Influence of Thyroid Hormones on Gluconeogenesis From Glycerol in Rat Hepatocytes: A Dose-Response Study Blandine Comte, Hubert Vidal, Martine Laville, and Jean-Paul Riou The role of ~-3.3’4 triiodothyronine (T31, in a pathophysiological range, on gluconeogenesis from low concentration of glycerol (2 mmol/L), was investigated in isolated liver cells from 24-hour fasted rats either thyroidectomized, normal, or treated by a T3 dose ranging from 1, 5, or 10 ~g/d/lOO g body weight (BW) during 3 days to 50 pg during 7 days. Gluconeogenesis from glycerol was decreased by 63% in hypothyroid rats and increased by 35% in severely hyperthyroid rats. However, in cells from mild hyperthyroid rats no increase of gluconeogenesis was observed. Nevertheless, in mild hyperthyroidism, a-glycerophosphate (G3P) was significantly decreased and gluconeogenesis from glycerol was not inhibited by the addition of ethanol (10 mmol/L). both of which have a drastic effect in cells from thyroidectomized rats. The decrease of gluconeogenesis observed in cells from thyroidectomized rats was reversed by the addition of pyruvate (10 mmol/L). Thus, when the cells were in a “reduced state” (addition of ethanol) the differences between the group were magnified, and when the cells were in an “oxidized state” (addition of pyruvate) the differences were suppressed. These findings suggest that alteration of the capacity of reducing equivalents transfer from the cytoplasmic compartment to the mitochondria is the main mechanism by which mild hyperthyroidism can stimulate gluconaogenesis. @ 1990 by W. B. Saunders Company.

T

HE EFFECTS OF THYROID STATUS on hepatic glucose production have been intensively investigated both in human and in rats.‘-6 It is usually admitted that thyroid hormone excess increases hepatic glucose production by acting both on glycogenolysis and gluconeogenesis. The gluconeogenic effect of L-3,3’-S-triiodothyronine (T3) has been related mainly to an increased activity of gluconeogenic enzymes such as pyruvate carboxylase, phosphoenol pyruvate carboxykinase, and glucose 6-phosphatase.7*9 The possible role of the induction by T3 of the mitochondrial glycerol 3-phosphate dehydrogenase (G3PDH), leading to an increase in the disposal of reducing equivalents also has been advocated.“-” Using isolated liver cells from hyperthyroid rats and nuclear magnetic resonance techniques, 4 Cohen et al demonstrated that T3 excess stimulates gluconeogenesis from glycerol and decreases glycerol 3-phosphate (G3P) accumulation. In spite of this convincing evidence, these findings could be argued on a pathophysiological basis. First, the glycerol concentration used (11 to 22 mmol/L) in this study4 could by itself interfere with the glycolytic gluconeogenic pathway.16*” More importantly, in most, if not all, studies published, the mode1 of hyperthyroidism was a pharmacological one since 10 to 100 rg per 100 g body weight (BW) of T3 was injected daily for 7 days or more. This treatment represents 10 to 100 times the physiological daily T3 requirement.” Thus, it is not yet clear whether mild hyperthyroidism, as observed in most patients with hyperthyroidism, will increase gluconeogenesis from glycerol and other gluconeogenic substrates. Therefore, in order to know whether or not T3 in the pathophysiological range affects gluconeogenesis from a relatively small concentration of glycerol (2 mmol/L), we studied this metabolic pathway in isolated liver cells from 24-hour fasted rats either thyroidectomized, normal, or with various degrees of hyperthyroidism. The role of a possible alteration in the transport of reducing equivalents was investigated by studying the effect of ethanol and pyruvate on gluconeogenesis from glycerol.

Metabolism, Vol39,

No 3 (March), 1990: pp 259-263

MATERIALS

AND

METHODS

All chemicals were obtained from Merck (Darmstadt, FRG) or Sigma Chemical (La Verpillitre, France). All enzymes were purchased from Boehringer (Meylan, France). T3 was from Sigma. Mini-osmotic pumps were a product of Scientific Marketing Associates (London, England). Male Sprague-Dawley rats (160 to 180 g) were obtained from IFFA CREDO (Dardilly, France). Four different groups of rats were studied (five to 10 per group). Normal rats were treated twice daily by intraperitoneal (IP) injection of normal saline containing 0.01 N NaOH. Hypothyroid rats were produced by surgical removal of the thyroid gland 8 weeks before use. As previously shown, I9 this treatment induced a tenfold increase in serum thyroid-stimulating hormone. Only rats in which growth rate was completely stopped were selected. These hypothyroid rats also were injected with saline. Mild to important byperthyroidism was produced by twice daily IP injection of T3 1, 5, or 10 pg/d/lOOg BW during 3 days. Pharmacological hyperthyroidism was induced by IP infusion of T3 through mini-osmotic pump of 50 rg/d/lOO g BW for 7 days. Plasma T3 concentration” in normal, hypothyroid, and T3 rats (1, 5, 10 rg and 50 pg) was, respectively, 60*8,15*3,94*8,216* 11,472*69,2,400+ 150ng/lOOmL (x * SEM). Hepatocytes were isolated using the method of Berry and Friend2’ as previously described.” Cell viability was routinely assessed by the trypan blue exclusion method, by measuring the adenosine triphosphate (ATP) concentration (2.1 * 0.1 rmol . g-‘, n = 20) and the linearity of glucose production from the substrate used. Incubations were performed in a well-gassed (95% 0,/5% CO,) Krebs Ringer bicarbonate buffer pH 7.45 containing 1% bovine serum albumin at a low cell concentration (5 to 10 mg wet weight [WW] per mL). Cells incubated in 20 mL polyethylene tubes were shaken continuFrom the Institut National de la SantC et de la Recherche M;dicale. Facultb de Medecine. Lyon, France. Supported by a grant from “Ministere de la Recherche” no 86025 to B.C. Address reprint requests to Blandine Comte. INSERM U 197, Facultb de Mkdecine A. Carrel, 8 rue G. Paradin. 69372 Lyon Ckdex 08-France. (D1990 by W.B. Saunders Company. 0026-0495/90/3903-00007%3.00/0

259

COMTE ET AL

260 ously (150 oscillations/min) until the incubation was stopped by the addition of ice cold perchloric acid (12% vol/vol). Metabolites (glucose, glycerol, G3P, lactate, pyruvate, ATP, ethanol) were determined in the neutralized deproteinized supernatant by standard enzymatic procedures. r’ For measurement of ethanol utilization, a standard curve was performed for every experiment. Food was withdrawn from each rat 24 hours before cell isolation. All results were expressed in micromoles of metabolites produced or utilized per gram-’ of cells (WW) and per minute-’ and presented as the mean * SEM of the number of cell preparations tested. Statistical analysis was performed using parametric tests (variance analysis and Student’s t test) and nonparametric tests as appropriate.

production from glycerol. Inhibition of gluconeogenesis by ethanol was dose-dependent, with an apparent inhibitory coefficient (Ki) of ethanol within normal range of T3 concentration. This finding explains why, when gluconeogenesis is measured in the presence of ethanol, even mild hyperthyroidism produces a significant increase of this pathway compared with cells from normal rats. When the same incubations were performed in the presence of pyruvate instead of ethanol, it was found that the addition of pyruvate completely suppresses the decrease of the gluconeogenic flux observed in cells from hypothyroid rats. This classical stimulating effect of pyruvate on gluconeogenesis from glycerol’6 was also observed, but to a lesser degree, in cells from rats with mild hyperthyroidism. The variation of G3P in cells incubated with ethanol or pyruvate confirms that these two metabolites are mainly acting through changes in [free NADH]/ [free NAD’] ratio. When NADH was provided by the addition of ethanol, G3P increased in each group of rats. When NADH was consumed by the addition of pyruvate, G3P was undetectable except in the cells from hypothyroid rats. As the thyroid status could have changed the metabolism of ethanol in the liver cells,24-26ethanol utilization was measured in cells from hypothyroid, normal, mild hyperthyroid, and severely hyperthyroid rats (Table 2). It was found to be similar in the first three groups and slightly increased in the group treated by 50 pg of T3. Table 2 also shows the change in the lactate/pyruvate ratio. In cells incubated with glycerol alone, there was no significant change with thyroid status. Ethanol addition led to a drastic increase of the lactate/pyruvate ratio only in cells from normal and hyperthyroid rats. The changes in glycerol utilization by the cells in the different thyroid states were grossly parallel to the changes in glucose production (Fig 1). Glycerol utilization was significantly decreased in cells from thyroidectomized rats and

RESULTS

Table 1 summarizes the results obtained in the different groups of rats. As previously shown by others,3-5gluconeogenesis from glycerol was significantly decreased in cells from hypothyroid rats and increased in cells from severely hyperthyroid rats (50 pg T3-treated rats). No parallel changes were observed in lactate production. G3P was increased in cells from hypothyroid rats and decreased in cells from hyperthyroid rats. When gluconeogenesis from glycerol was measured in cells from rats with mild hyperthyroidism (1 to 10 pg T3-treated rats), no significant difference was observed in glucose flux, even in the 10 pg T3-treated group in which G3P was significantly decreased. In order to investigate further this metabolic pathway, glucose production from glycerol was studied in the presence of ethanol used as an NADH donor and in the presence of pyruvate used as an NADH acceptor.24 Table 1 shows that in the presence of ethanol, gluconeogenesis from glycerol was completely suppressed in cells from hypothyroid rats, significantly decreased in cells from normal rats but hardly affected in cells from hyperthyroid rats. As little as 5 pg/d of T3 per 100 g BW for 3 days suppresses almost completely the inhibitory effect of ethanol on glucose

Table 1. Gluconeogenesis From Glycerol (2 mmol/LI f Ethanol (10 mmol/L) or Pyruvate (10 mmol/L) in Isolated Liver Cells From 24-Hour Fasted Rats T3

Thyroid Status TX

Substrates

N

1 PB

51rg

T3 50 fig

JOtie

. min ’ 0.7 2 0.05

Glucose Production pmol/g Glycerol

0.33

Glycerol + ethanol Glycerol + pyruvate

* 0.04’

0.7 * 0.03 0.40

0 + O.OOlS 1.16 + 0.061)

* 0.039

Glycerol

0.11

+ 0.01

0.15

k 0.01 (6)

(6)t

Glycerol + pyruvate

0.02

f 0.005

0.045

f 0.008

+ 0.1 f 0.07

0.59

1.42 k 0.14

1.30 + 0.0811

Glycerol + ethanol

0.76 0.51

(5)

1.11 r 0.1

G3P Production pmoljg -

(6)/l

min

Glycerol

0.14

f 0.04

(6)

0.19

+ 0.06 (7)

0.12

f 0.03

(6)

0.19

* 0.03 (7) 2.67 (21

0.79

-t 0.3

0.94

c 0.06f

0.63

+ 0.04

0.83

f 0.05%

1.24 + 0.035 0.004

+ 0.002

0.038

+ 0.02 (3)$

* 0.03

(3)

3.66 12)

NOTE. Rats were hypothyroid (TX), normal (NI, mildly hyperthyroid fT3, 1,5,

(4)’

ND (2) 0.034 (2)

ND (3)

0.3 * 0.07 (3) 0.39

1.71 * 0.115



-

ND (5)

Glycerol + ethanol

1.77 (2)

.

0.1 f 0.01 (5)$ Lactate Production pmol/g

Glycerol + pyruvate

+ 0.06

.

min

ND (2)



0.16 (2)

0.18

f 0.03 (4)

0.15

* 0.02 (5)

0.22 (2)

0.21

* 0.03 (3)

0.13

* 0.05 (3)

3.02 (2)

2.18 (2)

10 fig/d/l 00 g 8W for 3 days) or severely hyperthyroid (50 pg/d/lOO

g

8W for 7 days). Hyperthyroidism was induced as described in the Methods section. Cell incubations were parfwmed during 60 minutes. Metsbolites were measured as described in the Methods section. Results sre expressed in gmol/g &VW) and lactate concentrations, the number

of experiments is shown

- min- ’ f

SEM, for an sverageof six experiments. For G3P

in parentheses. ND, not detectable.

Comparison between cells from normal rats, incubated with glycerol alone and cells from hypothyroid or T3-treated rats: l, tP < .Ol, P < .05, respectively. Comparison in the same soup of rats, of cells incubated with glycerol alone versus cells incubated with glycerol + pyruvate or ethanol: $, 5, 1lP-c .Ol, Pc

.05, PC .OOl,respectively.

261

GLUCONEOGENESIS FROM GLYCEROL: EFFECTS OF T3

Table 2. Ethanol Utilization in isolated Liver Cells From 24-Hour Fasted Rats 0th~ Addition

ThyroidStatus

Ethand Utilization

None

Normal

Ethanol ( 10 mmol/L) Hypothyroid

10.1 + 1.8 (6)

1.87 f 0.11 (4)

2 1.3 f 0.7 (3)

-

12.2 f 2.7 (4)

None Ethanol (10 mmol/L)

Hyperthyroid (fig/d/ 100 g 8W

Lactate/PyruvateRatio

-

2.19

* 0.19 (3)

13.5 * 1.9 (4)

3-d LP

lOlr9

None

-

Ethanol ( 10 mmol/L) 50 peg

9.0 f 2.6 (5)

1.72 f 0.27

None

(5)

29.1

-

Ethanol (10 mmol/L)

2.57

f 8.5 (4)

5.3 f 0.4 (3)

i 0.06 (4)’

14.7 f 3.5 (3)

NOTE. Hepatocyte incubations with 2 mmol/L glycerol + 10 mmol/L ethanol, were performed as indicated in Table 1. Results are expressed in pmol/g (WW)

.

min-’

f SEM. The number of experiments are shown in parentheses.

and other substrates was decreased in cells from thyroidectomized animals and increased in cells from severely hyperthyroid rats. A striking finding was the lack of increment of gluconeogenesis from either glycerol, lactate, or pyruvate in cells from rats treated with either 1,5, or 10 pg/d T3 per 100 g BW for 3 days. The first question that arises is whether the rats were actually in an hyperthyroid state. The dose of 1 rg/d of T3 per 100 g BW is supposed to meet the physiologic daily requirement of a thyroidectomized rat.18 Therefore, the doses of 5 and 10 pg are far in excess and should have brought the rat in a clear hyperthyroid state. Keyes and Heimberg” have clearly shown that such treatment produces a definite hyperthyroid state with regard to ketone body metabolism. Moreover, in our study plasma T3 was clearly increased in the 5 and 10 pg T3-treated groups. Finally, and more importantly, it is clear that the metabolic state of the liver cells was affected by this treatment since glycerol flux in cells from mildly hyperthyroid rats was found to be clearly increased when incubations were performed in the presence of ethanol. This finding brings new insight into the possible mechanisms by which T3 exerts its gluconeogenic effect. At least two mechanisms could be hypothetized: changes in reducingequivalents transfer capacity25v27v29 secondary to induction of

further suppressed by the addition of ethanol. In cells from mildly hyperthyroid rats incubated with glycerol plus ethanol, the glycerol utilization rate was significantly increased compared with cells from normal rats, as was the gluconeogenie flux. The addition of pyruvate raises glycerol utilization to values identical to normal in cells from hypothyroid rats, but was devoid of any stimulating effect in cells from hyperthyroid rats. These results strongly suggest that the gluconeogenic effect of T3 is closely related to the redox state of the cells. In order to test further this hypothesis, we compared in the four groups of rats the gluconeogenic flux from 10 mmol/L lactate and 10 mmol/L pyruvate, which could be considered as reduced and oxidized substrates, respectively. Figure 2 shows that gluconeogenic flux from lactate compared with pyruvate was significantly reduced in cells from hypothyroid rats and significantly increased only in cells from the severely hyperthyroid rats. In the mildly hyperthyroid group neither gluconeogenesis from lactate nor from pyruvate was increased when compared with cells from normal rats. DISCUSSION

The results presented in this study confirm previous showing that gluconeogenesis from glycerol

findings3.4.26.27

c

.E

\

E

3

0

Hypothyroid

rats

n Normal rats

F :

I@ Hyperthyroid

rats

z

E

2

a c

t

0 ‘C c, : z Fig 1. Glycerol utilization in cells from 2Qhour starved hypothyroid rats, normal rats, and hyperthyoid rats (10 Ccg/d/lOO g BW for 3 days). Incubation condiiions were as described in Table 1. The number in brackets indicates the number of cell preparations tested. + indiccltes significant diisrence from ceils obtained from normal rats (++ P-z.01. +++ PC .OOl).

.C d 3

1

z

0 I z

(10)

(7)

2lnrl

glycerol

(8)

(10)

(7)

(8)

2mM glycerol+ 1 OmM ethenol

(8)

(7)

(9)

2mM glycerol+ 1 OmM pyruwte

COMTE ET AL

0.6

0*4

02

-,-

0 ~0

(8)

(7) TX

(5)

(7) I

(6)

(3)

(5)

T3 1Oug

mitochondrial G3PDH and increase in the activity of key gluconeogenic enzymes.7-9 Glycerol metabolism is directly related to reducingequivalent transfer.29-33 Mitochondrial G3PDH has long been recognized as having been induced by T3.‘“-‘5 The cytoplasmic enzyme is regulated by the availability of NAD+.16 In the hypothyroid state the suppression of gluconeogenic flux from glycerol (Table 1) and the increase of G3P concentration has been related to the suppression of mitochondrial G3PDH activity.14 When cells from hypothyroid rats were incubated with ethanol, the metabolism of which well-utilized NAD+,26S34-36 the gluconeogenic flux from glycerol was totally suppressed (Table l), a finding that strongly supports the lack of activity of mitochondrial G3PDH. The increase in the lactate/pyruvate ratio (which reflects the cytosolic NAD+/NADH ratio3’) and in the G3P concentration suggest that the capacity of transfer of reducing equivalents and the rate of mitochondrial NADH reoxydation were saturated. In hepatocytes from normal rats, in which the mitochondrial enzyme should be present, a significant flux of glycerol to glucose remains in the presence of ethanol, probably because the mitochondrial G3PDH is able to transfer inside the mitochondria the reduced equivalents. When NAD+ was provided by the addition of pyruvate, the glycerol flux in cells from hypothyroid rats was back to the control value (Table l), suggesting that when there is no need to transfer reducing equivalents, the cytoplasmic enzyme that is not affected by T3 is active enough to process the glycerol molecule through the pathway. The involvement of mitochondrial G3PDH is also of importance in the control of gluconeogenesis in liver cells from mildly hyperthyroid rats. Indeed, in this situation even though the addition of ethanol increased G3P levels, it was

(3)

T3 5Oug

Fig 2. Gluconeogenwis from lactate (10 mmol/ L) (ml and pyruvate (10 mmol/L) (ml in liver cells from 24-hour fasted hypothyroid (TX), normal IN), and hyperthyroid (T3.10 &. and (T3.60 w) rats. Comparison between cells from normal rats, incubated with lactate or pyruvate, and cells from hypothyroid or TB-treated rats: ‘,++,+‘+, P

Influence of thyroid hormones on gluconeogenesis from glycerol in rat hepatocytes: a dose-response study.

The role of L-3,3'-5 triiodothyronine (T3), in a pathophysiological range, on gluconeogenesis from low concentration of glycerol (2 mmol/L), was inves...
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