The burden of size and growth for the juveniles of large mammalian herbivores: Structural and functional constraints in the feeding biology of juveniles relative to adults in red kangaroos, Osphranter rufus.

Juvenile mammals in their postweaning developmental stages face many challenges in transitioning to adulthood. Among large grazing species such as ruminant bovids and cervids, an overarching challenge is acquiring and processing sufficient nutrients to survive and grow, with a gut that may not yet be fully developed. Marsupial kangaroos of Australia face similar challenges; they also digest vegetation by fermentation in a large foregut. In red kangaroos, Osphranter rufus (=Macropus rufus), the dominant species of Australia's arid interior, females may breed continuously; however, juvenile recruitment to the adult population is irregular and coincident with sporadic rainfall.As compared with adult females, the nutritional requirements of juvenile O. rufus are high in relation to their body mass (BM), largely due to the cost of their rapid growth. We examined processes that juveniles have in their morphology, physiology, and behaviors to meet their elevated nutritional needs, by comparing recently weaned juveniles of both sexes and adult female O. rufus in their desert habitat. Features studied include relative body sizes, relative dimensions, and capacities of principal gut regions, the foregut, small intestine, caecum, and large intestine with rectum. Also examined were digesta attributes and rates of digesta excretion. Additionally, the rates of change in skull parameters and dental characteristics to maturity were assessed. Field determinations of diet choice were made for both age classes.In juveniles, the content masses of major gut structures were related to body mass (BM), as were those of adult females, that is, ~BM1.0. In both age classes, the digesta mass of the foreguts exceeded 75% of the total digesta mass. Diets of both juvenile and adult O. rufus largely focused on grasses. Juveniles had higher rates of digesta excretion while foraging than adults. In addition, the foregut contents in juveniles occupy proportionally less of the total gut than in adult females. Together, the higher excretion rate and smaller relative foregut of juveniles suggest that they necessarily focus on forage that can be rapidly digested, such as young, green grasses, or herbage.Comparison of the skulls of juveniles and adults revealed how this harvest can occur. Relative to BM, juveniles had skulls of larger volume than adults. Additionally, during growth the skull lengthens proportionally faster than increasing BM. By weaning, the dimensions of the incisor bite of juveniles neared those of adult females. The area of wear on premolars/molars increased only slowly relative to the development of incisors, further pointing to juveniles selecting more digestible forage than adults. The intermittent availability of such forage, principally young grasses, appears key to the significant recruitment into the O. rufus population in their arid habitat.

Advances and unresolved challenges in the structural characterization of isomeric lipids.

As the field of lipidomics grows and its application becomes wide and varied it is important that we don't forget its foundation, i.e. the identification and measurement of molecular lipids. Advances in liquid chromatography and the emergence of ion mobility as a useful tool in lipid analysis are allowing greater separation of lipid isomers than ever before. At the same time, novel ion activation techniques, such as ozone-induced dissociation, are pushing lipid structural characterization by mass spectrometry to new levels. Nevertheless, the quantitative capacity of these techniques is yet to be proven and further refinements are required to unravel the high level of lipid complexity found in biological samples. At present there is no one technique capable of providing full structural characterization of lipids from a biological sample. There are however, numerous techniques now available (as discussed in this review) that could be deployed in a targeted approach. Moving forward, the combination of advanced separation and ion activation techniques is likely to provide mass spectrometry-based lipidomics with its best opportunity to achieve complete molecular-level lipid characterization and measurement from complex mixtures.

Fatty acid composition of the anterior cingulate cortex indicates a high susceptibility to lipid peroxidation in Parkinson's disease.

BACKGROUND: Oxidative stress contributes to Parkinson's disease (PD) etiology. Although previous studies have focused on sources of free radical formation in brain regions affected by PD, less is known regarding changes in lipid composition and the implications for susceptibility to peroxidation. OBJECTIVE: To assess fatty acid profiles from control and PD tissues that are susceptible to PD pathology but devoid of severe destruction. METHODS: We used gas chromatography methods to assess fatty acid profiles from control (n = 10) and PD (n = 9) postmortem tissues. We focused on the anterior cingulate cortex (ACC), a region that accumulates alpha-synuclein, but does not undergo severe destruction, and compared this to the occipital cortex, a region that is pathologically spared. RESULTS: Our data indicate a significant 33% increase in the proportion of polyunsaturated fatty acids (mol%) present in the PD ACC as compared to control ACC. Increases in highly unsaturated 22:5n-6 and 22:6n-3 fatty acids were particularly pronounced (109% and 73%, respectively). Calculation of a peroxidation index (accounting for total fatty acyl double bounds) indicated a 44% increase in susceptibility of the PD ACC to lipid peroxidation compared to control ACC. Such differences were not detected in the occipital cortex from the same donors. Assessment of F2-isprostane levels confirmed that PD tissue lipids were more oxidized than controls. CONCLUSIONS: The global composition of fatty acids in the PD ACC is altered in a way that increases susceptibility to peroxidation in a region-specific manner. This has important implications for PD, supporting the oxidative stress hypothesis of PD pathogenesis.

Lysosomal-associated membrane protein 2 isoforms are differentially affected in early Parkinson's disease.

Lysosomes are the primary catabolic compartment for the degradation of intracellular proteins through autophagy. The presence of abnormal intracellular α-synuclein-positive aggregates in Parkinson's disease (PD) indicates that the degradative capacity of lysosomes is impaired in PD. Specific dysfunction of chaperone-mediated autophagy (CMA) in PD is suggested by reductions in the CMA membrane receptor, lysosomal-associated membrane protein (LAMP) 2A, although whether LAMP2A is the only LAMP2 isoform affected by PD is unknown. Messenger RNA (mRNA) and protein expression of all three LAMP2 isoforms was assessed in brain extracts from regions with and without PD-related increases in α-synuclein in autopsy samples from subjects in the early pathological stage of PD (n = 9), compared to age- and postmortem delay-matched controls (n = 10). In the early stages of PD, mRNA expression of all LAMP2 isoforms was not different from controls, with LAMP2B and LAMP2C protein levels also unchanged in PD. The selective loss of LAMP2A protein directly correlated with the increased levels of α-synuclein and decreased levels of the CMA chaperone heat shock cognate protein 70 in the same PD samples, as well as with the accumulation of cytosolic CMA substrate proteins. Our data show that LAMP2 protein isoforms are differentially affected in the early stages of PD, with LAMP2A selectively reduced in association with increased α-synuclein, and suggests that dysregulation of CMA-mediated protein degradation occurs before substantial α-synuclein aggregation in PD.

Altered ceramide acyl chain length and ceramide synthase gene expression in Parkinson's disease.

Genetic studies have provided increasing evidence that ceramide homeostasis plays a role in neurodegenerative diseases including Parkinson's disease (PD). It is known that the relative amounts of different ceramide molecular species, as defined by their fatty acyl chain length, regulate ceramide function in lipid membranes and in signaling pathways. In the present study we used a comprehensive sphingolipidomic case-control approach to determine the effects of PD on ceramide composition in postmortem brain tissue from the anterior cingulate cortex (a region with significant PD pathology) and the occipital cortex (spared in PD), also assessing mRNA expression of the major ceramide synthase genes that regulate ceramide acyl chain composition in the same tissue using quantitative PCR. In PD anterior cingulate cortex but not occipital cortex, total ceramide and sphingomyelin levels were reduced from control levels by 53% (P < 0.001) and 42% (P < 0.001), respectively. Of the 13 ceramide and 15 sphingomyelin molecular lipid species identified and quantified, there was a significant shift in the ceramide acyl chain composition toward shorter acyl chain length in the PD anterior cingulate cortex. This PD-associated change in ceramide acyl chain composition was accompanied by an upregulation of ceramide synthase-1 gene expression, which we consider may represent a response to reduced ceramide levels. These data suggest a significant shift in ceramide function in lipid membranes and signaling pathways occurs in regions with PD pathology. Identifying the regulatory mechanisms precipitating this change may provide novel targets for future therapeutics.

Reduced glucocerebrosidase is associated with increased α-synuclein in sporadic Parkinson's disease.

Heterozygous mutations in GBA1, the gene encoding lysosomal glucocerebrosidase, are the most frequent known genetic risk factor for Parkinson's disease. Reduced glucocerebrosidase and α-synuclein accumulation are directly related in cell models of Parkinson's disease. We investigated relationships between Parkinson's disease-specific glucocerebrosidase deficits, glucocerebrosidase-related pathways, and α-synuclein levels in brain tissue from subjects with sporadic Parkinson's disease without GBA1 mutations. Brain regions with and without a Parkinson's disease-related increase in α-synuclein levels were assessed in autopsy samples from subjects with sporadic Parkinson's disease (n = 19) and age- and post-mortem delay-matched controls (n = 10). Levels of glucocerebrosidase, α-synuclein and related lysosomal and autophagic proteins were assessed by western blotting. Glucocerebrosidase enzyme activity was measured using a fluorimetric assay, and glucocerebrosidase and α-synuclein messenger RNA expression determined by quantitative polymerase chain reaction. Related sphingolipids were analysed by mass spectrometry. Multivariate statistical analyses were performed to identify differences between disease groups and regions, with non-parametric correlations used to identify relationships between variables. Glucocerebrosidase protein levels and enzyme activity were selectively reduced in the early stages of Parkinson's disease in regions with increased α-synuclein levels although limited inclusion formation, whereas GBA1 messenger RNA expression was non-selectively reduced in Parkinson's disease. The selective loss of lysosomal glucocerebrosidase was directly related to reduced lysosomal chaperone-mediated autophagy, increased α-synuclein and decreased ceramide. Glucocerebrosidase deficits in sporadic Parkinson's disease are related to the abnormal accumulation of α-synuclein and are associated with substantial alterations in lysosomal chaperone-mediated autophagy pathways and lipid metabolism. Our data suggest that the early selective Parkinson's disease changes are likely a result of the redistribution of cellular membrane proteins leading to a chronic reduction in lysosome function in brain regions vulnerable to Parkinson's disease pathology.

Polyunsaturated fats, membrane lipids and animal longevity.

Fatty acids are essential for life because they are essential components of cellular membranes. Lower animals can synthesize all four classes of fatty acids from non-lipid sources, but both omega-6 and omega-3 cannot be synthesized de novo by 'higher' animals and are therefore essential components of their diet. The relationship between normal variation in diet fatty acid composition and membrane fatty acid composition is little investigated. Studies in the rat show that, with respect to the general classes of fatty acids (saturated, monounsaturated and polyunsaturated) membrane fatty acid composition is homeostatically regulated despite diet variation. This is not the case for fatty acid composition of storage lipids, which responds to diet variation. Polyunsaturated fatty acids are important determinants of physical and chemical properties of membranes. They are the substrates for lipid peroxidation and it is possible to calculate a peroxidation index (PI) for a particular membrane composition. Membrane PI appears to be homeostatically regulated with respect to diet PI. Membrane fatty acid composition varies among species and membrane PI is inversely correlated to longevity in mammals, birds, bivalve molluscs, honeybees and the nematode Caenorhabditis elegans.

An improved high-throughput lipid extraction method for the analysis of human brain lipids.

We have developed a protocol suitable for high-throughput lipidomic analysis of human brain samples. The traditional Folch extraction (using chloroform and glass-glass homogenization) was compared to a high-throughput method combining methyl-tert-butyl ether (MTBE) extraction with mechanical homogenization utilizing ceramic beads. This high-throughput method significantly reduced sample handling time and increased efficiency compared to glass-glass homogenizing. Furthermore, replacing chloroform with MTBE is safer (less carcinogenic/toxic), with lipids dissolving in the upper phase, allowing for easier pipetting and the potential for automation (i.e., robotics). Both methods were applied to the analysis of human occipital cortex. Lipid species (including ceramides, sphingomyelins, choline glycerophospholipids, ethanolamine glycerophospholipids and phosphatidylserines) were analyzed via electrospray ionization mass spectrometry and sterol species were analyzed using gas chromatography mass spectrometry. No differences in lipid species composition were evident when the lipid extraction protocols were compared, indicating that MTBE extraction with mechanical bead homogenization provides an improved method for the lipidomic profiling of human brain tissue.

Fatty acid composition of membrane bilayers: importance of diet polyunsaturated fat balance.

In one of the most extensive analyses to date we show that the balance of diet n-3 and n-6 polyunsaturated fatty acids (PUFA) is the most important determinant of membrane composition in the rat under 'normal' conditions. Young adult male Sprague-Dawley rats were fed one of twelve moderate-fat diets (25% of total energy) for 8weeks. Diets differed only in fatty acid (FA) profiles, with saturate (SFA) content ranging 8-88% of total FAs, monounsaturate (MUFA) 6-65%, total PUFA 4-81%, n-6 PUFA 3-70% and n-3 PUFA 1-70%. Diet PUFA included only essential FAs 18:2n-6 and 18:3n-3. Balance between n-3 and n-6 PUFA is defined as the PUFA balance (n-3 PUFA as % of total PUFA) and ranged 1-86% in the diets. FA composition was measured for brain, heart, liver, skeletal muscle, erythrocytes and plasma phospholipids, as well as adipose tissue and plasma triglycerides. The conformer-regulator model was used (slope=1 indicates membrane composition completely conforming to diet). Extensive changes in diet SFA, MUFA and PUFA had minimal effect on membranes (average slopes 0.01, 0.07, 0.07 respectively), but considerable influence on adipose tissue and plasma triglycerides (average slopes 0.27, 0.53, 0.47 respectively). Diet balance between n-3 and n-6 PUFA had a biphasic influence on membrane composition. When n-3 PUFA<10% of total PUFA, membrane composition completely conformed to diet (average slope 0.95), while diet PUFA balance>10% had little influence (average slope 0.19). The modern human diet has an average PUFA balance ~10% and this will likely have significant health implications.

Membrane fatty acid composition of rat skeletal muscle is most responsive to the balance of dietary n-3 and n-6 PUFA.

The present study quantifies the relationships between diet fatty acid profile and fatty acid composition of rat skeletal muscle phospholipids. Young adult male Sprague-Dawley rats were fed, for 8 weeks, on one of twelve moderate-fat diets (25 % of total energy) differing only in fatty acid profile. SFA content ranged from 8-88 % of total fatty acids, MUFA 6-65 %, total PUFA 4-81 %, n-6 PUFA 3-70 % and n-3 PUFA 1-70 %. Diet PUFA included only essential fatty acids 18 : 2n-6 and 18 : 3n-3. The balance between n-3 and n-6 PUFA (PUFA balance) in the diet ranged from 1 : 99 to 86 : 14 % n-3 PUFA:n-6 PUFA. The slope of muscle phospholipid composition plotted against diet composition quantifies the response of muscle membrane composition to dietary fat (0, no response; 1, complete conformity with diet). The resulting slopes were 0.02 (SFA), 0.10 (PUFA), 0.11 (MUFA), 0.14 (n-3 PUFA) and 0.23 (n-6 PUFA). The response to PUFA balance was biphasic with a slope of 0.98 below 10 % diet PUFA balance and 0.16 above 10 %. Thus, low diet PUFA balance has greater influence on muscle composition than 18-carbon n-3 or n-6 PUFA individually. Equations provided may allow prediction of muscle composition for other diet studies. Diet PUFA balance dramatically affects muscle 20 : 4n-6 and 22 : 6n-3. This may have significant implications for some disease states in human subjects.

Phospholipid composition of the rat lens is independent of diet.

Dietary fatty acids are known to influence the phospholipid composition of many tissues in the body, with lipid turnover occurring rapidly. The aim of this study was to investigate whether changes in the fatty acid composition of the diet can affect the phospholipid composition of the lens. Male Sprague-Dawley rats were fed three diets with distinct profiles in both essential and non-essential fatty acids. After 8 weeks, lenses and skeletal muscle were removed, and the lenses sectioned into nuclear and cortical regions. In these experiments, the lens cortex was synthesised during the course of the variable lipid diet. Phospholipids were then identified by electrospray ionisation tandem mass spectrometry, and quantified via the use of internal standards. The phospholipid compositions of the nuclear and cortical regions of the lens differed slightly between the two regions, but comparison of the equivalent regions across the diet groups showed remarkable similarity. In contrast, the phospholipid composition of skeletal muscle (medial gastrocnemius) in these rats varied significantly. This study provides the first direct evidence to show that the phospholipid composition of the lens is tightly regulated and thus appears to be independent of diet. As phospholipids determine membrane fluidity and influence the activity and function of integral membrane proteins, regulation of their composition may be important for the function of the lens.