Organ-specific responses during brain death:increased aerobic metabolism in the liver and anaerobic metabolism with decreased perfusion in the kidneys

Hepatic and renal energy status prior to transplantation correlates with graft survival. However, effects of brain death (BD) on organ-specific energy status are largely unknown. We studied metabolism, perfusion, oxygen consumption, and mitochondrial function in the liver and kidneys following BD. BD was induced in mechanically-ventilated rats, inflating an epidurally-placed Fogarty-catheter, with sham-operated rats as controls. A 9.4T-preclinical MRI system measured hourly oxygen availability (BOLD-related R2*) and perfusion (T1-weighted). After 4 hrs, tissue was collected, mitochondria isolated and assessed with high-resolution respirometry. Quantitative proteomics, qPCR, and biochemistry was performed on stored tissue/plasma. Following BD, the liver increased glycolytic gene expression (Pfk-1) with decreased glycogen stores, while the kidneys increased anaerobic- (Ldha) and decreased gluconeogenic-related gene expression (Pck-1). Hepatic oxygen consumption increased, while renal perfusion decreased. ATP levels dropped in both organs while mitochondrial respiration and complex I/ATP synthase activity were unaffected. In conclusion, the liver responds to increased metabolic demands during BD, enhancing aerobic metabolism with functional mitochondria. The kidneys shift towards anaerobic energy production while renal perfusion decreases. Our findings highlight the need for an organ-specific approach to assess and optimise graft quality prior to transplantation, to optimise hepatic metabolic conditions and improve renal perfusion while supporting cellular detoxification

Obesity and type 2 diabetes : a systems biology perspective of a molecular mechanism

People who have excess body weight have a higher risk of insulin resistance and type 2 diabetes, because fat interferes with the body's ability to make and use insulin. Insulin, a hormone released by pancreatic beta-cells, is needed to move diet-derived glucose from blood into fat and muscle cells where it is used to produce energy. The precise ways of how fat interferes with insulin action are not yet known. Our hypothesis is that accumulation of fatty acids (break down product of fats) in these cells leads to increased levels of activated fatty acids, so called fatty acyl-CoA esters. The latter may impair cellular energy metabolism and stimulate the production of reactive oxygen species (ROS) due to inhibition of mitochondrial adenine nucleotide translocator (ANT) leading to impaired cell function, for example impaired insulin release from pancreatic beta-cells. In agreement with our hypothesis we have shown that addition of fatty acyl-CoA esters to mitochondria isolated from livers of normal rats leads to decreased activity of the ANT, decreased levels of extramitochondrial ATP and increased production of ROS. The effect of saturated fatty acyl-CoA ester (palmitoyl-CoA) was stronger than the effect of unsaturated fatty acyl-CoA ester (oleoyl-CoA). Interestingly, the observed effects depended on the working condition of these mitochondria. Next we showed, that long-term high fat diet feeding leads to higher blood glucose levels, as well as to oxidative stress and accumulation of fatty acyl-CoA esters in rat livers. However, long-term high fat diet feeding does not cause adaptive changes that would improve mitochondrial ability to deal with these challenges. This indicates that fatty acyl-CoA may exert same effects in intact liver as the ones observed in isolated mitochondria. Similarly to rat livers, long-term exposure of human endothelial cells (cells that line interior surface of blood vessels) to high levels of fatty acids leads to accumulation of fatty acyl-CoA ester and lower levels of ATP in these cells. This negatively affects the ability of these cells to survive and grow. Again, the effect of saturated fatty acid palmitate is stronger that the effect of unsaturated fatty acid oleate. In summary, our data indicate that the proposed mechanism linking obesity and type 2 diabetes (i.e. inhibition of the ANT by fatty acy-CoA esters) may indeed occur in cells leading to impaired cell function characteristic to type 2 diabetes.Heine, R.J. [Promotor]Westerhoff, H.V. [Promotor]Bakker, S.J.L. [Copromotor]Krab, K. [Copromotor

An in vivo magnetic resonance spectroscopy study of the effects of caloric and non-caloric sweeteners on liver lipid metabolism in rats

We aimed to elucidate the effects of caloric and non-caloric sweeteners on liver lipid metabolism in rats using in vivo magnetic resonance spectroscopy (MRS) and to determine their roles in the development of liver steatosis. Wistar rats received normal chow and either normal drinking water, or solutions containing 13% (w/v) glucose, 13% fructose, or 0.4% aspartame. After 7 weeks, in vivo hepatic dietary lipid uptake and de novo lipogenesis were assessed with proton-observed, carbon-13-edited MRS combined with13C-labeled lipids and13C-labeled glucose, respectively. The molecular basis of alterations in hepatic liver metabolism was analyzed in detail ex vivo using immunoblotting and targeted quantitative proteomics. Both glucose and fructose feeding increased adiposity, but only fructose induced hepatic lipid accumulation. In vivo MRS showed that this was not caused by increased hepatic uptake of dietary lipids, but could be attributed to an increase in de novo lipogenesis. Stimulation of lipogenesis by fructose was confirmed by a strong upregulation of lipogenic enzymes, which was more potent than with glucose. The non-caloric sweetener aspartame did not significantly affect liver lipid content or metabolism. In conclusion, liquid fructose more severely affected liver lipid metabolism in rats than glucose, while aspartame had no effect.</p

An In Vivo Magnetic Resonance Spectroscopy Study of the Effects of Caloric and Non-Caloric Sweeteners on Liver Lipid Metabolism in Rats

We aimed to elucidate the effects of caloric and non-caloric sweeteners on liver lipid metabolism in rats using in vivo magnetic resonance spectroscopy (MRS) and to determine their roles in the development of liver steatosis. Wistar rats received normal chow and either normal drinking water, or solutions containing 13% (w/v) glucose, 13% fructose, or 0.4% aspartame. After 7 weeks, in vivo hepatic dietary lipid uptake and de novo lipogenesis were assessed with proton-observed, carbon-13-edited MRS combined with C-13-labeled lipids and C-13-labeled glucose, respectively. The molecular basis of alterations in hepatic liver metabolism was analyzed in detail ex vivo using immunoblotting and targeted quantitative proteomics. Both glucose and fructose feeding increased adiposity, but only fructose induced hepatic lipid accumulation. In vivo MRS showed that this was not caused by increased hepatic uptake of dietary lipids, but could be attributed to an increase in de novo lipogenesis. Stimulation of lipogenesis by fructose was confirmed by a strong upregulation of lipogenic enzymes, which was more potent than with glucose. The non-caloric sweetener aspartame did not significantly affect liver lipid content or metabolism. In conclusion, liquid fructose more severely affected liver lipid metabolism in rats than glucose, while aspartame had no effect

Maintenance of cellular vitamin B 6 levels and mitochondrial oxidative function depend on pyridoxal 5′-phosphate homeostasis protein

Recently, biallelic variants in PLPBP coding for pyridoxal 5'-phosphate homeostasis protein (PLPHP) were identified as a novel cause of early-onset vitamin B 6-dependent epilepsy. The molecular function and precise role of PLPHP in vitamin B 6 metabolism are not well understood. To address these questions, we used PLPHP-deficient patient skin fibroblasts and HEK293 cells and YBL036C (PLPHP ortholog)-deficient yeast. We showed that independent of extracellular B 6 vitamer type (pyridoxine, pyridoxamine, or pyridoxal), intracellular pyridoxal 5'-phosphate (PLP) was lower in PLPHP-deficient fibroblasts and HEK293 cells than controls. Culturing cells with pyridoxine or pyridoxamine led to the concentration-dependent accumulation of pyridoxine 5'-phosphate and pyridoxamine 5'-phosphate (PMP), respectively, suggesting insufficient pyridox(am)ine 5'-phosphate oxidase activity. Experiments utilizing 13C 4-pyridoxine confirmed lower pyridox(am)ine 5'-phosphate oxidase activity and revealed increased fractional turnovers of PLP and pyridoxal, indicating increased PLP hydrolysis to pyridoxal in PLPHP-deficient cells. This effect could be partly counteracted by inactivation of pyridoxal phosphatase. PLPHP deficiency had a distinct effect on mitochondrial PLP and PMP, suggesting impaired activity of mitochondrial transaminases. Moreover, in YBL036C-deficient yeast, PLP was depleted and PMP accumulated only with carbon sources requiring mitochondrial metabolism. Lactate and pyruvate accumulation along with the decrease of tricarboxylic acid cycle intermediates downstream of α-ketoglutarate suggested impaired mitochondrial oxidative metabolism in PLPHP-deficient HEK293 cells. We hypothesize that impaired activity of mitochondrial transaminases may contribute to this depletion. Taken together, our study provides new insights into the pathomechanisms of PLPBP deficiency and reinforces the link between PLPHP function, vitamin B 6 metabolism, and mitochondrial oxidative metabolism

The malate-aspartate shuttle is important for de novo serine biosynthesis

The malate-aspartate shuttle (MAS) is a redox shuttle that transports reducing equivalents across the inner mitochondrial membrane while recycling cytosolic NADH to NAD +. We genetically disrupted each MAS component to generate a panel of MAS-deficient HEK293 cell lines in which we performed [U- 13C]-glucose tracing. MAS-deficient cells have reduced serine biosynthesis, which strongly correlates with the lactate M+3/pyruvate M+3 ratio (reflective of the cytosolic NAD +/NADH ratio), consistent with the NAD + dependency of phosphoglycerate dehydrogenase in the serine synthesis pathway. Among the MAS-deficient cells, those lacking malate dehydrogenase 1 (MDH1) show the most severe metabolic disruptions, whereas oxoglutarate-malate carrier (OGC)- and MDH2-deficient cells are less affected. Increasing the NAD +-regenerating capacity using pyruvate supplementation resolves most of the metabolic disturbances. Overall, we show that the MAS is important for de novo serine biosynthesis, implying that serine supplementation could be used as a therapeutic strategy for MAS defects and possibly other redox disorders

Mouse Studies to Shape Clinical Trials for Mitochondrial Diseases: High Fat Diet in Harlequin Mice

BACKGROUND: Therapeutic options in human mitochondrial oxidative phosphorylation (OXPHOS) diseases have been poorly evaluated mostly because of the scarcity of cohorts and the inter-individual variability of disease progression. Thus, while a high fat diet (HFD) is often recommended, data regarding efficacy are limited. Our objectives were 1) to determine our ability to evaluate therapeutic options in the Harlequin OXPHOS complex I (CI)-deficient mice, in the context of a mitochondrial disease with human hallmarks and 2) to assess the effects of a HFD. METHODS AND FINDINGS: Before launching long and expensive animal studies, we showed that palmitate afforded long-term death-protection in 3 CI-mutant human fibroblasts cell lines. We next demonstrated that using the Harlequin mouse, it was possible to draw solid conclusions on the efficacy of a 5-month-HFD on neurodegenerative symptoms. Moreover, we could identify a group of highly responsive animals, echoing the high variability of the disease progression in Harlequin mice. CONCLUSIONS: These results suggest that a reduced number of patients with identical genetic disease should be sufficient to reach firm conclusions as far as the potential existence of responders and non responders is recognized. They also positively prefigure HFD-trials in OXPHOS-deficient patients