Effects of a dietary ketone ester on hippocampal glycolytic and tricarboxylic acid cycle intermediates and amino acids in a 3xTgAD mouse model of Alzheimer's disease.

In patients with Alzheimer's disease (AD) and in a triple transgenic (3xTgAD) mouse model of AD low glucose metabolism in the brain precedes loss of memory and cognitive decline. The metabolism of ketones in the brain by-passes glycolysis and therefore may correct several deficiencies that are associated with glucose hypometabolism. A dietary supplement composed of an ester of D-ß-hydroxybutyrate and R-1,3 butane diol referred to as ketone ester (KE) was incorporated into a rodent diet and fed to 3xTgAD mice for 8 months. At 16.5 months of age animals were killed and brains dissected. Analyses were carried out on the hippocampus and frontal cortex for glycolytic and TCA (Tricarboxylic Acid) cycle intermediates, amino acids, oxidized lipids and proteins, and enzymes. There were higher concentrations of d-ß-hydroxybutyrate in the hippocampus of KE-fed mice where there were also higher concentrations of TCA cycle and glycolytic intermediates and the energy-linked biomarker, N-acetyl aspartate compared to controls. In the hippocampi of control-fed animals the free mitochondrial [NAD+ ]/[NADH] ratio were highly oxidized, whereas, in KE-fed animals the mitochondria were reduced. Also, the levels of oxidized protein and lipids were lower and the energy of ATP hydrolysis was greater compared to controls. 3xTgAD mice maintained on a KE-supplemented diet had higher concentrations of glycolytic and TCA cycle metabolites, a more reduced mitochondrial redox potential, and lower amounts of oxidized lipids and proteins in their hippocampi compared to controls. The KE offers a potential therapy to counter fundamental metabolic deficits common to patients and transgenic models. Read the Editorial Highlight for this article on page 162.

An Ester of ß-Hydroxybutyrate Regulates Cholesterol Biosynthesis in Rats and a Cholesterol Biomarker in Humans.

In response to carbohydrate deprivation or prolonged fasting the ketone bodies, ß-hydroxybutyrate (ßHB) and acetoacetate (AcAc), are produced from the incomplete ß-oxidation of fatty acids in the liver. Neither ßHB nor AcAc are well utilized for synthesis of sterols or fatty acids in human or rat liver. To study the effects of ketones on cholesterol homeostasis a novel ßHB ester (KE) ((R)-3-hydroxybutyl (R)-3-hydroxybutyrate) was synthesized and given orally to rats and humans as a partial dietary carbohydrate replacement. Rats maintained on a diet containing 30-energy % as KE with a concomitant reduction in carbohydrate had lower plasma cholesterol and mevalonate (-40 and -27 %, respectively) and in the liver had lower levels of the mevalonate precursors acetoacetyl-CoA and HMG-CoA (-33 and -54 %) compared to controls. Whole liver and membrane LDL-R as well as SREBP-2 protein levels were higher (+24, +67, and +91 %, respectively). When formulated into a beverage for human consumption subjects consuming a KE drink (30-energy %) had elevated plasma ßHB which correlated with decreased mevalonate, a liver cholesterol synthesis biomarker. Partial replacement of dietary carbohydrate with KE induced ketosis and altered cholesterol homeostasis in rats. In healthy individuals an elevated plasma ßHB correlated with lower plasma mevalonate.

Genomic and non-genomic regulation of PGC1 isoforms by estrogen to increase cerebral vascular mitochondrial biogenesis and reactive oxygen species protection.

We previously found that estrogen exerts a novel protective effect on mitochondria in brain vasculature. Here we demonstrate in rat cerebral blood vessels that 17ß-estradiol (estrogen), both in vivo and ex vivo, affects key transcriptional coactivators responsible for mitochondrial regulation. Treatment of ovariectomized rats with estrogen in vivo lowered mRNA levels of peroxisome proliferator-activated receptor-γ coactivator-1 alpha (PGC-1α) but increased levels of the other PGC-1 isoforms: PGC-1ß and PGC-1 related coactivator (PRC). In vessels ex vivo, estrogen decreased protein levels of PGC-1α via activation of phosphatidylinositol 3-kinase (PI3K). Estrogen treatment also increased phosphorylation of forkhead transcription factor, FoxO1, a known pathway for PGC-1α downregulation. In contrast to the decrease in PGC-1α, estrogen increased protein levels of nuclear respiratory factor 1, a known PGC target and mediator of mitochondrial biogenesis. The latter effect of estrogen was independent of PI3K, suggesting a separate mechanism consistent with increased expression of PGC-1ß and PRC. We demonstrated increased mitochondrial biogenesis following estrogen treatment in vivo; cerebrovascular levels of mitochondrial transcription factor A and electron transport chain subunits as well as the mitochondrial/nuclear DNA ratio were increased. We examined a downstream target of PGC-1ß, glutamate-cysteine ligase (GCL), the rate-limiting enzyme for glutathione synthesis. In vivo estrogen increased protein levels of both GCL subunits and total glutathione levels. Together these data show estrogen differentially regulates PGC-1 isoforms in brain vasculature, underscoring the importance of these coactivators in adapting mitochondria in specific tissues. By upregulating PGC-1ß and/or PRC, estrogen appears to enhance mitochondrial biogenesis, function and reactive oxygen species protection.

Endogenous ovarian hormones affect mitochondrial efficiency in cerebral endothelium via distinct regulation of PGC-1 isoforms.

Mitochondria support the energy-intensive functions of brain endothelium but also produce damaging-free radicals that lead to disease. Previously, we found that estrogen treatment protects cerebrovascular mitochondria, increasing capacity for ATP production while decreasing reactive oxygen species (ROS). To determine whether these effects occur specifically in endothelium in vivo and also explore underlying transcriptional mechanisms, we studied freshly isolated brain endothelial preparations from intact and ovariectomized female mice. This preparation reflects physiologic influences of circulating hormones, hemodynamic forces, and cell-cell interactions of the neurovascular unit. Loss of ovarian hormones affected endothelial expression of the key mitochondrial regulator family, peroxisome proliferator-activated receptor γ coactivator 1 (PGC-1), but in a unique way. Ovariectomy increased endothelial PGC-1α mRNA but decreased PGC-1ß mRNA. The change in PGC-1ß correlated with decreased mRNA for crucial downstream mitochondrial regulators, nuclear respiratory factor 1 and mitochondrial transcription factor A, as well as for ATP synthase and ROS protection enzymes, glutamate-cysteine ligase and manganese superoxide dismutase. Ovariectomy also decreased mitochondrial biogenesis (mitochondrial/nuclear DNA ratio). These results indicate ovarian hormones normally act through a distinctive regulatory pathway involving PGC-1ß to support cerebral endothelial mitochondrial content and guide mitochondrial function to favor ATP coupling and ROS protection.