently commercially available glucose-based PD solutions on the morphological integrity and function of the peritoneal membrane due to fibrosis. This is primarily driven by hyperglycaemia due to its effects, through multiple cytokine and transcription factor signalling-and their metabolic sequelae-on the synthesis of collagen and other extracellular membrane components. In this review, we outline these interactions and explore how novel PD solution formulations are aimed at utilizing this knowledge to minimise the complications associated with fibrosis, while maintaining adequate rates of ultrafiltration across the peritoneal membrane and preservation of patient urinary volumes. We discuss the development of a new generation of reduced-glucose PD solutions that employ a variety of osmotically active constituents and highlight the biochemical rationale underlying optimization of oxidative metabolism within the peritoneal membrane. They are aimed at achieving optimal clinical outcomes and improving the whole-body metabolic profile of patients, particularly those who are glucose-intolerant, insulin-resistant, or diabetic, and for whom daily exposure to high doses of glucose is contraindicated.

Hepatic VLDL secretion: DGAT1 determines particle size but not particle number, which can be supported entirely by DGAT2.

We investigated whether, in view of its activity being expressed on both aspects of the endoplasmic reticulum (ER; dual membrane topology), diacylglycerol acyltransferase 1 (DGAT1) plays a distinctive role in determining the triglyceride (TAG) content of VLDL particles secreted by the liver. Mice in which the DGAT1 gene was specifically ablated in hepatocytes (DGAT1-LKO mice) had the same number of VLDL particles (apoB concentration) in the plasma 1 h after Triton 1339 treatment, but these particles were approximately half the size of VLDL particles secreted by control mice and had a proportionately decreased content of TAG, with normal cholesterol and cholesteryl ester contents. Analyses of purified microsomal fractions prepared from 16 h fasted control and DAGT1-LKO mice showed that the TAG/protein ratio in the ER was significantly lower in the latter. Electron micrographs of these livers showed that those from DGAT1-LKO mice did not show the increased lipid content of the smooth ER shown by control livers. The effects of DGAT1- and DGAT2-specific inhibitors on apoB secretion by HepG2 cells showed that DGAT1 is not indispensable for apoB secretion and demonstrated redundancy in the ability of the two enzymes to support apoB secretion. Therefore, our findings show that DGAT1 is essential for the complete lipidation and maturation of VLDL particles within the lumen of the ER, consistent with its dual topology within the ER membrane. In the mouse, DGAT2 can support apoB secretion (particle number) even when TAG availability for full VLDL lipidation is restricted in the absence of DGAT1.

Current Opinion on Usage of L-Carnitine in End-Stage Renal Disease Patients on Peritoneal Dialysis.

The advantages of peritoneal dialysis (PD) over hemodialysis (HD) are well-documented. Notwithstanding, only a small proportion of patients with end-stage renal disease (ESRD) are managed with PD. This may be related to the high glucose load that PD solutions in current use have on the patient. The effects of such excess glucose include the relatively early limitation of the ultrafiltration capacity of the peritoneal membrane, and the metabolic effects associated with hyperglycemia, e.g., decreased insulin sensitivity. This article describes the advantages that may be realized by the glucose-sparing effects of substituting part of the glucose load with other osmotically active metabolites, particularly L-carnitine. The latter is anticipated to have metabolic advantages of its own, especially as in PD patients, high plasma concentrations can be achieved in the absence of renal clearance. Besides its better biocompatibility, L-carnitine demonstrates anti-anemia action due to its effects on erythropoiesis, and positive effects on the longevity and deformability of erythrocytes. Observations from our trials on the use of carnitine-enriched PD solutions have demonstrated the effectiveness of L-carnitine as an efficient osmolyte in PD, and its favorable effect on the insulin sensitivity of the patients. The significance of these findings for future developments in the use of PD in the management of patients with ESRD is discussed.

Diacylglycerol acyltransferase 2 links glucose utilization to fatty acid oxidation in the brown adipocytes.

Brown adipose tissue uptake of glucose and fatty acids is very high during nonshivering thermogenesis. Adrenergic stimulation markedly increases glucose uptake, de novo lipogenesis, and FA oxidation simultaneously. The mechanism that enables this concerted response has hitherto been unknown. Here, we find that in primary brown adipocytes and brown adipocyte-derived cell line (IMBAT-1), acute inhibition and longer-term knockdown of DGAT2 links the increased de novo synthesis of fatty acids from glucose to a pool of TAG that is simultaneously hydrolyzed, providing FA for mitochondrial oxidation. DGAT1 does not contribute to this pathway, but uses exogenous FA and glycerol to synthesize a functionally distinct pool of TAG to which DGAT2 also contributes. The DGAT2-dependent channelling of 14C from glucose into TAG and CO2 was reproduced in ß3-agonist-stimulated primary brown adipocytes. Knockdown of DGAT2 in IMBAT-1 affected the mRNA levels of UCP1 and genes important in FA activation and esterification. Therefore, in ß3-agonist activated brown adipocytes, DGAT2 specifically enables channelling of de novo synthesized FA into a rapidly mobilized pool of TAG, which is simultaneously hydrolyzed to provide substrates for mitochondrial fatty acid oxidation.

Extra-nuclear telomerase reverse transcriptase (TERT) regulates glucose transport in skeletal muscle cells.

Telomerase reverse transcriptase (TERT) is a key component of the telomerase complex. By lengthening telomeres in DNA strands, TERT increases senescent cell lifespan. Mice that lack TERT age much faster and exhibit age-related conditions such as osteoporosis, diabetes and neurodegeneration. Accelerated telomere shortening in both human and animal models has been documented in conditions associated with insulin resistance, including T2DM. We investigated the role of TERT, in regulating cellular glucose utilisation by using the myoblastoma cell line C2C12, as well as primary mouse and human skeletal muscle cells. Inhibition of TERT expression or activity by using siRNA (100nM) or specific inhibitors (100nM) reduced basal 2-deoxyglucose uptake by ~50%, in all cell types, without altering insulin responsiveness. In contrast, TERT over-expression increased glucose uptake by 3.25-fold. In C2C12 cells TERT protein was mostly localised intracellularly and stimulation of cells with insulin induced translocation to the plasma membrane. Furthermore, co-immunoprecipitation experiments in C2C12 cells showed that TERT was constitutively associated with glucose transporters (GLUTs) 1, 4 and 12 via an insulin insensitive interaction that also did not require intact PI3-K and mTOR pathways. Collectively, these findings identified a novel extra-nuclear function of TERT that regulates an insulin-insensitive pathway involved in glucose uptake in human and mouse skeletal muscle cells.

Hepatic triacylglycerol synthesis and secretion: DGAT2 as the link between glycaemia and triglyceridaemia.

lThe liver regulates both glycaemia and triglyceridaemia. Hyperglycaemia and hypertriglyceridaemia are both characteristic of (pre)diabetes. Recent observations on the specialised role of DGAT2 (diacylglycerol acyltransferase 2) in catalysing the de novo synthesis of triacylglycerols from newly synthesized fatty acids and nascent diacylglycerols identifies this enzyme as the link between the two. This places DGAT2 at the centre of carbohydrate-induced hypertriglyceridaemia and hepatic steatosis. This function is complemented, but not substituted for, by the ability of DGAT1 to rescue partial glycerides from complete hydrolysis. In peripheral tissues not normally considered to be lipogenic, synthesis of triacylglycerols may largely bypass DGAT2 except in hyperglycaemic/hyperinsulinaemic conditions, when induction of de novo fatty acid synthesis in these tissues may contribute towards increased triacylglycerol secretion (intestine) or insulin resistance (adipose tissue, and cardiac and skeletal muscle).

In Vivo Monitoring of Glycerolipid Metabolism in Animal Nutrition Biomodel-Fed Smart-Farm Eggs.

Although many studies have examined the biochemical metabolic pathways by which an egg (egg yolk) lowers blood lipid levels, data on the molecular biological mechanisms that regulate and induce the partitioning of hepatic glycerolipids are missing. The aim of this study was to investigate in vivo monitoring in four study groups using an animal nutrition biomodel fitted with a jugular-vein cannula after egg yolk intake: CON (control group, oral administration of 1.0 g of saline), T1 (oral administration of 1.0 g of pork belly fat), T2 (oral administration of 1.0 g of smart-farm egg yolk), and T3 (oral administration of T1 and T2 alternately every week). The eggs induced significant and reciprocal changes in incorporating 14C lipids into the total glycerolipids and releasing 14CO2, thereby regulating esterification and accelerating oxidation in vivo. The eggs increased phospholipid secretion from the liver into the blood and decreased triacylglycerol secretion by regulating the multiple cleavage of fatty acyl-CoA moieties' fluxes. In conclusion, the results of the current study reveal the novel fact that eggs can lower blood lipids by lowering triacylglycerol secretion in the biochemical metabolic pathway of hepatic glycerolipid partitioning while simultaneously increasing phospholipid secretion and 14CO2 emission.

Impact of the Combination of Probiotics and Digital Poultry System on Behavior, Welfare Parameters, and Growth Performance in Broiler Chicken.

Recently, applied technology in the form of the combination of a probiotics and a digital poultry system, with the convergence of Information and Communications Technology and farm animals, has enabled a new strategy to overcome the livestock production crisis caused by climate change, while maintaining sustainable poultry farming in terms of care, feeding, and environmental management systems for poultry. The aim of this study was to investigate the biological mechanisms of animal behavioral welfare and production improvement using the combination of a probiotics and a digital poultry system in broiler chickens. A total of 400 one-day-old male broilers (ROSS 308) were randomly divided into four treatment groups, with five replicates each (20 birds/replicate pen) in a completely randomized design: control group with a conventional poultry system without probiotics (CON), conventional poultry system with 500 ppm of probiotics (CON500), digital poultry control system without probiotics (DPCS), and digital poultry system with 500 ppm of probiotics (DPS500). All experimental animals were reared for 35 days under the same standard environmental conditions. The experimental results indicated that the animal behavioral welfare, which includes drinking, eating, locomotion, grooming, and resting, in addition to foot pads, knee burns, plumage, and gait scores, as well as the growth performance of the broiler chickens, were improved by maintaining immune function and cecal microbiota balance via interaction between the combination of a probiotics and a digital poultry system. In conclusion, it was found that the combined system showed improved broiler growth performance and animal behavioral welfare. Thus, further studies of molecular biological mechanisms by the use of such a combined system to improve the nutritional composition and quality of chicken meats are recommended.

Metformin Inefficiency to Lower Lipids in Vitamin B12 Deficient HepG2 Cells Is Alleviated via Adiponectin-AMPK Axis.

Background: Prolonged metformin treatment decreases vitamin B12 (B12) levels, whereas low B12 is associated with dyslipidaemia. Some studies have reported that metformin has no effect on intrahepatic triglyceride (TG) levels. Although AMP-activated protein kinase (AMPK) activation via adiponectin lowers hepatic TG content, its role in B12 deficiency and metformin has not been explored. We investigated whether low B12 impairs the beneficial effect of metformin on hepatic lipid metabolism via the AMPK-adiponectin axis. Methods: HepG2 was cultured using custom-made B12-deficient Eagle's Minimal Essential Medium (EMEM) in different B12-medium concentrations, followed by a 24-h metformin/adiponectin treatment. Gene and protein expressions and total intracellular TG were measured, and radiochemical analysis of TG synthesis and seahorse mitochondria stress assay were undertaken. Results: With low B12, total intracellular TG and synthesized radiolabelled TG were increased. Regulators of lipogenesis, cholesterol and genes regulating fatty acids (FAs; TG; and cholesterol biosynthesis were increased. FA oxidation (FAO) and mitochondrial function were decreased, with decreased pAMPKα and pACC levels. Following metformin treatment in hepatocytes with low B12, the gene and protein expression of the above targets were not alleviated. However, in the presence of adiponectin, intrahepatic lipid levels with low B12 decreased via upregulated pAMPKα and pACC levels. Again, combined adiponectin and metformin treatment ameliorated the low B12 effect and resulted in increased pAMPKα and pACC, with a subsequent reduction in lipogenesis, increased FAO and mitochondrion function. Conclusions: Adiponectin co-administration with metformin induced a higher intrahepatic lipid-lowering effect. Overall, we emphasize the potential therapeutic implications for hepatic AMPK activation via adiponectin for a clinical condition associated with B12 deficiency and metformin treatment.

Evidence that diacylglycerol acyltransferase 1 (DGAT1) has dual membrane topology in the endoplasmic reticulum of HepG2 cells.

Triacylglycerol (TAG) synthesis and secretion are important functions of the liver that have major impacts on health, as overaccumulation of TAG within the liver (steatosis) or hypersecretion of TAG within very low density lipoproteins (VLDL) both have deleterious metabolic consequences. Two diacylglycerol acyltransferases (DGATs 1 and 2) can catalyze the final step in the synthesis of TAG from diacylglycerol, which has been suggested to play an important role in the transfer of the glyceride moiety across the endoplasmic reticular membrane for (re)synthesis of TAG on the lumenal aspect of the endoplasmic reticular (ER) membrane (Owen, M., Corstorphine, C. C., and Zammit, V. A. (1997) Biochem. J. 323, 17-21). Recent topographical studies suggested that the oligomeric enzyme DGAT1 is exclusively lumen facing (latent) in the ER membrane. By contrast, in the present study, using two specific inhibitors of human DGAT1, we present evidence that DGAT1 has a dual topology within the ER of HepG2 cells, with approximately equal DGAT1 activities exposed on the cytosolic and lumenal aspects of the ER membrane. This was confirmed by the observation of the loss of both overt (partial) and latent (total) DGAT activity in microsomes prepared from livers of Dgat1(-/-) mice. Conformational differences between DGAT1 molecules having the different topologies were indicated by the markedly disparate sensitivities of the overt DGAT1 to one of the inhibitors. These data suggest that DGAT1 belongs to the family of oligomeric membrane proteins that adopt a dual membrane topology.

An environment-dependent structural switch underlies the regulation of carnitine palmitoyltransferase 1A.

The enzyme carnitine palmitoyltransferase 1 (CPT1), which is anchored in the outer mitochondrial membrane (OMM), controls the rate-limiting step in fatty acid ß-oxidation in mammalian tissues. It is inhibited by malonyl-CoA, the first intermediate of fatty acid synthesis, and it responds to OMM curvature and lipid characteristics, which reflect long term nutrient/hormone availability. Here, we show that the N-terminal regulatory domain (N) of CPT1A can adopt two complex amphiphilic structural states, termed Nα and Nß, that interchange in a switch-like manner in response to offered binding surface curvature. Structure-based site-directed mutageneses of native CPT1A suggest Nα to be inhibitory and Nß to be noninhibitory, with the relative Nα/Nß ratio setting the prevalent malonyl-CoA sensitivity of the enzyme. Based on the amphiphilic nature of N and molecular modeling, we propose malonyl-CoA sensitivity to be coupled to the properties of the OMM by Nα-OMM associations that alter the Nα/Nß ratio. For enzymes residing at the membrane-water interface, this constitutes an integrative regulatory mechanism of exceptional sophistication.

Packing of transmembrane domain 2 of carnitine palmitoyltransferase-1A affects oligomerization and malonyl-CoA sensitivity of the mitochondrial outer membrane protein.

The purpose of this study was to investigate the sequence-dependence of oligomerization of transmembrane domain 2 (TM2) of rat carnitine palmitoyltransferase 1A (rCPT1A), to elucidate the role of this domain in the function of the full-length enzyme. Oligomerization of TM2 was studied qualitatively using complementary genetic assays that facilitate measurement of helix-helix interactions in the Escherichia coli inner membrane, and multiple quantitative biophysical methods. The effects of TM2-mutations on oligomerization and malonyl-CoA inhibition of the full-length enzyme (expressed in the yeast Pichia pastoris) were quantified. Changes designed to disrupt close-packing of the GXXXG(A) motifs reduced the oligomeric state of the corresponding TM2 peptides from hexamer to trimer (or lower), a reduction also observed on mutation of the TM2 sequence in the full-length enzyme. Disruption of these GXXXG(A) motifs had a parallel effect on the malonyl-CoA sensitivity of rCPT1A, reducing the IC(50) from 30.3 ± 5.0 to 3.0 ± 0.6 µM. For all measurements, wild-type rCPT1A was used as a control alongside various appropriate (e.g., molecular mass) standards. Our results suggest that sequence-determined, TM2-mediated oligomerization is likely to be involved in the modulation of malonyl-CoA inhibition of CPT1A in response to short- and long-term changes in protein-protein and protein-lipid interactions that occur in vivo.

Association between fetal abdominal growth trajectories, maternal metabolite signatures early in pregnancy, and childhood growth and adiposity: prospective observational multinational INTERBIO-21st fetal study.

BACKGROUND: Obesity predominantly affects populations in high-income countries and those countries facing epidemiological transition. The risk of childhood obesity is increased among infants who had overweight or obesity at birth, but in low-resource settings one in five infants are born small for gestational age. We aimed to study the relationships between: (1) maternal metabolite signatures; (2) fetal abdominal growth; and (3) postnatal growth, adiposity, and neurodevelopment. METHODS: In the prospective, multinational, observational INTERBIO-21st fetal study, conducted in maternity units in Pelotas (Brazil), Nairobi (Kenya), Karachi (Pakistan), Soweto (South Africa), Mae Sot (Thailand), and Oxford (UK), we enrolled women (≥18 years, with a BMI of less than 35 kg/m2, natural conception, and a singleton pregnancy) who initiated antenatal care before 14 weeks' gestation. Ultrasound scans were performed every 5±1 weeks until delivery to measure fetal growth and feto-placental blood flow, and we used finite mixture models to derive growth trajectories of abdominal circumference. The infants' health, growth, and development were monitored from birth to age 2 years. Early pregnancy maternal blood and umbilical cord venous blood samples were collected for untargeted metabolomic analysis. FINDINGS: From Feb 8, 2012, to Nov 30, 2019, we enrolled 3598 pregnant women and followed up their infants to 2 years of age. We identified four ultrasound-derived trajectories of fetal abdominal circumference growth that accelerated or decelerated within a crucial 20-25 week gestational age window: faltering growth, early accelerating growth, late accelerating growth, and median growth tracking. These distinct phenotypes had matching feto-placental blood flow patterns throughout pregnancy, and different growth, adiposity, vision, and neurodevelopment outcomes in early childhood. There were 709 maternal metabolites with positive effect for the faltering growth phenotype and 54 for the early accelerating growth phenotype; 31 maternal metabolites had a negative effect for the faltering growth phenotype and 76 for the early accelerating growth phenotype. Metabolites associated with the faltering growth phenotype had statistically significant odds ratios close to 1·5 (ie, suggesting upregulation of metabolic pathways of impaired fetal growth). The metabolites had a reciprocal relationship with the early accelerating growth phenotype, with statistically significant odds ratios close to 0.6 (ie, suggesting downregulation of fetal growth acceleration). The maternal metabolite signatures included 5-hydroxy-eicosatetraenoic acid, and 11 phosphatidylcholines linked to oxylipin or saturated fatty acid sidechains. The fungicide, chlorothalonil, was highly abundant in the early accelerating growth phenotype group. INTERPRETATION: Early pregnancy lipid biology associated with fetal abdominal growth trajectories is an indicator of patterns of growth, adiposity, vision, and neurodevelopment up to the age of 2 years. Our findings could contribute to the earlier identification of infants at risk of obesity. FUNDING: Bill & Melinda Gates Foundation.

Alternative exon usage in the single CPT1 gene of Drosophila generates functional diversity in the kinetic properties of the enzyme: differential expression of alternatively spliced variants in Drosophila tissues.

The Drosophila melanogaster genome contains only one CPT1 gene (Jackson, V. N., Cameron, J. M., Zammit, V. A., and Price, N. T. (1999) Biochem. J. 341, 483-489). We have now extended our original observation to all insect genomes that have been sequenced, suggesting that a single CPT1 gene is a universal feature of insect genomes. We hypothesized that insects may be able to generate kinetically distinct variants by alternative splicing of their single CPT1 gene. Analysis of the insect genomes revealed that (a) the single CPT1 gene in each and every insect genome contains two alternative exons and (ii) in all cases, the putative alternative splicing site occurs within a small region corresponding to 21 amino acid residues that are known to be essential for the binding of substrates and of malonyl-CoA in mammalian CPT1A. We performed PCR analyses of mRNA from different Drosophila tissues; both of the anticipated splice variants of CPT1 mRNA were found to be expressed in all of the tissues tested (both in larvae and adults), with the expression level for one of the splice variants being significantly different between flight muscle and the fat body of adult Drosophila. Heterologous expression of the full-length cDNAs corresponding to the two putative variants of Drosophila CPT1 in the yeast Pichia pastoris revealed two important differences between the properties of the two variants: (i) their affinity (K(0.5)) for one of the substrates, palmitoyl-CoA, differed by 5-fold, and (ii) the sensitivity to inhibition by malonyl-CoA at fixed, higher palmitoyl-CoA concentrations was 2-fold different and associated with different kinetics of inhibition. These data indicate that alternative splicing that specifically affects a structurally crucial region of the protein is an important mechanism through which functional diversity of CPT1 kinetics is generated from the single gene that occurs in insects.