Melanie B. Gillingham

Maternal High Fat Diet Is Associated with Decreased Plasma n–3 Fatty Acids and Fetal Hepatic Apoptosis in Nonhuman Primates

To begin to understand the contributions of maternal obesity and over-nutrition to human development and the early origins of obesity, we utilized a non-human primate model to investigate the effects of maternal high-fat feeding and obesity on breast milk, maternal and fetal plasma fatty acid composition and fetal hepatic development. While the high-fat diet (HFD) contained equivalent levels of n-3 fatty acids (FAs) and higher levels of n-6 FAs than the control diet (CTR), we found significant decreases in docosahexaenoic acid (DHA) and total n-3 FAs in HFD maternal and fetal plasma. Furthermore, the HFD fetal plasma n-6∶n-3 ratio was elevated and was significantly correlated to the maternal plasma n-6∶n-3 ratio and maternal hyperinsulinemia. Hepatic apoptosis was also increased in the HFD fetal liver. Switching HFD females to a CTR diet during a subsequent pregnancy normalized fetal DHA, n-3 FAs and fetal hepatic apoptosis to CTR levels. Breast milk from HFD dams contained lower levels of eicosopentanoic acid (EPA) and DHA and lower levels of total protein than CTR breast milk. This study links chronic maternal consumption of a HFD with fetal hepatic apoptosis and suggests that a potentially pathological maternal fatty acid milieu is replicated in the developing fetal circulation in the nonhuman primate.

Current issues regarding treatment of mitochondrial fatty acid oxidation disorders.

Treatment recommendations in mitochondrial fatty acid oxidation (FAO) defects are diverse. With implementation of newborn screening and identification of asymptomatic patients, it is necessary to define whom to treat and how strictly. We here discuss critical questions that are currently under debate. For some asymptomatic long-chain defects, long-chain fat restriction plays a minor role, and a normal diet may be introduced. For patients presenting only with myopathic symptoms, e.g., during exercise, treatment may be adapted to energy demand. As a consequence, patients with exercise-induced myopathy may be able to return to normal activity when provided with medium-chain triglycerides (MCT) prior to exercise. There is no need to limit participation in sports. Progression of retinopathy in disorders of the mitochondrial trifunctional protein complex is closely associated with hydroxyacylcarnitine accumulation. A strict low-fat diet with MCT supplementation is recommended to slow or prevent progression of chorioretinopathy. Additional docosahexanoic acid does not prevent the decline in retinal function but does promote nonspecific improvement in visual acuity and is recommended. There is no evidence that L-carnitine supplementation is beneficial. Thus, supplementation with L-carnitine in a newborn identified by screening with either a medium-chain or long-chain defect is not supported. With respect to the use of the odd-chain medium-chain triglyceride triheptanoin in myopathic phenotypes, randomized trials are needed to establish whether triheptanoin is more effective than even-chain MCT. With increasing pathophysiological knowledge, new treatment options have been identified and are being clinically evaluated. These include the use of bezafibrates in myopathic long-chain defects.

Complete correction of hyperphenylalaninemia following liver-directed, recombinant AAV2/8 vector-mediated gene therapy in murine phenylketonuria.

Novel recombinant adeno-associated virus vectors pseudotyped with serotype 8 capsid (rAAV2/8) have recently shown exciting promise as effective liver-directed gene transfer reagents. We have produced a novel liver-specific rAAV2/8 vector expressing the mouse phenylalanine hydroxylase (Pah) cDNA and have administered this vector to hyperphenylalaninemic PAH-deficient Pahenu2 mice, a model of human phenylketonuria (PKU). Our hypothesis was that this vector would produce sufficient hepatocyte transduction frequency and PAH activity to correct blood phenylalanine levels in murine PKU. Portal vein injection of recombinant AAV2/8 vector into five adult Pahenu2 mice yielded complete and stable (up to 17 weeks) correction of serum phenylalanine levels. Liver PAH activity was corrected to 11.5±2.4% of wild type liver activity and was associated with a significant increase in phenylalanine clearance following parenteral phenylalanine challenge. Although questions of long-term safety and stability of expression remain, recombinant AAV2/8-mediated, liver-directed gene therapy is a promising novel treatment approach for PKU and allied inborn errors of metabolism.

Optimal dietary therapy of long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency.

Current dietary therapy for long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) or trifunctional protein (TFP) deficiency consists of fasting avoidance, and limiting long-chain fatty acid (LCFA) intake. This study reports the relationship of dietary intake and metabolic control as measured by plasma acylcarnitine and organic acid profiles in 10 children with LCHAD or TFP deficiency followed for 1 year. Subjects consumed an average of 11% of caloric intake as dietary LCFA, 11% as MCT, 12% as protein, and 66% as carbohydrate. Plasma levels of hydroxypalmitoleic acid, hydroxyoleic, and hydroxylinoleic carnitine esters positively correlated with total LCFA intake and negatively correlated with MCT intake suggesting that as dietary intake of LCFA decreases and MCT intake increases, there is a corresponding decrease in plasma hydroxyacylcarnitines. There was no correlation between plasma acylcarnitines and level of carnitine supplementation. Dietary intake of fat-soluble vitamins E and K was deficient. Dietary intake and plasma levels of essential fatty acids, linoleic and linolenic acid, were deficient. On this dietary regimen, the majority of subjects were healthy with no episodes of metabolic decompensation. Our data suggest that an LCHAD or TFP-deficient patient should adhere to a diet providing age-appropriate protein and limited LCFA intake (10% of total energy) while providing 10-20% of energy as MCT and a daily multi-vitamin and mineral (MVM) supplement that includes all of the fat-soluble vitamins. The diet should be supplemented with vegetable oils as part of the 10% total LCFA intake to provide essential fatty acids.

Beneficial and cautionary outcomes of resveratrol supplementation in pregnant nonhuman primates

Resveratrol has been proposed as a potential therapeutic to improve metabolic health during pregnancy, yet little is known about the fetal effects of this maternal dietary supplement. We hypothesized that when administered to pregnant nonhuman primates (NHPs), resveratrol would increase uterine blood flow and mitigate the harmful consequences of maternal Western‐style diet (WSD) consumption. NHPs were fed a WSD (36% fat) supplemented with 0.37% resveratrol throughout pregnancy. Outcomes were compared with cohorts fed WSD alone and control chow (14% fat) to distinguish between WSD and resveratrol‐specific effects in these animals. In the early third trimester, uterine blood flow was measured by Doppler ultrasound before fetal delivery and tissue collection. Resveratrol resulted in 30% maternal weight loss and improved glucose tolerance, increased uterine artery volume blood flow, and decreased placental inflammation and liver triglyceride deposition. In addition, fetal pancreatic mass was enlarged by 42%, with a 12‐fold increase in proliferation by Ki67 immunohistochemistry. These results demonstrate that resveratrol use during pregnancy yields improvements in maternal and placental phenotype with beneficial effects in the fetal liver but an unexplained and concerning alteration in fetal pancreatic development, which strongly cautions against the use of resveratrol by pregnant women.—Roberts, V. H. J., Pound, L. D., Thorn, S. R., Gillingham, M. B., Thornburg, K. L., Friedman, J. E., Frias, A. E., Grove, K. L. Beneficial and cautionary outcomes of resveratrol supplementation in pregnant nonhuman primates. FASEB J. 28, 2466–2477 (2014). www.fasebj.org

Substrate oxidation and cardiac performance during exercise in disorders of long chain fatty acid oxidation.

BACKGROUND The use of long-chain fatty acids (LCFAs) for energy is inhibited in inherited disorders of long-chain fatty acid oxidation (FAO). Increased energy demands during exercise can lead to cardiomyopathy and rhabdomyolysis. Medium-chain triglycerides (MCTs) bypass the block in long-chain FAO and may provide an alternative energy substrate to exercising muscle. OBJECTIVES To determine the influence of isocaloric MCT versus carbohydrate (CHO) supplementation prior to exercise on substrate oxidation and cardiac workload in participants with carnitine palmitoyltransferase 2 (CPT2), very long-chain acyl-CoA dehydrogenase (VLCAD) and long-chain 3-hydroxyacyl CoA dehydrogenase (LCHAD) deficiencies. DESIGN Eleven subjects completed two 45-minute, moderate intensity, treadmill exercise studies in a randomized crossover design. An isocaloric oral dose of CHO or MCT-oil was administered prior to exercise; hemodynamic and metabolic indices were assessed during exertion. RESULTS When exercise was pretreated with MCT, respiratory exchange ratio (RER), steady state heart rate and generation of glycolytic intermediates significantly decreased while circulating ketone bodies significantly increased. CONCLUSIONS MCT supplementation prior to exercise increases the oxidation of medium chain fats, decreases the oxidation of glucose and acutely lowers cardiac workload during exercise for the same amount of work performed when compared with CHO pre-supplementation. We propose that MCT may expand the usable energy supply, particularly in the form of ketone bodies, and improve the oxidative capacity of the heart in this population.

Observations regarding retinopathy in mitochondrial trifunctional protein deficiencies

Although the retina is thought to primarily rely on glucose for fuel, inherited deficiency of one or more activities of mitochondrial trifunctional protein results in a pigmentary retinopathy leading to vision loss. Many other enzymatic deficiencies in fatty acid oxidation pathways have been described, none of which results in retinal complications. The etiology of retinopathy among patients with defects in trifunctional protein is unknown. Trifunctional protein is a heteroctomer; two genes encode the alpha and beta subunits of TFP respectively, HADHA and HADHB. A common mutation in HADHA, c.1528G>C, leads to a single amino acid substitution, p. Glu474Gln, and impairs primarily long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) activity leading to LCHAD deficiency (LCHADD). Other mutations in HADHA or HADHB often lead to significant reduction in all three enzymatic activities and result in trifunctional protein deficiency (TFPD). Despite many similarities in clinical presentation and phenotype, there is growing evidence that they can result in different chronic complications. This review will outline the clinical similarities and differences between LCHADD and TFPD, describe the course of the associated retinopathy, propose a genotype/phenotype correlation with the severity of retinopathy, and discuss the current theories about the etiology of the retinopathy.

Evidence for an Association Between Infant Mortality and a Carnitine Palmitoyltransferase 1A Genetic Variant

OBJECTIVE: Alaska Native and other circumpolar indigenous populations have historically experienced high infant mortality rates, for unknown reasons. Through routine newborn screening, Alaskan and Canadian indigenous infants have been found to have a high frequency of a single sequence variant (c.1436C→T) in the gene coding for carnitine palmitoyltransferase type 1A (CPT1A). We sought to determine whether these 2 findings were related. METHODS: As part of a quality control exercise at the Alaskan Newborn Metabolic Screening Program, we conducted genotyping for 616 consecutively born, Alaska Native infants and reviewed their medical records. We conducted an ecological analysis comparing Census area–level variant CPT1A allele frequency and historical Alaska Native infant, postneonatal, and neonatal mortality rates. RESULTS: Infant death was identified for 5 of 152 infants homozygous for the c.1436C→T sequence variant (33 deaths per 1000 live births), 2 of 219 heterozygous infants (9 deaths per 1000 live births), and 0 of 245 infants carrying no copies of the variant allele (χ2 = 9.2; P = .01). All 7 cases of infant death had some evidence of an infectious process at the time of death, including 5 with respiratory infections. Census areas with the highest frequency of the variant allele had the highest historical infant, postneonatal, and neonatal mortality rates. CONCLUSIONS: Our data provide preliminary evidence that a highly prevalent CPT1A variant found among Alaska Native and other indigenous circumpolar populations may help explain historically high infant mortality rates. Larger definitive studies are needed.

Prevalence and Distribution of the c.1436C→T Sequence Variant of Carnitine Palmitoyltransferase 1A among Alaska Native Infants

OBJECTIVES To use genotype analysis to determine the prevalence of the c.1436C→T sequence variant in carnitine palmitoyltransferase 1A (CPT1A) among Alaskan infants, and evaluate the sensitivity of newborn screening by tandem mass spectrometry (MS/MS) to identify homozygous infants. STUDY DESIGN We compared MS/MS and DNA analyses of 2409 newborn blood spots collected over 3 consecutive months. RESULTS Of 2409 infants, 166 (6.9%) were homozygous for the variant, all but one of whom were of Alaska Native race. None of the homozygous infants was identified by MS/MS on the first newborn screen using a C0/C16 + C18 cutoff of 130. Among 633 Alaska Native infants, 165 (26.1%) were homozygous and 218 (34.4%) were heterozygous for the variant. The prevalence was highest in Alaskas northern/western regions (51.2% of 255 infants homozygous; allele frequency, 0.7). CONCLUSIONS The CPT1A c.1436C→T variant is prevalent among some Alaska Native peoples, but newborn screening using current MS/MS cutoffs is not an effective means to identify homozygous infants. The clinical consequences of the partial CPT1A deficiency associated with this variant are unknown. If effects are substantial, revision of newborn screening, including Alaska-specific MS/MS cutoffs and confirmatory genotyping, may be needed.

Influence of high fat diet and resveratrol supplementation on placental fatty acid uptake in the Japanese macaque

INTRODUCTION Adequate maternal supply and placental delivery of long chain polyunsaturated fatty acids (LCPUFA) is essential for normal fetal development. In humans, maternal obesity alters placental FA uptake, though the impact of diet remains uncertain. The fatty fetal liver observed in offspring of Japanese macaques fed a high fat diet (HFD) was prevented with resveratrol supplementation during pregnancy. We sought to determine the effect of HFD and resveratrol, a supplement with insulin-sensitizing properties, on placental LCPUFA uptake in this model. METHODS J. macaques were fed control chow (15% fat, n = 5), HFD (35% fat, n = 10) or HFD containing 0.37% resveratrol (n = 5) prior to- and throughout pregnancy. At ∼ 130 d gestation (term = 173 d), placentas were collected by caesarean section. Fatty acid uptake studies using (14)C-labeled oleic acid, arachidonic acid (AA) and docosahexanoic acid (DHA) were performed in placental explants. RESULTS Resveratrol supplementation increased placental uptake of DHA (P < 0.05), while HFD alone had no measurable effect. Resveratrol increased AMP-activated protein kinase activity and mRNA expression of the fatty acid transporters FATP-4, CD36 and FABPpm (P < 0.05). Placental DHA content was decreased in HFD dams; resveratrol had no effect on tissue fatty acid profiles. DISCUSSION Maternal HFD did not significantly affect placental LCPUFA uptake. Furthermore, resveratrol stimulated placental DHA uptake capacity, AMPK activation and transporter expression. Placental handling of DHA is particularly sensitive to the dramatic alterations in the maternal metabolic phenotype and placental AMPK activity associated with resveratrol supplementation.