Metabolomics (Non-Targeted) of Induced Type 2 Diabetic Sprague Dawley Rats Comorbid with a Tissue-Dwelling Nematode Parasite.

Type 2 diabetes is a non-communicable metabolic syndrome that is characterized by the dysfunction of pancreatic ß-cells and insulin resistance. Both animal and human studies have been conducted, demonstrating that helminth infections are associated with a decreased prevalence of type 2 diabetes mellitus (T2DM). However, there is a paucity of information on the impact that helminths have on the metabolome of the host and how the infection ameliorates T2DM or its progression. Therefore, this study aimed at using a non-targeted metabolomics approach to systematically identify differentiating metabolites from serum samples of T2DM-induced Sprague Dawley (SD) rats infected with a tissue-dwelling nematode, Trichinella zimbabwensis, and determine the metabolic pathways impacted during comorbidity. Forty-five male SD rats with a body weight between 160 g and 180 g were used, and these were randomly selected into control (non-diabetic and not infected with T. zimbabwensis) (n = 15) and T2DM rats infected with T. zimbabwensis (TzDM) (n = 30). The results showed metabolic separation between the two groups, where d-mannitol, d-fructose, and glucose were upregulated in the TzDM group, when compared to the control group. L-tyrosine, glycine, diglycerol, L-lysine, and L-hydroxyproline were downregulated in the TzDM group when compared to the control group. Metabolic pathways which were highly impacted in the TzDM group include biotin metabolism, carnitine synthesis, and lactose degradation. We conclude from our study that infecting T2DM rats with a tissue-dwelling nematode, T. zimbabwensis, causes a shift in the metabolome, causing changes in different metabolic pathways. Additionally, the infection showed the potential to regulate or improve diabetes complications by causing a decrease in the amino acid concentration that results in metabolic syndrome.

Metabolomics of Type 2 Diabetes Mellitus in Sprague Dawley Rats-In Search of Potential Metabolic Biomarkers.

Type 2 diabetes mellitus (T2DM) is an expanding global health concern, closely associated with the epidemic of obesity. Individuals with diabetes are at high risk for microvascular and macrovascular complications, which include retinopathy, neuropathy, and cardiovascular comorbidities. Despite the availability of diagnostic tools for T2DM, approximately 30-60% of people with T2DM in developed countries are never diagnosed or detected. Therefore, there is a strong need for a simpler and more reliable technique for the early detection of T2DM. This study aimed to use a non-targeted metabolomic approach to systematically identify novel biomarkers from the serum samples of T2DM-induced Sprague Dawley (SD) rats using a comprehensive two-dimensional gas chromatography coupled with a time-of-flight mass spectrometry (GCxGC-TOF/MS). Fifty-four male Sprague Dawley rats weighing between 160-180 g were randomly assigned into two experimental groups, namely the type 2 diabetes mellitus group (T2DM) (n = 36) and the non-diabetic control group (n = 18). Results from this study showed that the metabolite signature of the diabetic rats was different from that of the non-diabetic control group. The most significantly upregulated metabolic pathway was aminoacyl-t-RNA biosynthesis. Metabolite changes observed between the diabetic and non-diabetic control group was attributed to the increase in amino acids, such as glycine, L-asparagine, and L-serine. Aromatic amino acids, including L-tyrosine, were associated with the risk of future hyperglycemia and overt diabetes. The identified potential biomarkers depicted a good predictive value of more than 0.8. It was concluded from the results that amino acids that were associated with impaired insulin secretion were prospectively related to an increase in glucose levels. Moreover, amino acids that were associated with impaired insulin secretion were prospectively related to an increase in glucose levels.

Ion regulation in a freshwater crab, Potamonautes warreni: The effects of trace metal exposure.

Crustaceans inhabiting metal-contaminated freshwaters are susceptible to toxic insult to their osmoregulatory systems. The main osmoregulatory organs of decapod crustaceans, the gills, are continually bathed in freshwater and are therefore at risk from trace metal impacts. The effects of chronic (21 d) exposure to raised dissolved concentrations of Zn, Cd, Cu and Pb on aspects of hydromineral balance were investigated in Potamonautes warreni, a freshwater crab endemic to rivers in South Africa at potential risk from trace metal contamination from mining operations. Generally, hydromineral balance of P. warreni was tolerant to chronic metal exposures although sublethal cadmium exposure of 860 µg.l-1 for 21 days resulted in a reduced sodium concentration in the haemolymph. A chronic exposure to 43 µg.l-1 cadmium produced an elevated maximum unidirectional sodium uptake, possibly resulting from acclimation to the metal exposure. Branchial Na+/K+-ATPase and V-Type H+-ATPase activity were not affected by chronic in vivo Cd (43 µg.l-1) and Zn (500 µg.l-1) exposures. An important aspect of ameliorating metal toxicity may be through antioxidants and therefore the effects of applying a reducing agent were tested following in vitro metal treatment. Inhibition of Na+/K+-ATPase could be prevented by pre-incubation with a reducing agent, indicating the importance of antioxidants in reducing metal toxicity in this species. Although this study demonstrates the physiological resilience of P. warreni to dissolved trace metal impacts, the energetic consequences of long-term exposure are as yet not known.

The cross-tissue metabolic response of abalone (Haliotis midae) to functional hypoxia.

Functional hypoxia is a stress condition caused by the abalone itself as a result of increased muscle activity, which generally necessitates the employment of anaerobic metabolism if the activity is sustained for prolonged periods. With that being said, abalone are highly reliant on anaerobic metabolism to provide partial compensation for energy production during oxygen-deprived episodes. However, current knowledge on the holistic metabolic response for energy metabolism during functional hypoxia, and the contribution of different metabolic pathways and various abalone tissues towards the overall accumulation of anaerobic end-products in abalone are scarce. Metabolomics analysis of adductor muscle, foot muscle, left gill, right gill, haemolymph and epipodial tissue samples indicated that South African abalone (Haliotis midae) subjected to functional hypoxia utilises predominantly anaerobic metabolism, and depends on all of the main metabolite classes (proteins, carbohydrates and lipids) for energy supply. Functional hypoxia caused increased levels of anaerobic end-products: lactate, alanopine, tauropine, succinate and alanine. Also, elevation in arginine levels was detected, confirming that abalone use phosphoarginine to generate energy during functional hypoxia. Different tissues showed varied metabolic responses to hypoxia, with functional hypoxia showing excessive changes in the adductor muscle and gills. From this metabolomics investigation, it becomes evident that abalone are metabolically able to produce sufficient amounts of energy when functional hypoxia is experienced. Also, tissue interplay enables the adjustment of H. midae energy requirements as their metabolism shifts from aerobic to anaerobic respiration during functional hypoxia.This article has an associated First Person interview with the first author of the paper.

Uncovering the metabolic response of abalone (Haliotis midae) to environmental hypoxia through metabolomics.

INTRODUCTION: Oxygen is essential for metabolic processes and in the absence thereof alternative metabolic pathways are required for energy production, as seen in marine invertebrates like abalone. Even though hypoxia has been responsible for significant losses to the aquaculture industry, the overall metabolic adaptations of abalone in response to environmental hypoxia are as yet, not fully elucidated. OBJECTIVE: To use a multiplatform metabolomics approach to characterize the metabolic changes associated with energy production in abalone (Haliotis midae) when exposed to environmental hypoxia. METHODS: Metabolomics analysis of abalone adductor and foot muscle, left and right gill, hemolymph, and epipodial tissue samples were conducted using a multiplatform approach, which included untargeted NMR spectroscopy, untargeted and targeted LC-MS spectrometry, and untargeted and semi-targeted GC-MS spectrometric analyses. RESULTS: Increased levels of anaerobic end-products specific to marine animals were found which include alanopine, strombine, tauropine and octopine. These were accompanied by elevated lactate, succinate and arginine, of which the latter is a product of phosphoarginine breakdown in abalone. Primarily amino acid metabolism was affected, with carbohydrate and lipid metabolism assisting with anaerobic energy production to a lesser extent. Different tissues showed varied metabolic responses to hypoxia, with the largest metabolic changes in the adductor muscle. CONCLUSIONS: From this investigation, it becomes evident that abalone have well-developed (yet understudied) metabolic mechanisms for surviving hypoxic periods. Furthermore, metabolomics serves as a powerful tool for investigating the altered metabolic processes in abalone.

Possible maternal offloading of metals in the plasma, uterine and capsule fluid of pregnant ragged-tooth sharks (Carcharias taurus) on the east coast of South Africa.

We studied the possible metal offloading onto the progeny of three pregnant female ragged-tooth sharks (Carcharias taurus) (C. taurus). The presences of five metals, i.e. aluminium (Al), arsenic (As), cadmium (Cd), lead (Pb) and selenium (Se) were validated by mass spectrometry in the maternal plasma as well as the intracapsular and uterine fluids (UF) in which embryos develop. Metals were ranked in a decreasing concentration as follows: Plasma: As > Al > Se > Pb > Cd; ICF: As > Se > Al > Cd > Pb and UF: As > Se > Al > Cd > Pb. As was present in the highest concentration in all three sharks. Al, Pb and Cd were found to be the highest within the plasma, while concentrations of Se were similar in all three fluids. These results indicate that C. taurus embryos are exposed to metals during early development, but the impact of this exposure remains unknown. To the best of our knowledge, this is the first investigation to confirm the presence of metals in the fluids that surround the developing C. taurus embryos, a species that is already listed as vulnerable.

The involvement of the hypothalamopituitary-adrenocortical axis in stress physiology and its significance in the assessment of animal welfare in cattle.

The intensification of cattle production has raised concern for animal welfare due to the stress that is associated with farming practices. The welfare of an animal is determined by the animal's ability to cope with or adapt to its continuously changing environment and the biological cost that is associated with this adaptation and maintenance. Stressors arise from various psychological, physiological and physical aspects of farming practices due to management and human-cattle interactions. Measuring the activity of the hypothalamopituitary-adrenocortical (HPA) axis with plasma cortisol levels is a useful method for determining the effects of stress on animals as it is stimulated at the onset of a perceived stress. The activation of the HPA axis affects various target tissues or systems and can result in suppression of the immune system, increased susceptibility to disease and adverse effects on reproductive success in prenatal and neonatal calves. Although some levels of stress associated with farming practices are unavoidable, improvements in farming methods need to be implemented in order to maintain or increase the efficiency of cattle production in a way that does not compromise the welfare of the animal.

From untargeted LC-QTOF analysis to characterisation of opines in abalone adductor muscle: Theory meets practice.

Abalone have a unique ability to use pyruvate, various amino acids and dehydrogenases, to produce opines as means to prevent the accumulation of NADH during anaerobic conditions. In this study, the theoretical masses, formulae and fragment patterns of butylated opines were used to predict which of these compounds could be found in the abalone adductor muscle using untargeted liquid chromatography quadrupole time-of flight-mass spectrometry. These findings were validated using synthesised opine standards. In essence alanopine, lysopine, strombine and tauropine produced in abalone adductor muscle could be characterised using the highest identification confidence levels.

Differential responses of juvenile and adult South African abalone (Haliotis midae Linnaeus) to low and high oxygen levels.

Marine invertebrates have evolved multiple responses to naturally variable environmental oxygen, all aimed at either maintaining cellular oxygen homeostasis or limiting cellular damage during or after hypoxic or hyperoxic events. We assessed organismal (rates of oxygen consumption and ammonia excretion) and cellular (heat shock protein expression, anti-oxidant enzymes) responses of juvenile and adult abalone exposed to low (~83% of saturation), intermediate (~95% of saturation) and high (~115% of saturation) oxygen levels for one month. Using the Comet assay, we measured DNA damage to determine whether the observed trends in the protective responses were sufficient to prevent oxidative damage to cells. Juveniles were unaffected by moderately hypoxic and hyperoxic conditions. Elevated basal rates of superoxide dismutase, glutathione peroxidase and catalase were sufficient to prevent DNA fragmentation and protein damage. Adults, with their lower basal rate of anti-oxidant enzymes, had increased DNA damage under hypoxic and hyperoxic conditions, indicating that the antioxidant enzymes were unable to prevent oxidative damage under hypoxic and hyperoxic conditions. The apparent insensitivity of juvenile abalone to decreased and increased oxygen might be related to their life history and development in algal and diatom biofilms where they are exposed to extreme diurnal fluctuations in dissolved oxygen levels.

Acute responses of brown mussel (Perna perna) exposed to sub-lethal copper levels: integration of physiological and cellular responses.

This study examined the effect of sub-lethal copper levels on selected physiological and cellular responses of the marine bivalve Perna perna. Animals were exposed to five environmentally relevant concentrations of 12.5, 25.0, 37.5 and 50.0 µg L⁻¹ copper and metal accumulation was found to be significantly increased at the two higher copper concentrations after 24 h of exposure. Physiological responses found to increase during acute copper exposure included mucus secretion rate (at 25 and 50 µg L⁻¹ copper), nitrogen excretion rates and oxygen consumption rates (both at 25 and 50 µg L⁻¹ copper). Perna perna changed its substrate utilisation at 25, 37.5 and 50 µg L⁻¹ copper in favour of protein-based metabolism. A higher degree of ROS induced DNA damage was observed at acute exposure to 37.5 and 50 µg L⁻¹ copper. Filtration rate was unchanged during acute copper exposure. A model is proposed that integrates cellular and physiological responses to copper during short-term acute and long-term chronic exposures.

Molecules to migration: pressures of life.

The highly successful Fourth International Conference in Africa for Comparative Physiology and Biochemistry (ICA-CPB) was held in the Maasai Mara National Reserve in July 2008. The theme of the meeting was "Molecules to Migration: Pressures of Life." To enhance the theme, the venue and timing of the meeting were chosen to coincide with the arrival of approximately 1.4 million wildebeest on their annual migration from the Serengeti National Park in Tanzania. Like the three previous ICA-CPB meetings, the discussion topics and the resulting collection of synthesia presented here were very diverse. The articles in this special collection reflect the authors' interest in broadening our understanding of the field of comparative physiology and biochemistry and their commitment to engaging in global research with international colleagues. These articles are brief, synthetic reviews integrating information presented at and inspired by the meeting. From seasonal migration and reproduction in birds, to cardiovascular system development in vertebrates, to strategies for hypoxia survival, papers range from specific to broad interactions. What they all have in common: they increase our understanding of how animals are affected by and respond to the pressures of life.

Animals and environments: resisting schisms in comparative physiology and biochemistry.

The articles in this volume are a product of the enthusiasm shown by delegates to meet in a remote corner of southern Africa and to discuss comparative physiology and biochemistry in their wider interpretation and future course. This collection reflects a small but long-standing commitment to fostering the engagement of biological research with African issues and colleagues. Comparative physiology and biochemistry are evolving, but in this we must guard against fractionation of effort and purpose. Increasingly available molecular methods are seductive in encouraging work on model species and in employing these species in place of more appropriate comparative models. Concomitantly, the comparative approach is reaching out beyond the individual organism and organism-organism interactions to establish underlying principles at ecosystem and landscape levels. The integration of molecular methods into comparative studies will require judicious selection and use of such skills if it is to be achieved without abandoning nonmodel species. The physiological and metabolic bases of ecosystem and evolutionary approaches must be underpinned by relevant data, requiring comparative researchers to accommodate colleagues contributing this specialist knowledge. These articles report distinct symposia, prefaced by a plenary paper. While each paper is itself a review of an entire symposium, they all exhibit a common theme, that comparative physiology and biochemistry are about interactions. It is our hope that the Comparative Physiology and Biology in Africa meetings will continue to facilitate special interactions between the people who make this happen.

Effects of short-term copper exposure on gill structure, metallothionein and hypoxia-inducible factor-1alpha (HIF-1alpha) levels in rainbow trout (Oncorhynchus mykiss).

Rainbow trout (Oncorhynchus mykiss) were exposed to 1.65 microM of waterborne copper for 24 h. Fish were then transferred to metal-free water. Metallothionein mRNA induction in rainbow trout liver and gill tissue, hypoxia-inducible factor-1 (HIF-1alpha) accumulation in gill tissue and arithmetic mean thickness of gill epithelium (Har) were determined at 4 and 24h of exposure as well as 48 h after transfer to metal-free water. The arithmetic mean distance from water to blood was significantly elevated after both 4 and 24 h of exposure (Har was 4.67 and 4.66 microm, respectively in exposed fish, compared to 3.81 and 3.62 microm for the corresponding control fish). During the 48 h recovery Har returned towards the control values; the recovery value of 4.21 microm was significantly lower than values during exposures. There was also a significant increase in gill metallothionein mRNA levels after the 4 h exposure with MT/GAPDH ratio of 1.288 versus the control value of 0.988. In liver, metallothionein induction was not observed. HIF-1alpha protein showed an increased accumulation in gills after 4 h, with the HIF-1alpha/alpha-tubulin ratio of 0.562 being significantly higher than the 24 h exposure value of 0.232. These results suggest that exposure to copper for four hours causes hypoxia in the gill epithelium, which is adequate for the activation of HIF-1alpha.