PAPER I
Interaction between growth hormone and insulin in the regulation of lipoprotein metabolism in the rat.
GH affects many parameters involved in lipoprotein metabolism, and it also increases insulin secretion from β-cells [171]. To investigate the importance of increased serum insulin levels for in vivo effects of GH on lipoprotein metabolism, Hx rats were treated with GH or insulin alone or with GH and insulin in combination.
GH treatment of Hx rats decreased LDL cholesterol and increased HDL cholesterol levels independently of concomitant insulin treatment (Fig. 1A, Paper I). Similarly, GH decreased the level of LDL triglycerides and somewhat increased the VLDL triglyceride level, effects that were not affected by the presence of insulin (Fig. 1B, Paper I). The triglyceride secretion rate from the liver was increased by GH treatment, but this effect was suppressed by combined insulin treatment (Fig. 2B, Paper I). The hepatic triglyceride content changed in parallel with the secretion of triglycerides by the hormonal treatments (Fig. 2D, Paper I), suggesting that the rate of triglyceride secretion is dependent on triglyceride availability. The mRNA expression of two lipogenic enzymes was therefore measured. GH increased the mRNA levels of FAS and SCD-1 (Fig. 4, Paper I), which is likely to contribute to the increased hepatic triglyceride content and secretion after GH treatment. The effect of GH on FAS and SCD mRNA expression was similar in the presence of insulin, indicating that the GH-antagonistic action of insulin on triglyceride secretion rate and content is not via decreased mRNA expression of these genes. The mRNA level of SREBP-1c, a known regulator of lipogenic enzymes, was also increased by GH treatment (Fig. 4, Paper I).
In contrast to FAS and SCD, however, the effect of GH was counteracted by insulin, showing that upregulation of SREBP-1c mRNA is not needed for increased expression of FAS and SCD mRNA. Insulin treatment alone increased SCD and tended to increase FAS mRNA levels, suggesting that the stimulatory effect of GH on these genes is partly mediated by insulin. The serum level of apoE and editing of apoB mRNA were increased in GH-treated Hx rats (Fig. 3 and Table 2, Paper I). These findings indicate an increased turnover of VLDL particles after GH treatment, which agrees well with the calculated triglyceride clearance rate that is increased by GH treatment (Fig 7). ApoB mRNA editing was also elevated by insulin alone and the effect of GH was less marked in Hx rats given insulin compared to Hx rats not given insulin. This indicates that part of the effect of GH on apoB mRNA editing could be mediated by insulin.
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Figure 7. Effect of GH and insulin treatment on triglyceride clearance rate in Hx rats.
Values are based on 3-6 observations ±SEM (* p < 0.05 vs. Hx, one-way ANOVA followed by Bonferroni’s test).
In conclusion, GH treatment increased the hepatic triglyceride content and secretion, probably by enhancing lipogenesis (FAS and SCD mRNA). Upregulation of SREBP-1c mRNA was not required for increased FAS and SCD mRNA levels. Insulin antagonised the effects of GH on hepatic TG content and secretion, but this was not through decreased FAS and SCD mRNA expression. GH treatment also increased clearance of the secreted VLDL particles.
PAPER II
Effects of gender and growth hormone secretory pattern on sterol regulatory element binding protein-1c and its downstream genes in rat liver.
It is known that the hepatic triglyceride synthesis and VLDL secretion is higher in female rats compared to males due to the more continuous GH secretory pattern characteristic of the female rat [136, 137], but the mechanisms behind this are not fully known. As the availability of fatty acids limits triglyceride synthesis and hence formation and secretion of VLDL, the effects of gender and GH secretory pattern on the expression of genes involved in lipogenesis were investigated.
The mRNA levels of SREBP-1c, FAS and GPAT were higher in female rats than in male rats (Fig. 1, Paper II). Moreover, these genes were increased by GH administered as a continuous infusion to Hx female rats, thus mimicking the female GH secretory pattern, while GH given as two daily injections, thus mimicking the male GH secretory pattern, had no effect compared to Hx control rats (Fig. 2, Paper II). In contrast to the in vivo results, however, GH treatment in vitro decreased the mRNA levels of FAS and
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GPAT, and did not affect SREBP-1c mRNA expression (Fig. 3, Paper II). This suggests that the effect of GH on these genes in vivo is indirect. The GH plasma pattern of intact males was feminised by giving a low dose of GH as a continuous infusion. These rats were shown to be less insulin sensitive (Table 2, Paper II) and had higher levels of FAS and GPAT mRNA, indicating that continuous GH infusion could exert its stimulatory effect on FAS and GPAT mRNA expression through decreased insulin sensitivity. SREBP-1c mRNA expression, however, did not change in these animals, again showing that upregulation of SREBP-1c is not required for the stimulatory effect of the continuous GH infusion on FAS and GPAT mRNA (compare Paper I). The ACC-1 mRNA expression was not sex-differentiated but was specifically upregulated in Hx rats administered GH as a continuous infusion (Fig. 1 and 2, Paper II). However, ACC-1 mRNA levels were not affected in intact males given a low dose of GH as a continuous infusion, which suggests yet another regulation of this gene. In Paper I it was shown that the hepatic expression of SCD-1 increased after continuous infusion of GH to Hx rats. In this study, we extend those findings by showing that SCD-1 mRNA expression is upregulated by both female- and male-like GH administration to Hx rats (Fig. 2, Paper II) and that the mRNA level of SCD-1 was increased by GH in vitro (Fig. 3C, Paper II). Together these results show that SCD-1 is upregulated by a direct effect of GH on hepatocytes that is not dependent on the mode of GH exposure. The mRNA expression of liver X receptor (LXR) α, known to mediate the effects of insulin on SREBP-1c and lipogenesis, was decreased by GH both in vivo and in vitro (Fig. 2 and 3, Paper II) and could thus not explain the effects of GH on SREBP-1c and its downstream genes in vivo.
In conclusion, FAS and GPAT mRNA levels are specifically upregulated by the female GH secretory pattern, while SCD-1 mRNA expression is increased by GH irrespective of administration mode. Increased FAS and GPAT, but not SCD-1 mRNA levels, could thus explain the stimulatory effect of the female GH secretory pattern on hepatic triglyceride synthesis. Decreased insulin sensitivity, but not changed LXRα mRNA expression, could be responsible for these effects of GH in vivo.
PAPER III
Sex difference in hepatic microsomal triglyceride transfer protein expression is determined by the growth hormone secretory pattern in the rat.
In this paper, the mechanism behind the upregulation of VLDL secretion by the GH secretory pattern in female rats was further studied. As MTP is known to regulate the
assembly and secretion of VLDL particles, we investigated whether the expression of MTP also is influenced by gender and the sexually dimorphic GH secretory pattern.
The expression of MTP mRNA and protein was found to be higher in female rats than male rats (Fig. 1A and B, Paper III). This sex difference was abolished by gonadectomy, but restored by 17β-estradiol and testosterone treatment to Gx female and male rats, respectively (Fig. 1C, Paper III). The mRNA and protein expression of MTP was increased in male rats that were feminised with respect to their GH secretory pattern (Fig. 2, Paper III), while a continuous infusion of GH to female rats did not affect the expression of MTP. This suggests that the feminine, more continuous GH secretory pattern is responsible for the higher MTP expression in female rats. This is supported by the finding that Hx of female rats markedly decreased the level of MTP mRNA and protein, while Hx of male rats had no effect on MTP mRNA expression (Fig. 3, Paper III). Similarly, the mRNA and protein expression of MTP was specifically upregulated by the female-characteristic GH administration (continuous GH infusion) in Hx female rats (Fig. 4, Paper III). In Paper I it was shown that the hepatic triglyceride secretion was increased by a continuous infusion of GH and that this effect was diminished by conbined insulin treatment (Fig. 2B, Paper I). In this study, we investigated whether the inhibitory effect of insulin on triglyceride secretion could be due to a decrease in MTP levels. However, insulin did not affect the expression of MTP mRNA, neither alone nor in the presence of GH (Fig. 5, Paper III).
Thus, this indicates that insulin is not likely to mediate its inhibitory effect on GH-induced triglyceride secretion through decreased MTP mRNA expression.
In conclusion, the MTP expression is sexually differentiated with a higher expression in female rats due to the stimulatory effect of the feminine GH secretory pattern. These results might help to explain the effects of gender and GH on VLDL assembly and secretion.
PAPER IV
PPARα activation increases microsomal triglyceride transfer protein expression and activity in the liver.
PPARα agonists have previously been shown to enhance the secretion of apoB-100 in primary rat hepatocytes despite decreased triglyceride synthesis [92]. As the level of MTP is known to determine the secretion of apoB, we investigated whether the effect of PPARα activation on apoB-100 secretion could be explained by increased MTP expression.
Treatment of mice with the PPARα agonist WY 14,643 (WY) increased both expression and activity of MTP in the liver, but had no effect in the intestine (Fig. 1, Paper IV). WY treatment also increased MTP expression and activity in rat liver (Fig.
2, Paper IV), showing that the effect of WY was not specific to mice. Incubation of cultured mouse hepatocytes with WY also increased the mRNA expression of MTP, while a PPARγ agonist (rosiglitazone) did not have an effect on MTP mRNA levels (Fig. 3, Paper IV). This shows that MTP responds to PPARα, but not to PPARγ activation. Moreover, MTP mRNA expression in primary hepatocytes isolated from PPARα null mice was not changed by WY incubation, indicating that WY increases MTP expression specifically through PPARα activation (Fig. 4, Paper IV). Addition of the RXR ligand 9-cis-retinoic acid (cRA) to the medium, however, increased the expression of MTP both in primary hepatocytes from wildtype mice as well as PPARα null mice (Fig. 4, Paper IV), demonstrating that the effect of cRA is not dependent on PPARα. In line with the previous findings [92], WY increased the secretion of apoB-100 from primary rat hepatocytes but had no effect on apoB-48 secretion (Fig. 5C and D, Paper IV). In the present study, we extend these findings by showing the induction time for the increase in apoB secretion after WY treatment. MTP mRNA levels increased between 6 and 24 h, and MTP protein expression and apoB-100 secretion increased between 24 and 72 h (Fig. 5A and B, Paper IV). The similar time courses of these effects thus indicate that the stimulatory effect of the PPARα agonist on apoB-100 secretion could be mediated by an increased expression of MTP.
In conclusion, WY-induced PPARα activation stimulates MTP expression through a direct effect on hepatocytes. The increase in MTP levels is paralleled by a change in apoB-100 secretion, which indicates that MTP expression could mediate the stimulatory effect of WY on apoB-100 secretion.