Evaluating parental perceptions of written handbooks provided during shared decision making with parents anticipating extremely preterm birth.

OBJECTIVE: To explore parental perceptions of written handbooks provided to them during antenatal counseling for anticipated extremely preterm birth. STUDY DESIGN: This study involved a prospective convenience sample of parents anticipating delivery between 22 weeks + 0 days and 25 weeks + 6 days gestation. The antenatal counseling involved a shared decision-making process. In-person interviews were conducted using a semi-structured interview guide to gather feedback about new parent handbooks developed to support decision making. The questions during the semi-structured interview targeted seven main themes: overall impression, timing, graphs/tables, formatting, imagery, ease of use and understanding, and content. The interviews followed an antenatal consultation and provision of the appropriate handbook(s) by a neonatologist. Interviews were transcribed verbatim and thematic analysis of the data was completed. RESULTS: Eleven parents were interviewed. All parents described the provision of the handbook(s) following the consultation with a neonatologist as the ideal time. All parents considered a visual representation of the data to be invaluable. Parents considered the handbooks easy to understand and straightforward. Some parents were satisfied with simple information, which helped them feel less overwhelmed; others felt the depth of information was insufficient. Parents preferred a paper copy to electronic. Reactions to the photo of an infant receiving intensive care varied; some parents felt frightened, others felt comforted. CONCLUSION: Overall, parents positively evaluated the handbooks, supporting their utility for parents anticipating extremely preterm birth. Concrete suggestions for improvement were made; the handbooks will be modified accordingly. Parents at other perinatal centers may benefit from receiving such handbooks.

Autosomal dominant transmission of transient neonatal lactic acidosis: a case report.

BACKGROUND: Lactic acidosis is a common finding in neonates, in whom mitochondrial dysfunction is often secondary to tissue hypoperfusion, respiratory failure, and/or sepsis. Primary (non-physiological) lactic acidosis is comparatively rare, and suggests the presence of an inborn error of mitochondrial energy metabolism. Optimal medical management and accurate prognostication requires the correct determination of the etiology of lactic acidosis in a given patient. Unfortunately, genetic diagnoses are rare and highly variable for neonates presenting with primary lactic acidosis; individual case reports may offer the most promise for treatment considerations. The mitochondrion is a complex molecular machine incorporating the products of > 1000 distinct nuclear genes. Primary lactic acidoses are therefore characterized by high genetic heterogeneity and a specific genetic diagnosis currently remains out of reach in most cases. Most mitochondriopathies with neonatal onset follow autosomal recessive inheritance and carry a poor prognosis. Here we detail the case of a father and daughter with dominantly-inherited, resolving (i.e. transient) neonatal hyperlactatemia due to complex IV deficiency. We found no other published descriptions of benign transient complex IV deficiency with autosomal dominant inheritance. CASE PRESENTATION: Both individuals presented as neonates with unexplained, marked lactic acidosis suggesting a primary mitochondrial disorder. Within the first weeks of life, elevated blood lactate levels normalized. Their clinical and developmental outcomes were normal. Biochemical studies in the proband showed multiple abnormalities consistent with a complex IV respiratory chain defect. Cultured skin fibroblasts showed an elevated lactate-to-pyruvate ratio, deficient complex IV activity, and normal pyruvate dehydrogenase and pyruvate carboxylase activities. Whole-exome sequencing of the proband and both parents did not identify a causative mutation. CONCLUSION: We conclude that the proband and her father appear to have a dominant form of transient neonatal hyperlactatemia due to heterozygous changes in an as-yet unidentified gene. This transient neonatal complex IV deficiency should be considered in the differential diagnosis of primary neonatal hyperlactatemia; notable clinical features include autosomal-dominant inheritance and an apparently benign postnatal course. This report exemplifies the growing differential diagnosis for neonatal lactic acidosis and highlights the importance of both physician counselling and the use of family history in communicating with parents.

Lpaatδ/Agpat4 deficiency impairs maximal force contractility in soleus and alters fibre type in extensor digitorum longus muscle.

Lysophosphatidic acid acyltransferase (LPAAT) δ/acylglycerophosphate acyltransferase 4 is a mitochondrial enzyme and one of five homologues that catalyze the acyl-CoA-dependent synthesis of phosphatidic acid (PA) from lysophosphatidic acid. We studied skeletal muscle LPAATδ and found highest levels in soleus, a red oxidative fibre-type that is rich in mitochondria, and lower levels in extensor digitorum longus (EDL) (white glycolytic) and gastrocnemius (mixed fibre-type). Using Lpaatδ-deficient mice, we found no change in soleus or EDL mass, or in treadmill time-to-exhaustion compared to wildtype littermates. There was, however, a significant reduction in the proportion of type I and type IIA fibres in EDL but, surprisingly, not soleus, where these fibre-types predominate. Also unexpectedly, there was no impairment in force generation by EDL, but a significant reduction by soleus. Oxidative phosphorylation and activity of complexes I, I + II, III, and IV in soleus mitochondria was unchanged and therefore could not explain this effect. However, pyruvate dehydrogenase activity was significantly reduced in Lpaatδ-/- soleus and EDL. Analysis of cellular lipids indicated no difference in soleus triacylglycerol, but specific elevations in soleus PA and phosphatidylethanolamine levels, likely due to a compensatory upregulation of Lpaatß and Lpaatε in Lpaatδ-/- mice. An anabolic effect for PA as an activator of skeletal muscle mTOR has been reported, but we found no change in serine 2448 phosphorylation, indicating reduced soleus force generation is unlikely due to the loss of mTOR activation by a specific pool of LPAATδ-derived PA. Our results identify an important role for LPAATδ in soleus and EDL.

Agpat4/Lpaatδ deficiency highlights the molecular heterogeneity of epididymal and perirenal white adipose depots.

Acylglycerophosphate acyltransferase 4 (AGPAT4)/lysophosphatidic acid acyltransferase delta catalyzes the formation of phosphatidic acid (PA), a precursor of triacyl-glycerol (TAG). We investigated the effect of Agpat4 gene ablation on white adipose tissue (WAT) after finding consistent expression across depots. Epididymal WAT mass was 40% larger in male Agpat4-/- mice than wild-type littermates, but unchanged in perirenal, retroperitoneal, and inguinal WAT and subscapular brown adipose tissue. Metabolic changes were identified in epididymal WAT that were not evident in perirenal WAT, which was analyzed for comparison. The total epididymal TAG content doubled, increasing adipocyte cell size without changing markers of differentiation. Enzymes involved in de novo lipogenesis and complex lipid synthesis downstream of phosphatidic acid production were also unchanged. However, total epididymal TAG hydrolase activity was reduced, and there were significant decreases in total ATGL and reduced phosphorylation of hormone-sensitive lipase at the S563 and S660 PKA-activation sites. Analysis of Agpats 1, 2, 3, and 5, as well as Gpats 1, 2, 3, and 4, demonstrated compensatory upregulation in perirenal WAT that did not occur in epididymal WAT. Our findings therefore indicate depot-specific differences in the redundancy of Agpat4 and highlight the molecular and metabolic heterogeneity of individual visceral depots.

Mice Deficient in lysophosphatidic acid acyltransferase delta (Lpaatδ)/acylglycerophosphate acyltransferase 4 (Agpat4) Have Impaired Learning and Memory.

We previously characterized LPAATδ/AGPAT4 as a mitochondrial lysophosphatidic acid acyltransferase that regulates brain levels of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylinositol (PI). Here, we report that Lpaatδ-/- mice display impaired spatial learning and memory compared to wild-type littermates in the Morris water maze and our investigation of potential mechanisms associated with brain phospholipid changes. Marker protein immunoblotting suggested that the relative brain content of neurons, glia, and oligodendrocytes was unchanged. Relative abundance of the important brain fatty acid docosahexaenoic acid was also unchanged in phosphatidylserine, phosphatidylglycerol, and cardiolipin, in agreement with prior data on PC, PE and PI. In phosphatidic acid, it was increased. Specific decreases in ethanolamine-containing phospholipids were detected in mitochondrial lipids, but the function of brain mitochondria in Lpaatδ-/- mice was unchanged. Importantly, we found that Lpaatδ-/- mice have a significantly and drastically lower brain content of the N-methyl-d-asparate (NMDA) receptor subunits NR1, NR2A, and NR2B, as well as the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit GluR1, compared to wild-type mice. However, general dysregulation of PI-mediated signaling is not likely responsible, since phospho-AKT and phospho-mTOR pathway regulation was unaffected. Our findings indicate that Lpaatδ deficiency causes deficits in learning and memory associated with reduced NMDA and AMPA receptors.

Acute Fasting Induces Expression of Acylglycerophosphate Acyltransferase (AGPAT) Enzymes in Murine Liver, Heart, and Brain.

During fasting, cells increase uptake of non-esterified fatty acids (NEFA) and esterify excess into phosphatidic acid (PtdOH), the common precursor of both triacylglycerols and phospholipids, using acylglycerophosphate acyltransferases/lysophosphatidic acid acyltransferases (AGPAT/LPAAT). Knowledge of the regulation of AGPAT enzymes is important for understanding fasting adaptations. Total RNA was isolated from liver, heart, and whole brain tissue of C57BL/6J mice fed ad libitum, or fasted for 16 h. Following fasting, induction of Agpat2, 3, 4, and 5 was observed in the liver, Agpat2 and 3 in heart tissue, and Agpat1, 2, and 3 in whole brain tissue. As a result, the relative abundance profile of the individual homologues within specific tissues was found to be significantly altered depending on the nutritive state of the animal. These data demonstrate tissue-specific effects of fasting on the regulation of different Agpat that are implicated in supporting unique downstream glycerolipid synthesis pathways.

Data on hepatic lipolysis, adipose triglyceride lipase, and hormone-sensitive lipase in fasted and non-fasted C57BL/6J female mice.

Liver homogenates produced from fasted and non-fasted C57BL/6J female mice were assayed for total lipolytic activity measured as hydrolysis of [9,10-(3)H(N)]-triolein into [(3)H] free fatty acids (FFA). Liver homogenates were also used for immunoblotting to determine levels of the lipolytic enzymes adipose-triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL), as well as site specific phosphorylation at the 14-3-3 binding site of ATGL and the serine 565 and serine 660 sites of HSL. Significantly higher triolein hydrolysis activity was observed in fasted liver samples, as well as a significant increase in total ATGL and a significant decrease in HSL phosphorylation at the S565 site.

Data on oxygen consumption rate, respiratory exchange ratio, and movement in C57BL/6J female mice on the third day of consuming a high-fat diet.

Whole animal physiological measures were assessed following three days of either standard diet or high fat diet, in either the fasted or non-fasted states. Our data shows that acute 3-day high fat feeding increases whole body lipid oxidation. When this feeding protocol is followed by an overnight fast, oxygen consumption (VO2) in the light phase is reduced in both dietary groups, but oxygen consumption in the dark phase is only reduced in mice fed the high-fat diet. Furthermore, the fasting-induced rise in dark cycle activity level observed in mice maintained on a standard diet is abolished when mice are fed a high-fat diet.

Data on acylglycerophosphate acyltransferase 4 (AGPAT4) during murine embryogenesis and in embryo-derived cultured primary neurons and glia.

Whole mouse embryos at three developmental timepoints, embryonic (E) day E10.5, E14.5, and E18.5, were analyzed for Agpat4 mRNA expression. Primary cortical mouse cultures prepared from E18.5 mouse brains were used for immunohistochemistry. Our data show that Agpat4 is differentially expressed at three timepoints in murine embryogenesis and is immunodetectable in both neurons and glial cells derived from the developing mouse brain. This paper contains data related to research concurrently published in Bradley et al. (2015) [1].

The HRASLS (PLA/AT) subfamily of enzymes.

The H-RAS-like suppressor (HRASLS) subfamily consists of five enzymes (1-5) in humans and three (1, 3, and 5) in mice and rats that share sequence homology with lecithin:retinol acyltransferase (LRAT). All HRASLS family members possess in vitro phospholipid metabolizing abilities including phospholipase A1/2 (PLA1/2) activities and O-acyltransferase activities for the remodeling of glycerophospholipid acyl chains, as well as N-acyltransferase activities for the production of N-acylphosphatidylethanolamines. The in vivo biological activities of the HRASLS enzymes have not yet been fully investigated. Research to date indicates involvement of this subfamily in a wide array of biological processes and, as a consequence, these five enzymes have undergone extensive rediscovery and renaming within different fields of research. This review briefly describes the discovery of each of the HRASLS enzymes and their role in cancer, and discusses the biochemical function of each enzyme, as well as the biological role, if known. Gaps in current understanding are highlighted and suggestions for future research directions are discussed.

Acylglycerophosphate acyltransferase 4 (AGPAT4) is a mitochondrial lysophosphatidic acid acyltransferase that regulates brain phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol levels.

The acylglycerophosphate acyltransferase/lysophosphatidic acid acyltransferase (AGPAT/LPAAT) family is a group of homologous acyl-CoA-dependent lysophospholipid acyltransferases. We performed studies to better understand the subcellular localization, activity, and in vivo function of AGPAT4/LPAATδ, which we found is expressed in multiple mouse brain regions. Endogenous brain AGPAT4 and AGPAT4 overexpressed in HEK293 or Sf9 insect cells localizes to mitochondria and is resident on the outer mitochondrial membrane. Further fractionation showed that AGPAT4 is present specifically in the mitochondria and not in the mitochondria-associated endoplasmic reticulum membrane (i.e. MAM). Lysates from Sf9 cells infected with baculoviral Agpat4 were tested with eight lysophospholipid species but showed an increased activity only with lysophosphatidic acid as an acyl acceptor. Analysis of Sf9 phospholipid species, however, indicated a significant 72% increase in phosphatidylinositol (PI) content. We examined the content of major phospholipid species in brains of Agpat4(-/-) mice and found also a >50% decrease in total levels of PI relative to wildtype mice, as well as significant decreases in phosphatidylcholine (PC) and phosphatidylethanolamine (PE), but no significant differences in phosphatidylserine, phosphatidylglycerol, cardiolipin, or phosphatidic acid (PA). A compensatory upregulation of Agpats 1, 2, 3, 5, and 9 may help to explain the lack of difference in PA. Our findings indicate that AGPAT4 is a mitochondrial AGPAT/LPAAT that specifically supports synthesis of brain PI, PC, and PE. This understanding may help to explain apparent redundancies in the AGPAT/LPAAT family.

Fasting upregulates adipose triglyceride lipase and hormone-sensitive lipase levels and phosphorylation in mouse kidney.

Circulating non-esterified fatty acids (NEFA) rise during fasting and are taken up by the kidneys, either directly from the plasma or during re-uptake of albumin from glomerular filtrate, and are stored as triacylglycerol (TAG). Subsequent utilization of stored fatty acids requires their hydrolytic release from cellular lipid droplets, but relatively little is known about renal lipolysis. We found that total [(3)H]triolein hydrolase activity of kidney lysates was significantly increased by 15% in the fasted state. Adipose triglyceride lipase (Atgl) and hormone-sensitive lipase (Hsl) mRNA expression was time-dependently increased by fasting, along with other fatty acid metabolism genes (Pparα, Cd36, and Aox). ATGL and HSL protein levels were also significantly induced (by 239 ± 7% and 322 ± 8%, respectively). Concomitant with changes in total protein levels, there was an increase in ATGL phosphorylation at the AMPK-regulated serine 406 site in the 14-3-3 binding motif, and an increase in HSL phosphorylation at serines 565 and 660 that are regulated by AMPK and PKA, respectively. Using immunofluorescence, we further demonstrate nearly ubiquitous expression of ATGL in the renal cortex with a concentration on the apical/lumenal surface of some cortical tubules. Our findings suggest a role for ATGL and HSL in kidney lipolysis.