Lois M. Roeder

Glucose Transport in Astrocytes: Regulation by Thyroid Hormone

Abstract: Primary cultures of astrocytes from newborn rat brain showed evidence of a substrate‐saturable process for glucose transport. The system shows a relatively high affinity for the substrate, with an apparent Km of approximately 1 mM. Maintenance of the cells in medium containing thyroid‐hormone‐free serum for 3, 6, or 9 days resulted in significantly reduced rates of hexose transport. Addition of exogenous triiodothyronine to the transport incubation medium of these “hypothyroid′’ cells markedly increased the net rate of 2‐deoxyglucose uptake within 60 s to values equal to or above those of control cultures (cells maintained in normal serum). These findings support a key role for thyroid hormone in the transport of glucose across plasma membranes of brain cells and demonstrate the presence of this regulatory system in astrocytes.

Transport of 2-deoxy-D-glucose by dissociated brain cells.

The characteristics of glucose transport into dissociated cells from rat brain were determined using [1,2-3H]2-deoxyglucose as substrate. The rate of net uptake exhibited biphasic saturation kinetics with increasing substrate concentration; two values each for Km (8.85 and 1.05 mM) and Vmax (20.41 +/- 5.99 nmol/min/mg protein) were obtained, indicating the presence of two transport systems. D-glucose competed with [1,2-3H]2-deoxyglucose as shown by increasing degrees of inhibition of uptake of labeled substrate with increasing concentrations of D-glucose. The presence of an accelerative exchange mechanism was demonstrated by enhanced rates of uptake of labeled substrate by cells pre-loaded with high levels of unlabeled 2-deoxyglucose. Transport was inhibited by cytochalasin B, phloretin and phloridzin in a manner suggesting that the system is sodium-independent. Transport was also inhibited by sodium cyanide, potassium cyanide and dinitrophenol, but not by sodium arsenite or ouabain. Insulin status of the animals had no effect on the rate of transport of this substrate. Net transport was significantly lower in neonatal (4-day-old) rats than in either older sucklings (14-16-day-old) or adult animals; no significant difference between the latter two groups was observed. These findings demonstrate that two carrier-mediated systems for glucose transport are present on the membranes of these mixed brain cells suggesting that the kinetic characteristics of glucose transport may differ between neurons and glial cells. The age change in transport rate may reflect age-associated glial cell proliferation and/or an age-dependent increase in the number of transporters per cell in one brain cell type.

Ketone body enzyme activities in purified neurons, astrocytes and oligodendroglia

Ketone bodies serve as sources for energy and as precursors for lipid synthesis in developing brain. Whether neurons and neuroglia are equally capable of using ketone bodies during differentiation is not known. Using purified populations of neurons and astrocytes from developing rat brain and purified oligodendroglia from bovine brain, the activities for the three enzymes involved in ketone body metabolism were evaluated. Enzymatic activities were found in all three cell types. Surprisingly, astrocytes had the highest levels of activity for both 3-ketoacid-CoA transferase and acetoacetyl-CoA thiolase; these activites showed dramatic changes during development. Nonetheless, neurons, astrocytes and oligodendroglia are all quite capable of using ketone bodies as metabolic fuels.

Thyroid hormone action on glucose transporter activity in astrocytes

In astrocytes from rat brain cultured in thyroid hormone-deficient media cytochalasin B-binding was decreased 80%; addition of L-T3 increased binding to 75% of control levels. Saponin-treatment of controls increased accessibility of binding sites to 60% above untreated cells. Saponin also increased binding in deficient cells; however, the level was less than in treated controls, suggesting L-T3 deficiency decreases total glucose transporters. Addition of L-T3 appeared to convert most (90%) of the binding sites from unavailable to accessible status. Changes in binding to plasma membranes in response to L-T3 level were similar to those in intact cells. No binding to Golgi was detectable, thus no evidence for translocation of carriers was obtained. L-T3 may activate the glucose transporter by increasing its accessibility in brain cells.

Interleukin (IL)-1β Increases Glucose Uptake and Induces Glycolysis in Aerobically Cultured Rat Ovarian Cells: Evidence That IL-1β May Mediate the Gonadotropin-Induced Midcycle Metabolic Shift1

This communication explores the possibility that interleukin (IL)-1beta, a putative intermediary in the ovulatory process, may take part in the gonadotropin-driven midcycle diversion of ovarian carbohydrate metabolism toward glycolysis. We examined the effect of treatment with IL-1beta on glucose metabolism in aerobically cultured whole ovarian dispersates from immature rats. Treatment with IL-1beta increased cellular glucose consumption/uptake, stimulated extracellular lactate accumulation and media acidification, and decreased extracellular pyruvate accumulation in a receptor-mediated, time-, dose- and cell density-dependent manner. Endogenous IL-1beta-like bioactivity was shown to mediate the ability of gonadotropins to exert these same metabolic effects. The IL-1beta effect was also (1) apparent over a broad range of glucose concentrations, inclusive of the putative physiological window; (2) relatively specific, because tumor necrosis factor-alpha and insulin were inactive; (3) contingent upon cell-cell cooperation (4) and reliant on de novo protein synthesis. Comparison of the molar ratios of lactate accumulation to glucose consumption in IL-1beta-replete vs. IL-1beta-deplete cultures suggests that IL-beta promotes the conversion of all available glucose to lactate but that other substrates for lactate production may also exist. However, no lactate was generated by cells grown under glucose-free conditions. Taken together, our data suggest that IL-1beta may act as a metabolic hormone in the ovary. Subject to the limitations of the in vitro paradigm, our data also suggest that IL-1beta may mediate the gonadotropin-associated midcycle shift in ovarian carbohydrate metabolism. By converting the somatic ovarian cells into a glucose-consuming glycolytic machinery, IL-1beta may establish glycolysis as the main energy source of the relatively hypoxic preovulatory follicle and the resultant cumulus-oocyte complex. The consequent oxygen sparing may conserve the limited supply of oxygen needed for vital biosynthetic processes such as steroidogenesis. This adaptational response may also provide the glycolytically incompetent oocyte with the obligatory tricarboxylic cycle precursors it depends on to meet the increased energy demands imposed upon it by the resumption of meiosis.

Serum effects on substrate oxidation by dissociated brain cells: possible sites of action

This report is an extension of recent studies indicating the presence of a factor in serum that preferentially inhibits 14CO2 production from labeled glucose. Experiments with dissociated cells revealed that the inhibitory effects of serum were only slightly changed over more than a 50-fold range in initial glucose concentration. Serum had no effect on the rate of glucose transport (uptake of 1,3[3H]2-deoxyglucose). The inhibitory effect of serum was greater on 14CO2 production from [6-14C]glucose than [1-14C]glucose. Other studies revealed that 14CO2 production from [1-14C]pyruvate was more than 5 times the rate obtained using [3-14C]pyruvate; however, the inhibitory effect of serum was much greater on the latter (20% vs 60% inhibition respectively) at 2 mM pyruvate and in the presence of 1% fetal bovine serum. Attempts to characterize the factor using Amicon filtration showed the highest inhibitory activity in a 10,000 mol. wt. fraction, although some inhibitory activity was found in commercial preparations of bovine serum albumin. Delipidation of serum had no effect. Based on these results, we postulate that the observed decrease in labeled CO2 production reflects the regulation of substrate utilization at the pyruvate carboxylase step by one or more factors in serum (with a mol. wt. of approximately 10,000).

Differential effects of adrenocorticotropin(1–24) on [3H]2-deoxy-d-glucose uptake in cultured cells derived from different brain regions

The effect of adrenocorticotropin(1-24) (ACTH(1-24)) on the uptake of [3H]2-deoxy-D-glucose ([3H]2-DG) was compared in cell cultures derived from two regions (hypothalamus, and extrahypothalamic forebrain) of fetal rat brain. Under control conditions, [3H]2-DG uptake was similar in extrahypothalamic (10.9 +/- 1.1 nmol/mg protein/5 min) and hypothalamic (11.9 +/- 1.3) cells. No significant effect of ACTH (1-24) (10(-7) to 10(-5) M) was found on uptake of [3H]2-DG in extrahypothalamic cells. In contrast, in hypothalamic cells, a potent stimulatory effect (P less than 0.0001) up to 174% over the control value of [3H]2-DG uptake was produced by these concentrations of ACTH(1-24). This study suggests that ACTH may be a stimulator of brain glucose uptake, and that this effect varies in different brain regions.

Scalp Hair Morphology in Normal and Diabetic Children and Adolescents

The morphological characteristics of samples of scalp hair from normal children and adolescents (8-17 years) and from a group of diabetic children (8-11 years) were determined. In normal children, there were no sex differences. Significant increases in the diameters of both bulb and shaft were found when prepubertal (8-11 years) and pubertal (12-17 years) groups were compared. Diabetic females had smaller bulb diameters and diabetics of both sexes had reduced shaft diameters in comparison to normal children of similar age. These findings suggest previous nutritional deficits in male and female diabetics and the possibility of continued nutritional problems in the females.

The effects of ketone bodies, bicarbonate, and calcium on hepatic mitochondrial ketogenesis☆

Abstract The effect of various factors on hepatic mitochondrial ketogenesis was investigated in the rat. A comparison of three different incubation media revealed that bicarbonate ion inhibited the rate of ketone body production and decreased the ratio of 3-hydroxybutyrate/acetoacetate. The addition of 0.8 m m calcium caused significant inhibition of ketogenesis from both octanoate (40–50%) and palmitate (25–30%) and no change in the ratio of 3-hydroxybutyrate/acetoacetate. In the presence of components of the malate/aspartate shuttle, the inhibition by calcium was 80% or more with both substrates. Experimental alteration of the respiratory state of the mitochondria from state 3 to state 4 was associated with an enhanced rate of ketogenesis. The addition of ketone bodies themselves had marked effects on the rate of ketone body production. Increasing amounts of exogenously added acetoacetate were accompanied by increasing rates of total ketone body production reflecting enhanced 3-hydroxybutyrate synthesis. In the presence of added 3-hydroxybutyrate, there was striking inhibition of ketogenesis. Rotenone, which prevents oxidation of NADH 2 via the electron transport chain, almost completely inhibited ketone body synthesis. This inhibition was partially overcome by the addition of acetoacetate which regenerates NAD + from NADH 2 during conversion to 3-hydroxybutyrate. These observations provide evidence for additional sites of metabolic control over hepatic ketogenesis.

Metabolic Regulation in Brain Cells

Recent studies of metabolic regulation in brain have focused on the complexity and fine-tuning involved in the control of metabolic processes. Gaps in our knowledge are rapidly being filled as previously unidentified “factors” are purified and characterized, new compounds are identified, and new functions for familiar compounds are discovered. Often new information is obtained initially from tests of substances that support survival and growth of neural tissue in vitro, and the mechanisms involved are not fully investigated until much later. Such studies also have revealed the importance of cell-cell interactions (Mandel et al., 1977; Seeds and Hawkins, 1985), and several workers have identified specific metabolic processes involved in these exchanges (Cummins et al., 1979; Hertz, 1979; Pentreath and Kai-Kai, 1982; Sykora, 1983; Hertz and Richardson, 1984). Different factors seem to be involved in central nervous system (CNS) and peripheral nervous system (PNS) (Seifert and Muller, 1984). However, nerve growth factor (NGF), which is essential for the survival, both in vivo and in vitro, of sympathetic and sensory neurons (Levi-Montalcini and Angeletti, 1968) may also have a role in brain (Honegger and Lenoir, 1982). The proposed mechanisms of the neurotrophic action of NGF (and of some new putative growth factors) were recently reviewed by Thoenen and Edgar (1985). Kauffman and coworkers have begun to identify some of the specific metabolic alterations that accompany the growth effect of NGF on the superior cervical ganglion (Dumbrowski et al., 1983).