Molecular and Cellular Endocrinology, 9 (1977) 133-144 0 Elscvier/North-Holland Scientific Publishers, Ltd.

THYROID HORMONES AND LIPOGENESIS FROM GLUCOSE IN RAT FAT CELLS

C. CORREZE, J. NUNEZ and A. GORDON Unit6 de Recherche sur la Glande Thyroide et la RPgulation Hormonale, INSERM, Equipe de Recherche Associee No. 449, C.N.R.S., 78 avenue du G&&al Leclerc, 94270 Bice^tre, France Received

4 May 1977; accepted

IS July

1977

The effect of thyroidectomy on lipogenesis from glucose was investigated in rat fat cells. It was shown that thyrordectomy resulted in a clear increase in the oxidation of glucose to CO? and also m its conversion to fatty acids. Such an increase in lipogenesis from glucose after thyroidectomy was not due to a change in the cell size or to a modtficatron of the cell surface. The diffusion of L-glucose and the facilitated dtffusion transport of 3-O methyl glucose were not increased; the increase in glucose oxidation observed after thyroidectomy is therefore not the result of an increased transfer of glucose. In contrast, the uptake of deoxyglucosc, a sugar which enters the cell and is phosphorylated by the hexokinase system. but is then not further metabolized, was markedly enhanced in cells from the thyroidectomized ammals. A 44S-fold increase over control in the Vmax, but no change in the Km, were observed. Since no effects were noted on sugar diffusion or transport, this result demonstrates that a maJor consequence of thyroidectomy is an increase in glucose phosphorylation. Previous observations (Correze et al., 1974; Van Inwegen et al., 1975) showed how fat cells from thyroidectomized rats lose theu hpolytic capacity; in the present study we provide evidence for an increase in lipogenesis in these same cells. Thyroid hormones might therefore modulate a critical regulatory stcpts) common to lipolysis and hpogenesis. Keywords:

adipocytes;

glucose

oxidation;

thyroid

hormones.

It is well established that several lipolytic hormones stimulate lipolysis, i.e. the breakdown of triglycerides of the epididymal fat cells to free fatty acids and glycerol, through a regulatory pathway where the key intermediate is cyclic AMP. Some observations (Blecher, 1967) support the hypothesis that the stimulatory effect of insulin on lipogenesis might also be related to tissue levels of cyclic AMP. This hypothesis, which is far from being proven, would imply that lipolysis and lipogenesis, like glycogenolysis and glycogenesis in the muscle (Krebs and Fisher, 1962), are regulated by a ‘double-barrier mechanism’: opening of one pathway implies the closing of the other one, and vice versa. We have previously shown (Correze et al., 1974) that fat cells from thyroidectomized animals do not respond to lipolytic hormones because the cyclic AMP, which is formed in these cells, is destroyed as soon as it is produced. Similar results have been also obtained by others (Van Inwegen et al., 1975). If, as noted above, 133

134

C. Corrkze et al.

lipolysis and lipogenesis are regulated by a ‘double-barrier mechanism’, one should therefore expect fat cells from thyroidectomized rats to be highly lipogenic. This study provides some arguments in favour of this hypothesis.

MATERIALS

AND METHODS

Products Collagenase CLS (161 U/mg) was purchased from Worthington; bovine serum albumin (Fraction V), 3-O methyl glucose, 2-deoxy-D-glucose, L-glucose and D-glucose were from Sigma; Hepes buffer was from Calbiochem; theophylline was from BDH Biochemicals; 3-O methyl-D-l -[ 1-3H]glucose (3 Ci/mmol), 2-deoxy-D-[ l-3H] glucose (19 Ci/mmol), L-[ 14C]glucose (4 mCi/mmol) and [3H]inulin (300 mCi/ mmol) were from Amersham; D-[U-‘4C]glucose (200-300 mCi/mmol) was from Commissariat a I’Energie Atomique, France. General Wistar rats weighing 60-80 g were used at least 15 days after thyroidectomy. Fat cells were isolated from epididymal fat pads of normal and thyroidectomized rats by the method of Rodbell (1967) with collagenase (5 mg/g tissue). The buffer used was Krebs-Ringer bicarbonate, pH 7.4, containing 4% ofdefatted bovine serum albumin (albumin bicarbonate buffer). Glucose oxidation The washed cells (100-150 pmol of triglycerides per ml of suspension) were incubated in albumin bicarbonate buffer containing 3 ~mol/ml of D-[‘4C]glucose (0.3 PCi per assay). Incubations for varying periods of time were performed as described by Rodbell (1964). After the incubation, the medium was acidified, CO2 was collected on pieces of filter paper moistened with Hyamine 10X (0.25 ml), and counted for radioactivity in a liquid scintillation spectrometer (Intertechnique). Labelled lipids were extracted by the method of Dole and Meinertz (1960). Aliquots of the upper phase were analyzed for triglyceride content by the procedure of Rapport and Alonzo (1955), and total lipid radioactivity determined. A 1 ml sample of the upper phase was also evaporated to dryness, saponified with 4% ethanolic KOH (w/v) and the liberated fatty acids extracted with hexane. The hexane extracts were dried and counted for radioactivity. The difference between the radioactivity measured in total lipids, and that measured in fatty acids, represents the radioactivity in the glycerol moiety. Results are expressed as micromoles of glucose per mmol of triglyceride in the cell suspension. Glucose transport studies 1 ml of isolated fat cells were suspended in Krebs-Ringer buffer containing 1% albumin and 15 mM Hepes, pH 7.4, and incubated at 37°C for varying periods of

Thyroxine

regulation of lipogerwsis

135

time. In the 3-O methyl glucose uptake studies the incubation lasted between 20120 set while in the L-glucose and 2-deoxyglucose experiments the cells were mcubated for 3-15 min. At the end of the incubation period, uptake of sugars by isolated fat cells was measured by the procedure of Gliemann et al. (1972). Samples of 200 ~1 of the cell suspension were taken and placed in a plastic microcentrifuge tube containing 200 ~1 of dinonyl phthalate. The tubes were immediately centrifuged for 30 set in a Beckman microfuge and the assay was considered terminated when centrifugation began. The dinonyl phthalate oil works as a specific gravity intermediate between cells and buffer. The cells on top of the oil were removed and their radioactivity was determined. The amount of trapped sugar at 15 set time was considered as extracellular space water, or extracellular space water was calculated with [ 3H] inulin. Estimation of cell diameter The samples of cell suspensions were observed by optical microscopy (X500) with a calibrated grid. The diameter of 80-100 cells from several fields was measured and the mean value was calculated +SEM. Cell volume and cell surface area were calculated according to Hirsch and Gallian (1968). 5’-Nucleotidase determination 5’-nucleotidase activity was measured (1965).

as described

by Heppel

and Hilmoe

RESULTS Efiect of thyroidectomy on the metabolism of /14C]glucose in isolated fat cells Fat cells from the adipose tissue of normal and thyroidectomized rats were incubated in Krebs-Ringer bicarbonate buffer (pH 7.4) containing 4% bovine albumin, 3 mM D-glucose labelled with 0.25 PCi [‘4C]glucose for varying periods of time. The quantities of glucose carbon converted to CO;?, to glyceride-glycerol and to fatty acids during the course of the incubation were measured. Glucose oxidation proceeded at a constant rate for at least 2 h in both types of adipocytes. Fig. 1 represents a typical experiment: thyroidectomy resulted in a clear increase in the conversion of glucose to CO* (fig. 1) and also in its conversion to fatty acids (fig. 2a), but only in a slight enhancement of the production ofglyceride-glycerol from glucose (fig. 2b). Table 1 represents the mean values of triplicates obtained in 3 experiments employing the same sample of Sigma bovine albumin (Fraction V). A clear increase in the oxidation of glucose was observed. The enhancement of glucose conversion to CO* was 70% and to glycero-lipids 50% (comprising a 68% increase in the FFA fraction with a 33% rise for the glyceride-glycerol). Experiments with different batches of albumin gave different figures; such a variation in response has also been noted by others, and attributed to the albumin (Lockwood and East, 1974).

C. Codze

5

v



et al.

I’ 1 30

1 60

i 120

Fig. 1. Time course of oxidation of [U- 14C]glucose to CO2 by isolated fat cells. Each point represents the mean of 3 determinations. The vertical bar represents standard error. Cells were incubated for varying times at 37°C in 1 ml of albumin bicarbonate buffer, pH 7.4, containing 3 nmol of [ t4Cjglucose per ml and an initial specific activity of 0.8 /.Ki/mol. Fat cell conor thycentration was 80 nmol of triglycerides per ml. Fat cells from normal rats f* +) roidectom~ed rats fo---o).

MINUTES

MINUTES

Fig. 2. Time course of glucose conversion to fatty acids (a) and to glyceride-glycerol (b). Conor thyroidectomized rats to -0). ditions as in fig. 1; fat ceils from normal rats (0 -0)

Thyroxine

regulation of lipogenesis

Table 1 Effect of thyroidectomy on adipocyte and lipids over a 1 h incubation

Adipocytes from normal rats Adipocytes from thyroidectomized rats ~___

137

glucose

metabolism.

conversion

of

[ 14C)glucose

to CO2

No. of experrments

CO2

Total lipids

Glycerideglycerol

Fatty acids

3

1.44 + 0.07

1.45 f 0.03

0.75 f 0.05

0.70 i 0.04

1.00 t 0.04 **

1 18 * 0.14 **

3 2.45 + 0.15 * 2.18 * 0.1 * _._- -.-__~_~_..__... ~______~_

Conversion of [U-14C]glucose (timol/mmol TG/h). Values are the means * SEM of 3 separate experiments each of which was performed in quadruplicate. The rats were used 15-20 days after surgery. Incubation conditions as described in fig. 1. * Significantly different from the corresponding values from control cells (P < 0.001) by Student’s t-test. ** P < 0.005.

Glucose oxidation to CO, was nonetheless calculated, and gave an average value +SEM for 9 different experiments of 0.587 + 0.130 I_mmol/mmol TG in the normal, and of 1.399 + 0.255 pmol/mmol TG in cells from thyroidectomized animals, P being

Thyroid hormones and lipogenesis from glucose in rat fat cells.

Molecular and Cellular Endocrinology, 9 (1977) 133-144 0 Elscvier/North-Holland Scientific Publishers, Ltd. THYROID HORMONES AND LIPOGENESIS FROM GLU...
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