Fatty Acid Oxidation and Ketogenesis by Astrocytes in Primary Culture

Abstract

The oxidation of the fatty acids octanoate and palmitate to CO2 and the ketone bodies acetoacetate and D-(-)-3-hydroxybutyrate was examined in astrocytes that were prepared from cortex of 2-day-old rat brain and grown in primary culture to confluence. Accumulation of acetoacetate (by mass) in the culture medium of astrocytes incubated with octanoate (0.3-0.5 mM) was 50-90 nmol C2 units h-1 mg of protein-1. A similar rate was obtained using radiolabeled tracer methodology with [1-14C]octanoate as labeled substrate. The results from the radiolabeled tracer studies using [1-14C]- and [7-14C]octanoate and [1-14C]-, [13-14C]-, and [15-14C]palmitate indicated that a substantial proportion of the omega-terminal four-carbon unit of these fatty acids bypassed the beta-ketothiolase step of the beta-oxidation pathway and the 3-hydroxy-3-methylglutaryl (HMG)-CoA cycle of the classic ketogenic pathway. The [14C]acetoacetate formed from the 1-14C-labeled fatty acids, obligated to pass through the acetyl-CoA pool, contained 50% of the label at carbon 3 and 50% at carbon 1. By contrast, the [14C]acetoacetate formed from (omega-1)-labeled fatty acids contained 90% of the label at carbon 3 and 10% at carbon 1, whereas that formed from the (omega-3)-labeled fatty acid contained 20% of the label at carbon 3 and 80% at carbon 1. These results indicate that acetoacetate is primarily formed either by the action of 3-oxo-acid-CoA transferase (EC 2.8.3.5) or acetoacetyl-CoA deacylase (EC 3.1.2.11) or both on acetoacetyl-CoA and not by the action of the mitochondrial HMG-CoA cycle involving HMG-CoA lyase (EC 4.1.3.4), which was readily detected, and HMG-CoA synthase (EC 4.1.3.5), which was barely measurable.

Neuregulins Increase Mitochondrial Oxidative Capacity and Insulin Sensitivity in Skeletal Muscle Cells

“…Cells were incubated for 3 h, and 14 CO 2 was trapped and measured as described (23). A commercially available kit (Sigma 826-UV) was used to measure lactate release in 1 ml incubation medium from cells incubated in the presence or absence of Hrg for 48 h. Glycogen synthesis was measured as the incorporation of glucose into glycogen, as described elsewhere (24).…”

Molecular cloning of rat mitochondrial 3-hydroxy-3-methylglutaryl-CoA lyase and detection of the corresponding mRNA and of those encoding the remaining enzymes comprising the ketogenic 3-hydroxy-3-methylglutaryl-CoA cycle in central nervous system of suckling rat

“…HMG-CoA synthase mRNA when compared with tissue in the corresponding brain subregion. Astrocytes are a major neural cell type involved in energy provision in brain [12] ; cortical astrocytes can be prepared to a high degree of purity (more than 95 %) in primary culture. Thus RPAs were used to investigate the abundances of HMGCoA cycle mRNA species in neonatal and adult cortical astrocytes ( Figure 6).…”

“…HMG-CoA synthase mRNA or protein in the brains of suckling rats [4] or adult mice [7]. In addition, studies attempting to demonstrate the presence of the three enzyme activities necessary to execute the HMG-CoA cycle in mitochondria of whole brain [8][9][10][11] or cultured astrocytes [12] are generally equivocal. In contrast, the liver is an overall provider of ketone bodies as it converts most diet-derived fatty acids to ketone bodies that are then released into the blood for consumption by other organs such as heart and brain [13].…”

Isolation of mitochondria with high respiratory control from primary cultures of neurons and astrocytes using nitrogen cavitation

“…As some evidence indicates that specific metabolic enzyme activities are distinctly different in neuronal and astrocytic mitochondria Clark, 1979, 1989;McKenna et al, 2000a,b;Sonnewald et al, 2004), additional comparisons of bioenergetic and physiologic characteristics are needed. In response to this need, attempts were made to isolate mitochondria with good functional integrity from primary cultures of pure neurons and astrocytes (Auestad et al, 1991;Almeida and Medina, 1997b). The approach used to isolate mitochondria from these cells employs mechanical disruption of cell membranes by homogenization, a technique normally also applied to various tissues.…”