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Endocrinology Vol. 144, No. 9 3914-3921
Copyright © 2003 by The Endocrine Society

Sex Difference in Hepatic Microsomal Triglyceride Transfer Protein Expression Is Determined by the Growth Hormone Secretory Pattern in the Rat

Caroline Améen and Jan Oscarsson

Department of Physiology and Pharmacology and Wallenberg Laboratory for Cardiovascular Research, Sahlgrenska University Hospital, SE-413 45 Göteborg, Sweden

Address all correspondence and requests for reprints to: Caroline Améen, Wallenberg Laboratory for Cardiovascular Research, Sahlgrenska University Hospital, SE-413 45 Göteborg, Sweden. E-mail: caroline.ameen{at}wlab.gu.se.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Microsomal triglyceride transfer protein (MTP) is essential and rate limiting for the assembly and secretion of apoB-containing lipoproteins. The aim of this study was to investigate whether gender and GH influence hepatic MTP expression. We used intact, gonadectomized, or hypophysectomized (Hx) adult Sprague Dawley rats. Gonadal steroids and insulin were given as a daily sc injection for 7 d. GH was given for 7 d either as a continuous infusion or as two daily injections (2 x GH) to mimic the feminine and masculine GH secretory patterns, respectively. MTP mRNA and MTP and protein disulfide isomerase protein expression was measured. MTP mRNA, and protein expression was higher in females than in males. Gonadectomy abolished the sex difference, and treatment with gonadal steroids restored the sex difference in MTP mRNA levels. MTP mRNA expression was not influenced in either sex by 2 wk of cholesterol (1% wt/wt) feeding. Hx decreased MTP mRNA in females but not in males. A continuous GH infusion increased MTP mRNA and protein expression in intact males but not in females. A continuous GH infusion to Hx females normalized MTP mRNA and protein expression, but 2 x GH had no effect. Also, insulin treatment had no effect. In summary, MTP expression is sex differentiated and regulated by the sexually dimorphic secretory pattern of GH at the level of mRNA. These results are important for the understanding of the effects of gender and GH in the regulation of very low-density lipoprotein assembly and secretion.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
MICROSOMAL TRIGLYCERIDE TRANSFER protein (MTP) is a heterodimeric lipid transfer protein that consists of a 97-kDa catalytic subunit (further referred to as MTP) associated with a 58-kDa multifunctional chaperone called protein disulfide isomerase (PDI). MTP is predominantly found in the lumen of microsomes isolated from liver and intestine. PDI is ubiquitously expressed in high concentrations (for review see Ref.1, 2).

MTP is crucial for the assembly and secretion of apolipoprotein B (apoB)-containing lipoproteins. It transports lipids, preferentially triglyceride and cholesterol esters, onto developing apoB in the lumen of the endoplasmic reticulum (1, 2). A defect in the large catalytic subunit of MTP causes abetalipoproteinemia, a rare autosomal recessive disorder that results in an inability to secrete apoB-containing lipoproteins from the liver and intestine (2).

Recent studies have shown that the level of MTP expression is rate limiting for the very low-density lipoprotein (VLDL) assembly and secretion. Adenoviral-mediated overexpression of MTP results in higher secretion of VLDL-triglycerides and VLDL-apoB from chow-fed mice (3, 4) and increased secretion of apoB100-containing lipoproteins from HepG2 cells (3). Conversely, heterozygous MTP knockout mice, showing a 50% reduction in MTP expression, have decreased secretion and plasma levels of apoB-containing lipoproteins (5, 6).

Not much is known about the hormonal regulation of MTP expression. The human MTP promoter contains an insulin-responsive element, which negatively regulates MTP expression (7). Similarly, high concentrations of glucose also reduce MTP gene transcription in HepG2 cells (8). In contrast, insulin resistance models, such as obese OLETF rats, (fa/fa) Zucker rats, ob/ob mice, and fructose-fed Syrian hamsters, have increased hepatic expression of MTP (9, 10, 11, 12). Cholesterol has been found to increase MTP mRNA expression in hamsters (13) and HepG2 cells (7). Conversely, cholesterol depletion of HepG2 cells lowered the level of MTP mRNA and protein via up-regulation of the sterol regulatory element binding protein-2 (14). Other manipulations, such as high-fat and high-sucrose diets in hamster, have been shown to increase hepatic MTP mRNA expression (15). Moreover, endotoxins and cytokines (TNF{alpha}, IL-1, and IL-6) are negative regulators of MTP mRNA expression (16).

Another putative regulator of MTP is GH because GH is known to influence the assembly and secretion of VLDL (17, 18, 19, 20, 21). The hepatic triglyceride synthesis and VLDL secretion are higher in female rats, compared with male rats (22, 23, 24). These sex differences have been shown to be dependent on the sexually dimorphic secretory pattern of GH (17, 20). In female rats, GH is released in a near continuous fashion with high basal levels, but male rats release GH intermittently with low or undetectable GH levels between peaks (25). Experimentally, the feminine and masculine GH secretory patterns can be mimicked by administering GH as a continuous infusion via miniosmotic pumps and as two daily sc injections, respectively (26, 27).

The aim of this study was to investigate whether MTP is regulated by gender and GH. We found a higher MTP expression in females, which was explained by the feminine GH secretory pattern. These results indicate that MTP expression is of importance for the sex difference in VLDL secretion and that GH is an important regulator of this protein.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
All chemicals used were from Sigma Chemicals (St. Louis, MO) if not stated otherwise.

Animals and hormonal treatment
Normal and hypophysectomized (Hx) Sprague Dawley rats were purchased from Møllegaard Breeding Center (Ejby, Denmark). They were maintained under standardized conditions of temperature (24–26 C) and humidity (50–60%) and with lights on between 0500 and 1900 h. The rats had free access to standard 4% fat (wt/wt) laboratory chow (rat and mouse standard diet, B&K Universal, Sollentuna, Sweden) and water. Alternatively, normal male and female rats were given a semisynthetic Western diet containing 20.8% (wt/wt) fat (mainly cacao butter) and 1% (wt/wt) cholesterol (R-638, Lactamin, Kimstad, Sweden) for 2 wk.

Male and female rats were gonadectomized at 37 d of age (Fig. 1CGo). Treatment of gonadectomized female rats with 17ß-estradiol (0.1 mg/kg·d) and gonadectomized male rats with testosterone (0.5 mg/kg·d) (28, 29, 30) was initiated 7–8 d after surgery. The hormones were diluted in propylene glycol and given as a daily sc injection for 7 d. Control rats were given vehicle alone.



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  		FIG. 1. Hepatic MTP mRNA (A) and protein (B) expression levels in intact female and male rats, and the effects of gonadal steroids given to gonadectomized (Gx) female (F) and male rats (M) (C) as well as MTP mRNA levels in cholesterol-fed rats (D). The rats (C) were gonadectomized at 37 d of age. Treatment with 17ß-estradiol (E2; 0.1 mg/kg·d) and testosterone (T; 0.5 mg/kg·d), dissolved in propylene glycol, started 7–8 d later. After 7 d of treatment, the rats were killed and the liver taken out. D, Male and female rats were given a high-fat diet containing 1% cholesterol for 2 wk. MTP mRNA and protein levels were determined with gel RPA and Western blot, respectively, as described in Materials and Methods. Representative blots are shown under each bar graph. Levels of MTP mRNA were normalized to ß-actin mRNA values (A and C) and cyclophilin mRNA levels (D). Both mRNA and protein levels are given as the percentage of the mean value in the female group. Values represent the mean of four to six observations ± SE. Bars with different superscripts denote statistically significant differences between groups (P < 0.05, t test).

 
Forty-day-old intact female and male rats were given recombinant bovine GH (0.5 mg/kg·d) as a continuous infusion by means of Alzet osmotic minipumps (model 2001, Alza Corp., Palo Alto, CA) implanted sc between the scapulae (Fig. 2Go) (31). The rats were anesthetized with a combination of ketamine hydrochloride (77 mg/kg; Ketalar, Parke-Davis, Detroit, MI) and xylazine (9 mg/kg; Rompun, Bayer, Lever-Kusen, Germany) during implantation of the osmotic minipumps. The recombinant bovine GH was a generous gift from Dr. Parlow (National Hormone and Peptide Program, Torrance, CA). The hormone was diluted in 0.05 M phosphate buffer (pH 8.6) with 1.6% glycerol and 0.02% sodium azide. The hormonal treatment continued for 7 d.



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  		FIG. 2. Hepatic MTP mRNA (A) and protein (B) expression levels in normal female and male rats after treatment with a continuous infusion of GH. Normal female and male rats were administered GH as a continuous infusion by means of implanted osmotic minipumps (GHc; 0.5 mg/kg·d). Age-matched female and male rats served as controls. Hormonal treatment continued for 7 d. Hepatic MTP mRNA and protein levels were determined with gel RPA and Western blot, respectively, as described in Materials and Methods. Representative blots are shown under each bar graph. Levels of MTP mRNA were normalized to ß-actin mRNA values. mRNA and protein levels are given as the percentage of the mean value in the female group and male group, respectively. Values represent the mean of five observations ± SE. Bars with different superscripts denote statistically significant differences between groups [P < 0.05, one-way ANOVA followed by Bonferroni’s test (A) and t test (B)].

 
Female and male rats were hypophysectomized with a temporal approach at 42–43 d of age (Figs. 3Go and 4Go). A weight gain of more than 0.5 g/d among Hx rats during a 7- to 10-d observation period was regarded as a sign of remaining pituitary tissue and used as an exclusion criterion (32). All of the Hx rats were substituted with cortisol phosphate (400 µg/kg·d; Solu-Cortef, Upjohn, Puurs, Belgium) and L-thyroxine (10 µg/kg·d); Nycomed, Oslo, Norway) diluted in saline and given as a daily sc injection at 0800 h (32, 33). Recombinant bovine GH (0.7 mg/kg·d) was given to female Hx rats either continuously by means of Alzet osmotic minipumps (GHc) or as two daily sc injections at 12-h intervals (0800 and 2000 h) (2 x GH) (31, 32). Hormonal treatment started 7–10 d after Hx and continued for 7 d. Age-matched rats served as controls.



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  		FIG. 3. Hepatic MTP mRNA (A) and protein (B) expression levels in normal and Hx female and male rats. All Hx rats were substituted with cortisol phosphate (400 µg/kg·d) and L-thyroxine (10 µg/kg·d) for 7 d (Hx). Age-matched female and male rats served as controls. MTP mRNA and protein levels were determined with gel RPA and Western blot, respectively, as described in Materials and Methods. Representative blots are shown under each bar graph. Levels of MTP mRNA were normalized to ß-actin mRNA values. Both mRNA and protein levels are given as the percentage of the mean value in the female group. Values represent the mean of five to six observations ± SE. Bars with different superscripts denote statistically significant differences between groups [P < 0.05, one-way ANOVA, followed by Bonferroni’s test].

 


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  		FIG. 4. Hepatic MTP mRNA (A) and protein (B) expression levels in Hx female rats administered GH as a continuous infusion or as two daily injections. Hx female rats were administered GH (0.7 mg/kg·d) either continuously (GHc) or as two daily injections (2 x GH). All Hx rats were treated with cortisol phosphate (400 µg/kg·d) and L-thyroxine (10 µg/kg·d). The hormonal treatments continued for 7 d. Those rats given GH injections were killed either 2 or 6 h after the last GH injection; in B, only the 6-h group is analyzed. Age-matched normal and Hx female rats served as controls. MTP mRNA and protein levels were determined with gel RPA and Western blot, respectively, as described in Materials and Methods. Representative blots are shown under each bar graph. Levels of MTP mRNA were normalized to ß-actin mRNA values. mRNA and protein levels are given as the percentage of the mean value in the female group and Hx group, respectively. Values in A represent the mean of four to nine observations ± SE and in B three to four observations ± SE. Bars with different superscripts denote statistically significant differences between groups [P < 0.05, one-way ANOVA followed by Bonferroni’s test].

 
Female rats were hypophysectomized at 50 d of age and substituted as above (Fig. 5Go). The rats were treated either with a continuous infusion of GH (1.5 mg/kg·d) via miniosmotic pumps or with a slow-release form of insulin (Insulatard, 100 IU/ml, Novo Nordisk, Gentofte, Denmark) (34, 35, 36). Alternatively, these two hormones were given in combination. Insulin was diluted in saline and given as a daily sc injection at 1600 h. To avoid insulin-induced fatal hypoglycemia, the insulin dose was gradually increased from d 1–4 (1.0 IU/d) to d 5–7 (2.0 IU/d) (34, 35, 36).



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  		FIG. 5. Hepatic MTP mRNA expression levels in Hx female rats treated with GH or insulin alone or with GH and insulin in combination. Hx female rats were treated with continuous GH (1.5 mg/kg·d) and/or a daily injection of insulin. The insulin dose was gradually increased from d 1–4 (1.0 IU/d) to d 5–7 (2.0 IU/d). All of the Hx rats were given cortisol phosphate (400 µg/kg·d) and L-thyroxine (10 µg/kg·d). The hormonal treatments continued for 7 d. Age-matched normal and Hx female rats served as controls. MTP mRNA and protein levels were determined with gel RPA and Western blot, respectively, as described in Materials and Methods. Representative blots are shown under each bar graph. Levels of MTP mRNA were normalized to ß-actin mRNA values. mRNA levels are given as the percentage of the mean value in the female group. Values represent the mean of four observations ± SE. Bars with different superscripts denote statistically significant differences between groups [P < 0.05, one-way ANOVA, followed by Bonferroni’s test].

 
At the end of the experiments, rats were decapitated and the livers were taken out between 0900 and 1100 h. The only exception was the experiment when the rats were given GH injections. These rats were killed either 2 h (1000 h) or 6 h (1400 h) after the last GH injection at 0800 h. The livers were immediately frozen in liquid nitrogen and stored at -70 C until analysis. The Ethics Committee of Göteborg University approved this study. All animal experimentation was conducted in accord with accepted standards of humane animal care.

Quantification of mRNA
Total RNA was isolated with TriReagent according to the manufacturer’s protocol (Ambion, Austin, TX) (37). The concentration of RNA was determined spectrophotometrically at 260 nm.

MTP.
A 179-bp-long fragment of rat MTP cDNA was amplified with specific primers (5'-AGTGTCTGTAAAGGCTGTCC-3' and 5'-CTTCTTTCTTCTCTGCCTTCAG-3') (9) and inserted into a pCR II-TOPO vector according to the manufacturer’s protocol (TOPO TA cloning kit, Invitrogen, Carlsbad, CA). The vector was linearized with HindIII, and a biotin-labeled antisense MTP RNA probe was generated by use of Biotin-16-UTP (Enzo, Roche, Indianapolis, IN) and T7 RNA polymerase (Maxiscript, Ambion).

ß-Actin and cyclophilin.
A 126-bp-long biotin-labeled fragment of rat ß-actin cDNA (Ambion) was used as an internal control in the gel ribonuclease protection assays (RPAs). The levels of ß-actin mRNA was not regulated by the various hormonal treatments used in this study, except for cholesterol feeding in female rats that increased ß-actin mRNA. We therefore used a 103-bp-long biotin-labeled fragment of rat cyclophilin cDNA (Ambion) as an internal standard in that analysis. Cyclophilin mRNA was not influenced by the cholesterol diet or gender.

The RNA probes were hybridized to the sample RNA in an RPA using a RPA III kit (Ambion). Protected fragments were separated on denaturing 6% polyacrylamide Tris-boric acid-EDTA-urea gels (Novex, San Diego, CA) and transferred to Bright Star-Plus membranes (Ambion) by a transfer system (Transblot cell; Bio-Rad Laboratories, Hercules, CA). After the transfer, the protected fragments were cross-linked to the membrane by UV irradiation (UVC Crosslinker, Hoefer Pharmacia BioTech, San Francisco, CA). The detection was carried out using the Bright Star BioDetect kit as described by the manufacturer (Ambion). The chemiluminescence was detected using a Fluor-S-Multimager (Bio-Rad) and the band intensity was quantified with ImageQuant software (Molecular Dynamics, Sunnyvale, CA). The amounts of the transcripts are expressed as the ratio between the MTP and the internal control band.

Protein analysis
Western blots were performed with total protein extracts from frozen livers. In brief, liver tissue was homogenized in homogenization buffer (0.05 M Tris-HCl, 2% sodium dodecyl sulfate, 15% sucrose, 5 mM dithiothreitol with supplemented protease inhibitors, Complete mini, Boehringer Mannheim, Mannheim, Germany), followed by denaturation at 95 C and centrifugation at 100,000 x g for 1 h. Proteins were recovered from the supernatant and concentrations were determined with RC DC protein assay kit II (Bio-Rad).

Thirty micrograms of protein were separated on 10% polyacrylamide Tris-glycine gels (Novex) and transferred to Hybond-P polyvinylidene difluoride transfer membrane (Amersham Pharmacia Biotech, Amersham, Buckinghamshire, UK) by a transfer system (Transblot cell, Bio-Rad) in transfer buffer [25 mM Bis-Tris (pH 7.6) with 192 mM glycine and 25% methanol] for 2–2.5 h at 400 mA. Using 30 µg protein, the amount of loaded protein was in a linear interval with respect to measured band intensity (data not shown). Equal loading was confirmed by staining the membranes with 0.2% Ponceau S (Serva, Heidelberg, Germany). The membrane-bound proteins were blocked overnight at 4 C in 50 mM Tris-buffered saline (pH 7.6) containing 0.1% Tween 20 (TBS-T) and 5% nonfat milk. This procedure was followed by incubation for 1 h at room temperature with polyclonal anti-MTP antibodies [kindly provided by Carol Shoulders (38)] diluted 1/2000 in TBS-T and 5% nonfat milk. The membrane was incubated for 1 h at room temperature with peroxidase-labeled antirabbit IgG secondary antibody (Amersham Life Science, Arlington Heights, IL) diluted 1/50,000 in TBS-T and 5% nonfat milk, followed by detection and development using the enhanced chemiluminescence detection system (Amersham Pharmacia Biotech) according to the manufacturer. Washing between incubations was performed with TBS-T. The chemiluminescence was measured using a Fluor-S-Multimager (Bio-Rad) and the band intensity was quantified with ImageQuant software (Molecular Dynamics). The molecular mass standard Full Range Rainbow marker (Amersham) was used to indicate the molecular mass of the proteins.

The anti-MTP antibody is raised against the whole MTP complex, and it therefore binds PDI in addition to MTP (38). The antibody also showed to bind to another unknown protein with a molecular mass of about 25 kDa. To identify this protein, the corresponding band was located on the gel by staining with Coomassie (0.1% Coomassie brilliant blue R-250, 10% acetic acid, and 50% ethanol). After excision of the band, its identification was established by in-gel trypsic digestion followed by mass spectrometry (MALDI-TOF and MS/MS) as described (39).

Statistical analysis
Values are expressed as means ± SE. Comparisons between groups were made by either t test or one-way ANOVA followed by Bonferroni’s test between individual groups. Values were transformed to logarithms when appropriate. P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Gender difference in MTP levels
Female rats have a higher VLDL secretion than male rats (22, 23, 24). Because the level of MTP expression regulates VLDL assembly and secretion, the rationale was to compare MTP levels in female and male rats. Livers were dissected from normal rats of both sexes at about 50 d of age, and MTP mRNA and protein levels were analyzed with RPA and Western blot, respectively. Female rats had 79% higher levels of MTP mRNA, compared with male rats (Fig. 1AGo). A comparable difference in MTP expression between female and male rats was also observed at protein level (Fig. 1BGo). There was no difference in PDI protein expression between female and male rats (females 100 ± 2.6%, males 92.1 ± 2.6%; n = 5–6).

The antibody we used was found to bind an unknown protein with a molecular mass of about 25 kDa in addition to MTP and PDI. This protein was also sexually differentiated but with a higher expression in males than females (Fig. 1BGo). Western blot and Coomassie staining revealed that this protein was abundant. The protein was identified as carbonic anhydrase III (CAIII) by in-gel trypsic digestion followed by mass spectrometry (MALDI-TOF and MS/MS) (39). CAIII is known to be more highly expressed in the livers of male rats than female rats (40).

To determine the importance of the gonads and gonadal steroids for the sex difference in MTP, male and female rats were gonadectomized (Fig. 1CGo). The sex difference in MTP mRNA was abolished in gonadectomized rats, but treatment of gonadectomized male rats with testosterone and gonadectomized female rats with 17ß-estradiol restored the sex difference in MTP mRNA expression (Fig. 1CGo).

Cholesterol has been shown to be a positive regulator of MTP in hamster (13) and HepG2 cells (7). To see whether cholesterol regulates MTP levels also in the rat and whether the response to cholesterol is different in males and females, rats of both sexes were given a cholesterol-enriched diet for 2 wk. This treatment did not alter MTP mRNA levels in either sex (Fig. 1DGo). The body weight gain did not vary between the different diets (data not shown). The livers from cholesterol-treated animals were yellowish in color, indicating an extensive loading of lipids, and serum cholesterol levels increased in cholesterol-fed rats of both sexes, compared with rats given standard laboratory chow (data not shown). Hence, the rats were fed a sufficiently cholesterol-enriched diet to have effects on the liver. Thus, cholesterol feeding does not change the sex-differentiated MTP mRNA levels in the rat.

Continuous infusion of GH to male rats increases MTP
VLDL assembly and secretion has been shown to be up-regulated by the feminine GH secretion pattern (18, 20, 36). Because elevated MTP expression increases the secretion of VLDL, we hypothesized that the higher MTP of female rats could be an effect of the more continuous secretory pattern of female rats. To investigate this, normal male rats were feminized with respect to the GH secretory pattern by giving a low dose (0.5 mg/kg·d) of GH as a continuous infusion. A low dose of GH given as a continuous infusion to male rats increased the levels of MTP mRNA to levels seen in normal female rats (Fig. 2AGo). Continuous infusion of GH to female rats did not affect their MTP mRNA levels. A continuous infusion of GH to males also increased MTP protein levels (Fig. 2BGo), although this treatment had no effect on MTP expression in females (data not shown). The expression of PDI protein did not differ between normal and GH-treated male rats (normal males 100 ± 6.1,% GHc males 87 ± 12.4%; n = 5). The reduction in CAIII protein levels in males given a continuous infusion of GH shows that CAIII protein expression also was feminized by this treatment (Fig. 2BGo).

Hypophysectomy of female rats results in a decrease in MTP
Removal of the pituitary abolishes the sex differentiated endogenous GH secretion and would therefore show the relative importance of the continuous and intermittent GH secretion pattern in the regulation of MTP expression. MTP mRNA expression decreased markedly by hypophysectomy of female rats (Fig. 3AGo). Hypophysectomy of male rats, on the other hand, had no effect on MTP mRNA expression. These findings indicate that the GH secretion of females is more important for MTP mRNA expression than the GH secretion of males. In parallel with mRNA data, the expression of MTP protein clearly decreased in Hx female rats (Fig. 3BGo). In contrast to mRNA levels, MTP protein levels decreased after hypophysectomy of male rats (Fig. 3BGo). No differences in PDI protein expression levels were observed (normal females 100 ± 2.6%, Hx females 92.7 ± 1.5%, normal males 92.1 ± 2.6%, Hx males 90.4 ± 4.3%; n = 5). As seen in Fig 3BGo, CAIII expression was regulated by hypophysectomy opposite to MTP (i.e. it was markedly up-regulated by hypophysectomy of female rats), but hypophysectomy of male rats had a minor effect.

Increase in MTP by continuous but not intermittent GH administration
To decisively establish the role of the female GH secretion pattern in controlling MTP expression, female rats were hypophysectomized and administered GH either continuously or as two injections daily. This regimen imitates the feminine and masculine pattern of GH secretion, respectively (26, 27). The level of MTP mRNA decreased after hypophysectomy in female rats and was normalized after treatment with a continuous infusion of GH (Fig. 4AGo). Two daily injections of GH to Hx female rats, on the other hand, had no effect, compared with Hx controls, irrespective of measuring the MTP levels 2 or 6 h after the last GH injection (Fig. 4AGo). The changes in MTP mRNA levels were mirrored by similar variations in protein levels (Fig. 4BGo). Thus, a continuous infusion of GH resulted in increased MTP protein expression, but two daily GH injections had no effect. No significant effects on PDI protein levels were detected (Hx 100 ± 40.1%, GHc 90 ± 17.2%, 2 x GH (6 h) 133.0 ± 16.2%; n = 3–4). Finally, CAIII protein expression decreased in Hx rats given GH as a continuous infusion, but two daily GH injections had no effect, compared with Hx rats (Fig. 4BGo).

Insulin does not antagonize the effect of GH on MTP mRNA
We have previously shown that the hepatic triglyceride secretion rate increased after 7 d of continuous infusion of GH to Hx rats and that this increase was blunted by concomitant insulin treatment for 7 d (36). Because it has been shown that MTP expression is up-regulated in insulin resistance models (9, 10, 11, 12) and that the human MTP promoter is under negative insulin regulation (7), we hypothesized that the inhibitory effect of insulin on hepatic triglyceride secretion could be due to decreased MTP expression. We therefore used liver RNA from the experiment described in the article by Frick et al. (36) and measured MTP mRNA. A continuous GH infusion enhanced MTP mRNA levels in Hx female rats (cf. Fig. 4AGo and 5Go). Insulin alone to Hx female rats had no effect on MTP mRNA expression (Fig. 5Go) and insulin did not inhibit the increased MTP mRNA levels seen after administration of GH as a continuous infusion.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Previous studies have shown that gender and gonadal steroids regulate VLDL secretion in man (41), rats (22, 23, 24), and apoE*3-Leiden transgenic mice (42). Thus, female gender and estrogens stimulate VLDL secretion and male sex and testosterone have the opposite effect. The results of this study suggest that a higher expression of MTP helps to explain the higher VLDL secretion in females, compared with males. Because we found that the sex difference in MTP expression could be restored in gonadectomized rats by giving sex steroids, we conclude that the gonadal steroids are important determinants for the sex difference in MTP expression. Because gonadal hormones have been found to regulate the sex-differentiated GH secretory pattern (29), our findings suggest that gonadal steroids regulate hepatic MTP expression via their effects on the sexually dimorphic GH secretory pattern.

GH seems to affect MTP expression mainly by changing the mRNA expression. The only discrepant effects on MTP mRNA and protein were observed in Hx male rats. The reason for this finding is unclear but may be a general effect on pituitary deficiency on hepatic protein synthesis.

Cholesterol has been shown to up-regulate MTP mRNA levels in hamster (13) and HepG2 cells (7). In rats, however, cholesterol feeding had no effect on MTP mRNA expression in either male or female rats. This finding indicates a species difference and, moreover, that factors regulated by cholesterol metabolism is not involved in the sex difference in MTP expression in the rat.

Insulin had no effect on MTP mRNA expression, either alone or after a continuous GH infusion. Therefore, the inhibitory action of 7 d of insulin treatment on the GH stimulated hepatic triglyceride secretion rate in Hx rats (36) is not likely to be mediated by decreased MTP expression.

Many functions in the liver are sex differentiated, and several of those have been shown to be regulated by gonadal steroids via their influence on the secretory pattern of GH. Gene products that are sexually dimorphic and regulated by the GH secretory pattern include several P-450 enzymes (26, 27) and other proteins such as major urinary proteins (43), apolipoprotein E (17), S-adenosylmethionine synthetase (31), and CAIII (44). Using the MTP/PDI antiserum, we could also detect CAIII. The reason for this is not clear, but the proteins may have epitopes in common, although no obvious amino acid sequence similarities could be detected by database homology search. We did not have the preimmune serum from these rabbits, but other preimmune rabbit serum could not detect CAIII. However, the observed regulation of CAIII protein was in line with previously published results regarding the regulation of CAIII (40, 44), including the finding that a continuous infusion of GH partially feminized the high CAIII expression in males (44).

We have shown that the higher hepatic triglyceride synthesis in females, compared with males, could be explained by the more continuous secretion of GH in females (20). Increased hepatic triglyceride synthesis (20, 21) and accumulation of triglycerides (21, 36) can help to explain a higher VLDL assembly and secretion after GH treatment because an increased supply of triglycerides decreases the co- and posttranslational degradation of apoB. However, increased MTP expression also decreases cotranslational degradation of apoB and increases the VLDL assembly and secretion by enhancing the supply of triglycerides and cholesterol esters to the growing apoB chain (2, 45). Recent studies also suggest that MTP takes part in the assembly of the apoB-free lipid droplets found in the endoplasmic reticulum (46) that in a second step of VLDL assembly form mature VLDL particles (2, 45). The feminine GH secretory pattern also increases the hepatic production of apoE, probably by increasing the translation of apoE (17). Because apoE production is of importance for VLDL secretion (47), three main factors may contribute to the increased VLDL assembly and secretion following continuous GH infusion: 1) increased apoE production, 2) increased triglyceride synthesis, and 3) increased MTP expression.

There is evidence to suggest that the sex difference in GH secretion in man (48) also takes part in the regulation of hepatic metabolism in general (49) and in lipoprotein metabolism in particular (50, 51). Lipoprotein (a) is secreted from the liver via assembly of the apo(a)-protein with apoB-containing lipoproteins. A continuous infusion of GH to GH-deficient hypopituitary adults resulted in a 80% greater increase in lipoprotein (a) levels than daily sc injections of GH (51). Mice expressing a 370-kb human genomic fragment containing the apo(a) gene and a large part of the promoter [YAC-apo(a) transgenic mice] showed a marked sex difference in apo(a) mRNA expression and apo(a) plasma levels (50). A continuous infusion of GH markedly enhanced apo(a) mRNA and apo(a) plasma levels, but GH as daily injections had no effect in these mice. Together these studies (49, 50, 51) suggest that the human hepatic functions, including lipoprotein production, are also sensitive to the sexually dimorphic secretory pattern of GH.

In summary, we show that MTP expression is sex differentiated and that the feminine GH secretory pattern is an important determinant of MTP expression. Thus, GH is the only hormone that so far has been shown to increase MTP expression.


   Acknowledgments
 
We thank Dr. Carol Shoulders and Dr. Penny Ritchie at Imperial College School of Medicine, Hammersmith Hospital (London, UK) for providing the MTP antibody and Dr. Björn Dahllöf and Oleg Panfilov at Cell Biology and Biochemistry AstraZeneca R&D (Mölndal, Sweden) for their help with the identification of carbonic anhydrase III.


   Footnotes
 
This work was supported by Grant 14291 from the Swedish Medical Research Council, King Gustav V’s and Queen Victorias Foundation, Novo Nordisk Foundation, and the Swedish Heart and Lung foundation.

Abbreviations: apoB, Apolipoprotein B; CAIII, carbonic anhydrase III; Hx, hypophysectomized; MTP, microsomal triglyceride transfer protein; PDI, protein disulfide isomerase; RPA, ribonuclease protection assay; TBS-T, Tris-buffered saline containing Tween 20; VLDL, very low-density lipoprotein.

Received April 24, 2003.

Accepted for publication May 27, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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