Interaction between growth hormone and insulin in the regulation of lipoprotein metabolism in the rat

Fredrik Frick1,*, Daniel Lindén1,*, Caroline Améen1, Staffan Edén1, Agneta Mode2, and Jan Oscarsson1

1 Department of Physiology, Göteborg University, S-405 30 Göteborg; and 2 Department of Medical Nutrition, Karolinska Institutet, Novum, S-141 86 Huddinge, Sweden


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The importance of insulin for the in vivo effects of growth hormone (GH) on lipid and lipoprotein metabolism was investigated by examining the effects of GH treatment of hypophysectomized (Hx) female rats with and without concomitant insulin treatment. Hypophysectomy-induced changes of HDL, apolipoprotein (apo)E, LDL, and apoB levels were normalized by GH treatment but not affected by insulin treatment. The hepatic triglyceride secretion rate was lower in Hx rats than in normal rats and increased by GH treatment. This effect of GH was blunted by insulin treatment. The triglyceride content in the liver changed in parallel with the changes in triglyceride secretion rate, indicating that the effect of the hormones on triglyceride secretion was dependent on changed availability of triglycerides for VLDL assembly. GH and insulin independently increased editing of apoB mRNA, but the effects were not additive. The expression of fatty-acid synthase (FAS), stearoyl-CoA desaturase-1 (SCD-1), and sterol regulatory element-binding protein-1c (SREBP-1c) was increased by GH treatment. Insulin and GH had no additive effects on these genes; instead, insulin blunted the effect of GH on SREBP-1c mRNA. In contrast to the liver, adipose tissue expression of SREBP-1c, FAS, or SCD-1 mRNA was not influenced by GH. In conclusion, the increased hepatic expression of lipogenic enzymes after GH treatment may be explained by increased expression of SREBP-1c. Insulin does not mediate the effects of GH but inhibits the stimulatory effect of GH on hepatic SREBP-1c expression and triglyceride secretion rate.

apolipoprotein B; apolipoprotein E; apoB mRNA editing; triglyceride secretion; sterol regulatory element-binding protein-1; fatty-acid synthase; stearoyl-CoA desaturase; liver; adipose tissue


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

IT IS WELL KNOWN THAT GROWTH HORMONE (GH) has marked effects on lipid and lipoprotein metabolism (1, 30). GH also increases secretion (27, 32) and plasma levels of insulin in humans and rats (31, 32, 37, 44). Moreover, GH increases DNA synthesis and proliferation of beta -cells and insulin secretion in vitro (27), showing that GH enhances beta -cell function independently of its insulin-antagonistic action (34, 35, 45). The increased serum insulin levels and the insulin-antagonistic effect of GH may be of importance for several effects of GH in vivo, but few studies have addressed this question (31, 36). Treatment of normal rats with the combination of insulin and GH results in an additive effect on body weight gain. However, GH treatment antagonizes the stimulatory effects of insulin on food intake and adipose tissue weight, indicating a complex interaction between GH and insulin (36).

The interaction of insulin and GH in the regulation of lipid and lipoprotein metabolism is of special interest, because similar effects of GH and chronic hyperinsulinemia have been observed (3, 4, 25, 42, 46, 47). GH treatment in vivo increases editing of apoB mRNA, production of apolipoprotein (apo)B-48, and very low-density lipoprotein (VLDL) secretion from isolated hepatocytes (42, 43) and perfused liver (10). Furthermore, triglyceride synthesis and secretion have been shown to increase in hepatocytes after GH treatment in vivo (7, 42) and in vitro (25). GH therapy of GH-deficient adults also increased VLDL-apoB secretion, an effect accompanied by increased serum insulin levels and insulin resistance, as indicated by increased hemoglobin A1c (3).

Insulin treatment in vivo has been shown to either increase or decrease the VLDL triglyceride secretion (18, 46). The different effects of insulin in vivo could be attributed to the nutritional and hormonal status of the subjects, including degree of insulin resistance (18, 23). In vitro, the duration of insulin incubation has been shown to be of importance. Short-term incubations of hepatocytes with insulin (up to 16 h) decrease VLDL secretion. However, exposure to insulin for longer periods of time (24-48 h) results in increased VLDL secretion (18, 23, 46, 49) and editing of apoB mRNA (47), i.e., effects similar to those of GH in vitro (25).

GH treatment was recently shown to increase hepatic gene expression of two key enzymes involved in fatty acid synthesis: fatty-acid synthase (FAS) and stearoyl-CoA desaturase-1 (SCD-1) (12, 48). Insulin has been shown to induce FAS mRNA (38) and SCD-1 mRNA expression (28) in diabetic rodents. The effect of insulin on theses enzymes has been attributed to increased gene expression of the transcription factor sterol regulatory element-binding protein (SREBP)-1 (13, 40) and has been reviewed (20, 39). SREBP-1 exists in two forms, SREBP-1a and SREBP-1c, which are transcribed from a single gene by the use of alternate promoters (20, 39). SREBP-1c predominates in most organs, including the liver. It has been demonstrated that hepatic SREBP-1 mRNA expression is increased in hyperinsulinemic insulin-resistant rodent models concomitant with increased hepatic lipogenesis (8, 41) and increased VLDL secretion (8).

In the present study, we used hypophysectomized (Hx) female rats that had decreased insulin secretion (26) as a nondiabetogenic model to study the interplay of insulin and GH on lipid and lipoprotein metabolism. Moreover, we wanted to investigate whether the GH effects on lipogenic enzymes could be mediated by increased SREBP-1 gene expression.


    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

Female Sprague-Dawley rats (Møllegaard Breeding Center, Ejby, Denmark) were hypophysectomized with a temporal approach at 50 days of age and maintained under standardized conditions of temperature (24-26°C), humidity (50-60%), and with lights on between 0500 and 1900. The rats had free access to standard laboratory chow (rat and mouse standard diet, B&K Universal, Sollentuna, Sweden) and water. Hormonal treatment started 7-10 days after hypophysectomy. All of the Hx rats were given cortisol phosphate (400 µg · kg-1 · day-1; Solu-Cortef, Upjohn, Puurs, Belgium) and L-thyroxine (10 µg · kg-1 · day-1; Nycomed, Oslo, Norway) diluted in saline as a daily subcutaneous injection (0800) (29, 43). Recombinant bovine GH was a generous gift from American Cyanamide (Princeton, NJ). The hormone was diluted in 0.05 M phosphate buffer, pH 8.6, with 1.6% glycerol and 0.02% sodium azide. Bovine GH (1.5 mg · kg-1 · day-1) was given by means of Alzet osmotic minipumps (model 2001, Alza, Palo Alto, CA) implanted subcutaneously between the scapulae by use of xylazine (9 mg/kg; Rompun, Bayer, Lever-Kusen, Germany) and ketamine hydrochloride (77 mg/kg; Ketalar, Parke-Davis, Detroit, MI) anesthesia (29, 31). The GH dose given (1.5 mg · kg-1 · day-1) is based on the calculated secretion of GH in these rats (230 µg/day or ~1.3 mg · kg-1 · day-1) (16, 21). A slow-release form of insulin (Insulatard, 100 IU/ml, Novo Nordisk, Denmark) was diluted in saline and given as a daily subcutaneous injection at 1600. To avoid insulin-induced fatal hypoglycemia, the insulin dose was gradually increased from days 1-4 (1.0 IU/day) to days 5-7 (2.0 IU/day) (15, 17, 31). The hormonal treatment continued for 7 days. In one of the experiments, the rats were killed between 0900 and 1100 by decapitation without prior fasting. Trunk blood was collected, and retroperitoneal as well as ovarian adipose tissues were blotted and weighed. The livers and adipose tissue depots were snap-frozen in liquid nitrogen and stored at -70°C until analysis. In the other experiment, the rats were without food for 5 h before measurement of hepatic triglyceride secretion (see Serum Lipoprotein Profiles). The Ethics Committee of Göteborg University approved this study.

Serum Analyses

Triglyceride and cholesterol concentrations in both serum and fast protein liquid chromatography (FPLC) fractions were determined by enzymatic colorimetric assays (Roche, Mannheim, Germany). The intra-assay coefficient of variation (CV) was 4% for the triglyceride assay and 3% for the cholesterol assay. Serum apoB and apoE concentrations were determined by electroimmunoassays as previously described (44). Serum glucose concentrations were measured by the glucose-6-phosphate dehydrogenase method (Merck, Darmstadt, Germany). Serum insulin concentration was determined by an RIA (human insulin RIA, Phedebas, Pharmacia Upjohn, Uppsala, Sweden) (31, 44).

Serum Lipoprotein Profiles

Lipoprotein profiles were obtained by gel filtration using FPLC equipment (Pharmacia Upjohn) (14). Briefly, 250 µl of serum from 6 rats in each group were pooled to give a total volume of 1.5 ml, and the density was adjusted to 1.215 g/ml with KBr. After ultracentrifugation at 35,000 g for 24 h at 4°C, the removed supernatant, containing the total lipoprotein fraction, was adjusted with FPLC buffer (0.15 M NaCl, 0.01% EDTA, 0.02% sodium azide, pH 7.3) to 2 ml. After filtration through a 0.45-µm filter, the sample was loaded on a 25-ml Superose 6B column (Pharmacia Upjohn). The sample was eluted at a constant flow rate of 0.35 ml/min, and 0.5-ml fractions were collected. Triglyceride and cholesterol concentrations were determined with enzymatic colorimetric assays, as described above. The density (d) classes of lipoproteins obtained by sequential ultracentrifugation, VLDL (d <1.006 g/ml), intermediate/low-density lipoprotein (d 1.006-1.063 g/ml), and HDL (d 1.063-1.21 g/ml) are indicated in Fig. 1.


View larger version (26K):
[in this window]
[in a new window]
 
Fig. 1.   Effects of growth hormone (GH) and insulin treatment on size distribution of serum lipoproteins. Cholesterol and triglyceride contents of fractions are shown in A and B, respectively. Hypophysectomized (Hx) female rats were treated with GH (1.5 mg · kg-1 · day-1) or insulin (INS) alone and in combination (GH+INS) for 7 days. Normal female (N) and Hx rats not treated with GH or insulin served as controls. The dose of insulin was gradually increased from days 1-4: 1.0 U/day to days 5-7: 2.0 U/day. Animals were killed between 0900 and 1100 without prior fasting. Density (d) classes of lipoproteins obtained by sequential ultracentrifugation are shown as VLDL (d <1.006 g/ml), IDL/LDL (d 1.006-1.063 g/ml), and HDL (d 1.063-1.21 g/ml). Results are from pooled serum from 6 rats in each group. Similar results were obtained in 2 separate experiments.

Direct Primer Extension Analysis

To analyze the extent of apoB mRNA editing, direct primer extension was performed as described previously (25, 43).

In Vivo Hepatic Triglyceride Secretion

Triglyceride secretion rate in vivo was measured by intravenous administration of Triton WR-1339 (24). The animals were fasted for 5 h (0700-1200) to avoid the influence of ongoing production of chylomicrons from the intestine. Thereafter, the rats were anesthetized and injected intravenously with Triton WR-1339 diluted in saline (200 mg/ml) via the tail vein (500 mg/kg body wt). Blood samples were taken before the injection (0 min) and 60, 120, and 180 min after Triton WR-1339 administration. Serum triglyceride levels were analyzed as described above, and hepatic triglyceride secretion rate was calculated from the slope of the curve and expressed as micromoles per hour per gram of body weight. The plasma volume was estimated to be 3.2% of the body weight, as described in normal rats (Jackson Laboratories, www.jax.org).

Quantification of mRNA

Total liver RNA was prepared using the Tri Reagent system.

FAS. Specific primers (5'-CTGAGACTCTTCTGGGCTACA-3' and 5'-CGTTCCCTGAATCATCAAAGG-3') amplified a 291-bp-long fragment of rat FAS cDNA (nt 250-540, accession no. X13415), which was inserted into a pCR II-TOPO vector according to the manufacturer's protocol (TOPO TA Cloning kit, Invitrogen). The vector was linearized with EcoRV, and a biotin-labeled antisense FAS RNA probe was generated by use of Biotin-16-UTP (Enzo, Roche) and T7 RNA polymerase (Strip-EZ RNA, RNA probe synthesis kit, Ambion, Austin, TX).

SREBP-1. A 257-bp fragment of rat SREBP-1 subcloned into PGEM 3Zf(+) (Promega, Madison, WI) was kindly provided by Dr. Joseph Goldstein, University of Texas Southwestern Medical Center, Dallas, TX. This probe allows measurement of both SREBP-1a and SREBP-1c (40). The vector was linearized with HindIII, and a biotin-labeled antisense SREBP RNA probe was generated using Biotin-16-UTP and T7 RNA polymerase, as described above.

SCD-1. A 200-bp fragment of rat SCD-1 (nt 4283-4482, accession no. JO2585) was subcloned into PGEM 3Z (Promega, Madison, WI). We thank Cissi Gardmo for making this construct. The vector was linearized with EcoRI and biotin-labeled as described for FAS and SREBP-1, except for use of Sp6 RNA polymerase.

A biotin-labeled fragment of rat beta -actin cDNA (Ambion) was used as an internal control in the gel ribonuclease protection assays (RPA). The levels of beta -actin mRNA did not change as a result of the various hormonal treatments; beta -actin was therefore regarded as an appropriate control. RPA was performed as described by the manufacturer (RPA III kit, Ambion). Protected fragments were separated on denaturing 6% polyacrylamide Tris-boric acid-EDTA-urea gels (Novex, San Diego, CA). For detection of biotin-labeled probes, protected fragments were transferred to Bright Star-Plus membranes (Ambion). After the transfer, the protected fragments were cross-linked by UV irradiation to the membrane, and detection was carried out using the Bright Star BioDetect Kit as described by the manufacturer (Ambion). The chemiluminescence was detected and quantified using the Fluor-S-Multimager. The amounts of the mRNA are expressed as a percentage, the ratio between the respective mRNA and beta -actin mRNA.

Statistical Analysis

Values are expressed as means ± SE. Comparisons between groups were made by one-way analysis of variance (ANOVA) followed by Bonferroni's test between individual groups. Values were transformed to logarithms when appropriate. A P value <0.05 was considered significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Weights and Serum Analyses

Treatment with GH normalized the reduced weight gain of the Hx rats (Table 1). Insulin treatment increased the weight gain of Hx rats, but not to the level seen after GH treatment. Combined treatment with GH and insulin tended to increase the weight gain further compared with GH treatment alone (Table 1). The retroperitoneal adipose tissue weight was increased by insulin treatment, but this effect was blunted by concomitant GH treatment, indicating an insulin-antagonistic effect of GH. The ovarian adipose tissue weight was not significantly affected by GH or insulin treatment (Table 1). In this experiment, the rats were killed at 0900-1100. The serum level of insulin was 33.4 ± 10 mU/l in the group of Hx rats given insulin treatment compared with 8.0 ± 0.9 mU/l in Hx control rats when measured with a human insulin RIA. In Hx rats killed during the same diurnal period, serum insulin levels measured with a rat insulin RIA have been shown to be 13.7 ± 2.5 mU/l (15). Because the rats were killed 17-19 h after the last insulin injection, these results indicate that treatment with this slow-release form of insulin resulted in increased diurnal insulin levels. Serum glucose concentrations were not affected by Hx or GH treatment but decreased after insulin treatment (Table 1). Combined treatment with GH and insulin restored the serum levels of glucose, indicating an insulin-antagonistic effect of GH on glucose metabolism. Neither GH nor insulin had any effect on serum free fatty acid (FFA) levels in this experiment (Table 1).

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Weight gain, retroperitoneal and ovarian adipose tissue weights, serum glucose, and FFA levels

To investigate the interaction between GH and insulin in the regulation of lipoprotein metabolism, we measured serum lipids, lipoproteins, and apolipoproteins that are known to be influenced by GH (29, 44) (Table 2, Fig. 1). Serum cholesterol concentrations decreased in Hx rats compared with normal rats but were not significantly affected by GH or insulin treatment (Table 2). Serum triglyceride concentrations tended to decrease in Hx rats compared with normal rats but were not significantly affected by GH or insulin treatment (Table 2).

                              
View this table:
[in this window]
[in a new window]
 
Table 2.   Serum levels of cholesterol, triglyceride, apoB, and apoE

Cholesterol and triglyceride content in different lipoprotein fractions was measured by separating lipoproteins according to size by FPLC (Fig. 1). Hx rats had lower HDL cholesterol levels and increased LDL cholesterol levels compared with normal rats. GH normalized LDL cholesterol and increased HDL cholesterol to the same degree with or without insulin treatment (Fig. 1A). Hx rats had decreased VLDL triglyceride levels and increased LDL triglyceride levels compared with normal rats (Fig. 1B). GH slightly increased VLDL triglyceride levels and decreased LDL triglyceride levels. This effect of GH was similar with or without insulin treatment (Fig. 1B). Insulin alone increased the cholesterol and triglyceride content of the LDL fraction, an effect of insulin that was not observed in the presence of GH (Fig. 1, A and B).

GH treatment normalized the increased serum apoB levels and the decreased serum apoE levels observed in the Hx rats (Table 2). Insulin treatment alone had no effect on serum levels of apoB or apoE, and insulin treatment did not influence the effects of GH treatment (Table 2).

Hepatic Triglyceride Content and Secretion

To study the interaction between the effects of GH and insulin on hepatic triglyceride secretion in vivo, the rats were injected with Triton WR-1339 after 5 h of fasting (Fig. 2, A and B). The accumulation of triglycerides in serum after injection of Triton WR-1399 in the different groups of rats is illustrated in Fig. 2A. The hepatic triglyceride secretion rate was calculated and shown to be lower in Hx rats compared with normal rats. The hepatic triglyceride secretion rate increased ~70% by GH treatment (Fig. 2B). On the other hand, insulin treatment of Hx rats resulted in a trend toward lower hepatic triglyceride secretion. When GH and insulin were given together, the hepatic triglyceride secretion was markedly lower than when GH was given alone (Fig. 2B). Triglycerides and FFA concentrations in the serum samples taken before injection of Triton WR-1339 are shown in Fig. 2C. No significant effects of the various hormonal treatments on serum FFA levels were observed, in line with the results in the previous experiment. However, serum triglycerides were significantly lower when GH and insulin were given in combination (Fig. 2C). This result indicates that a short-term fast may influence the effect of combined GH and insulin treatment on serum triglycerides (see for example Table 2 and Fig. 2C).


View larger version (30K):
[in this window]
[in a new window]
 
Fig. 2.   A and B: hepatic triglyceride secretion in vivo from normal female rats (N), Hx female rats treated with GH or insulin (INS) or GH and insulin in combination (GH+INS). C: serum levels of free fatty acids (FFA) and triglycerides (TG). D: hepatic triglyceride content. Rats were fasted for 5 h before these samples were taken. Triglyceride secretion rate was measured after administration of Triton WR-1339 (500 mg/kg). Serum triglycerides were determined before (0 min) and 60, 120, and 180 min after Triton administration (A). Triglyceride secretion rate was calculated from the slope of the curves (B). Serum levels of triglycerides, FFA, and hepatic triglyceride content were measured as described in MATERIALS AND METHODS. Values are means ± SE for 5-6 rats/group. Values with different superscripts are significantly different from each other (P < 0.05, one-way ANOVA followed by Bonferroni's test).

To investigate whether the triglyceride content of the liver changed in parallel with the hepatic triglyceride secretion, hepatic triglyceride content was measured (Fig. 2D). Hepatic triglyceride content was influenced in a manner similar to triglyceride secretion after the hormonal treatments. GH treatment increased and insulin treatment decreased the hepatic triglyceride content. Moreover, when GH and insulin were given together, the hepatic triglyceride content was markedly lower than when GH was given alone (Fig. 2D).

ApoB mRNA Editing, SREBP-1, and Downstream Genes

ApoB mRNA editing in hepatocytes has been shown to increase by GH treatment in vivo (43) and in vitro (25). Moreover, editing of apoB mRNA has been shown to increase in the hyperinsulinemic Zucker rats (9) and by long-term insulin incubation of hepatocytes (25, 47). Therefore, apoB mRNA editing was measured (Fig. 3). All mRNA levels were determined in the rats that were not fasted before they were killed, i.e., those described in Tables 1 and 2. The Hx rats had decreased apoB mRNA editing compared with normal rats. GH treatment increased the editing of apoB mRNA without (apoB-100 to apoB-48 mRNA from 1:0.6 to 1:1.3) and with combined insulin treatment (from 1:0.85 to 1:1.3). Insulin treatment also slightly increased the editing of apoB mRNA (from 1:0.6 to 1:0.85) (Fig. 3).


View larger version (21K):
[in this window]
[in a new window]
 
Fig. 3.   Hepatic apolipoprotein (apo)B mRNA editing in normal female rats and in Hx female rats treated with GH or insulin alone or with GH and insulin in combination. Animals were killed between 0900 and 1100 without prior fasting. Editing of apoB mRNA was measured by direct primer extension and calculated as edited mRNA/(edited and nonedited mRNA). Values are means ± SE for 5-6 rats/group. Values with different superscripts are significantly different from each other (P < 0.05, one-way ANOVA followed by Bonferroni's test).

Both GH (12, 48) and insulin (28, 38) have been shown to increase FAS and SCD-1 gene expression in the liver. To address the question of an interaction between GH and insulin, we measured the expression of these gene products. Compared with normal female rats, Hx rats expressed lower levels of FAS and SCD-1 mRNA (Fig. 4, A and B). GH treatment normalized both FAS and SCD-1 mRNA expression. Insulin treatment tended to increase FAS mRNA and significantly increased SCD-1 mRNA, but the effects of GH and insulin were not additive. Because increased FAS and SCD-1 gene expression has been shown to be mediated by increased expression of SREBP-1 (20, 39), we investigated the effect of GH on SREBP-1 expression. Compared with normal female rats, there was a trend toward a decreased expression of SREBP-1c mRNA in Hx rats. GH treatment resulted in an 85% increase in SREBP-1c mRNA expression (Fig. 4C). Insulin had no effect alone, but the effect of GH was blunted by concomitant insulin treatment (Fig. 4C). We also measured hepatic expression of SREBP-1a mRNA, but none of the hormonal treatments affected SREBP-1a gene expression (data not shown). Because GH may also affect SREBP-1 gene expression in adipose tissue, the effect of Hx and GH on adipose tissue expression of SREBP-1 mRNA, FAS mRNA, and SCD-1 mRNA was determined. There was no significant effect of Hx or GH treatment on SREBP-1c, FAS, or SCD-1 mRNA in adipose tissue (Table 3). As in the liver, there was no effect of Hx or GH treatment on SREBP-1a mRNA expression in adipose tissue (data not shown). Thus GH had no effect on SREBP-1c, FAS, or SCD-1 mRNA expression in adipose tissue, in contrast to the stimulatory effect of GH in the liver (Fig. 4C).


View larger version (23K):
[in this window]
[in a new window]
 
Fig. 4.   Hepatic mRNA expression of fatty-acid synthase (FAS, A), stearoyl-CoA desaturase-1 (SCD-1, B), and sterol regulatory element-binding protein (SREBP)-1c (C) in normal female rats and in Hx female rats treated with GH or insulin or the combination of GH and insulin. Animals were killed between 0900 and 1100 without prior fasting. mRNA levels were measured with gel ribonuclease protection assays, as described in MATERIALS AND METHODS. FAS and SCD-1 mRNA were run in the same gel, whereas SREBP-1c mRNA was run separately. Representative autoradiograms are shown. mRNA expression values are shown relative to those of beta -actin mRNA, given as a percentage of values in the N group. Results are presented as means ± SE of 5-6 rats in each group. Values with different superscripts are significantly different from each other (P < 0.05, one-way ANOVA followed by Bonferroni's test).


                              
View this table:
[in this window]
[in a new window]
 
Table 3.   Effects of hypophysectomy and GH treatment on SREBP-1c, FAS, and SCD-1 mRNA levels in ovarian adipose tissue


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The present study was designed to investigate the interaction between GH and insulin in vivo on lipid and lipoprotein metabolism in a nondiabetogenic rat model. We found that insulin treatment did not affect most of the observed effects of GH on serum lipoproteins or apolipoprotein levels, indicating that these GH effects are not a result of changed insulin action. In contrast, the increased hepatic triglyceride secretion observed after GH treatment was blunted by concomitant insulin treatment, indicating that insulin and GH have antagonistic effects in terms of hepatic triglyceride secretion in vivo. Changed hepatic triglyceride content and expression of SREBP-1c mRNA paralleled the effect of GH on triglyceride secretion, indicating that the increased triglyceride secretion is at least partly mediated by increased triglyceride synthesis as a result of increased SREBP-1c expression. Compared with the effect of GH alone, concomitant GH and insulin treatment resulted in lower hepatic triglyceride content, lower triglyceride secretion, and lower gene expression of SREBP-1c. These findings suggest that the effect of GH on hepatic triglyceride secretion and SREBP-1c mRNA expression is dependent on decreased insulin action. Thus the increased expression of SREBP-1c and increased triglyceride secretion after GH treatment resemble the insulin-resistant state in, e.g., the obese JCR:LA-cp rats (8). In contrast to the effect of insulin treatment on hepatic content and secretion of triglycerides and SREBP-1 c mRNA expression, insulin treatment of the Hx rats increased apoB mRNA editing and SCD-1 mRNA expression. GH had a smaller effect on apoB mRNA editing and SCD-1 mRNA when GH treatment was combined with insulin treatment. This finding indicates either that insulin antagonizes the effect of GH or that part of the effect of GH on apoB mRNA editing and SCD-1 mRNA expression is mediated by increased insulin secretion. In contrast to the findings in the liver, GH had no effect on SREBP-1, FAS, or SCD-1 mRNA in adipose tissue, showing a differential regulation of these genes by GH in the liver and adipose tissue. These results emphasize that GH has a liver-specific lipogenic effect.

The insulin treatment regimen used in this study has been shown to increase lipoprotein lipase activity in adipose tissue (31) and to increase incorporation of glucose into triglycerides in isolated adipose tissue segments (15). Thus the adipose tissue responded to this insulin treatment with an increased triglyceride accumulation, in contrast to what was observed in the liver. In line with this observation, infusion of 1.4 U/day of insulin for 7 days to intact rats has been shown to increase lipogenesis in adipose tissue but not in the liver (22). Together, these results emphasize that administration of insulin specifically enhances lipogenesis in adipose tissue.

Our observation of increased hepatic triglyceride secretion rate after GH treatment is in line with previous observations that GH increases triglyceride secretion ex vivo (10, 42) and in vitro (25). We extend these findings by showing that GH increases hepatic triglyceride secretion in vivo. Moreover, GH treatment in vivo increased the hepatic triglyceride content, which is in line with our previous finding that GH incubation of hepatocytes increased the triglyceride content of the cultured cells (25). Thus the effect of GH on hepatic triglyceride secretion in vivo is, at least partly, due to an increased triglyceride production.

Despite an increased triglyceride secretion from the liver after GH treatment, VLDL triglyceride levels were modestly affected. This finding may be explained by the increase in apoB mRNA editing. Increased apoB mRNA editing enhances the turnover of VLDL due to decreased proportion of apoB-100 containing VLDL (24, 25, 43). Moreover, lipoprotein lipase activity in skeletal muscle and heart is of major importance for the turnover of VLDL and serum VLDL levels (50), and these activities are increased by GH treatment (31). We have observed that the combined GH and insulin treatment of Hx rats results in increased lipoprotein activity in both adipose tissue and muscle tissues (J. Oscarsson and S. Edén, unpublished results). This finding, together with the blunted hepatic triglyceride secretion, could be the reason for the low serum triglyceride levels after combined GH and insulin treatment in the fasted animals. Our finding that insulin treatment had small or no effects on serum lipid and lipoprotein levels is in line with previous studies in which intact rats were treated with a similar dose of insulin (22).

The increased gene expression of FAS and SCD-1 after GH treatment most likely contributed to the increased hepatic triglyceride content and secretion. Because the expression levels of FAS and SCD-1 mRNA were not different between the group of rats given GH alone and the rats given GH in combination with insulin, the inhibitory effect of insulin on hepatic triglyceride secretion could not be explained by changed gene expression of these enzymes. The inhibitory effect of insulin on GH-induced triglyceride secretion could be due to a decreased flux of FFA to the liver and/or inhibition of other enzyme activities that are upregulated by GH. Indeed, incubation with insulin has been shown to counteract the stimulatory effect of GH on phosphatidate phosphohydrolase activity in cultured rat hepatocytes (33). Thus insulin treatment may reduce the effect of GH on triglyceride secretion via a decreased supply of FFA for triglyceride synthesis and by inhibiting the effect of GH on phosphatidate phosphohydrolase activity.

Insulin treatment has been shown to increase both SCD-1 mRNA (28) and FAS mRNA expression (38) in diabetic animal models. The rather small effects of insulin in this study could be due to several factors. One obvious reason could be that the effect of insulin was studied in Hx rats that are not deficient in insulin (28, 38). Another reason could be increased levels of insulin-antagonistic hormones such as glucagon and catecholamines, as indicated by the lower serum glucose levels observed after insulin treatment. These hormones increase the hepatocyte content of cAMP, which has been shown to antagonize the effect of insulin on both SCD mRNA (28) and FAS mRNA expression (38). Because glucose is a powerful regulator of SREBP-1c mRNA expression (13, 39), the low glucose levels could have contributed to the low SREBP-1c, SCD-1, and FAS mRNA expression after insulin treatment. Furthermore, glucose is regulating FAS mRNA levels by increasing FAS mRNA stability (38).

The effect of GH on hepatic SCD-1 mRNA levels is in line with the observation that a continuous infusion of GH increased SCD-1 mRNA in Hx male rats (12). However, divergent results exist concerning the effect of GH on hepatic FAS mRNA expression. GH given as a continuous infusion, thus mimicking the female secretory pattern of GH (6), resulted in increased FAS mRNA levels as observed in this study and in intact male rats (48). On the contrary, GH given as daily injections to ovariectomized female rats, mimicking the male secretory pattern of GH, decreased FAS mRNA expression (5). In this context, it is interesting to note that only GH given as a continuous infusion could fully restore triglyceride synthesis and VLDL secretion in Hx rats to those of normal rats (42).

We observed no effect of GH on gene expression of SREBP-1, FAS, or SCD-1 mRNA in adipose tissue. This finding is in line with the observation that GH does not influence SREBP-1 expression in cultured rat preadipocytes (19). To the best of our knowledge, the effect of GH on adipose tissue SCD-1 mRNA has not previously been studied. However, GH has been shown to decrease FAS mRNA in cultured rat preadipocytes (19), cultured 3T3-F442A cells, and pig adipose tissue (11). The reason for these discrepant data is not clear but may depend on differences between species or experimental conditions.

In summary, GH treatment increased triglyceride content and secretion and increased hepatic gene expression of SREBP-1c, FAS, and SCD-1 mRNA. In contrast, Hx and GH treatment had no effect on the expression of SREBP-1c or its downstream genes in adipose tissue. The most prominent effect of insulin was to mitigate the effect of GH on hepatic lipogenesis, as indicated by the finding that insulin inhibited the effect of GH on hepatic triglyceride secretion, triglyceride content, and SREBP-1c mRNA expression. Thus the effects of insulin in GH-treated Hx rats resemble the previously observed inhibitory action of insulin on hepatic triglyceride secretion (2, 18, 46, 49). Furthermore, it is noteworthy that the effect of insulin on SREBP-1c mRNA expression differs between the nondiabetogenic rat model used in this study and diabetogenic rat models (40).


    ACKNOWLEDGEMENTS

This work was supported by Grants 8269 and 14291 from the Swedish Medical Research Council, the Novo Nordisk Foundation, and the King Gustav V and Queen Victoria Foundation.


    FOOTNOTES

* Both authors contributed equally to this article.

Address for reprint requests and other correspondence: J. Oscarsson, Dept. of Physiology, Göteborg Univ., Box 434, S-405 30 Göteborg, Sweden (E-mail: Jan.Oscarsson{at}fysiologi.gu.se).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

July 30, 2002;10.1152/ajpendo.00260.2002

Received 12 June 2002; accepted in final form 23 July 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Angelin, B, and Rudling M. Growth hormone and hepatic lipoprotein metabolism. Curr Opin Lipidol 5: 160-165, 1994[Medline].

2.   Chirieac, DV, Chirieac LR, Corsetti JP, Cianci J, Sparks CE, and Sparks JD. Glucose-stimulated insulin secretion suppresses hepatic triglyceride-rich lipoprotein and apoB production. Am J Physiol Endocrinol Metab 279: E1003-E1011, 2000[Abstract/Free Full Text].

3.   Christ, ER, Cummings MH, Albany E, Umpleby AM, Lumb PJ, Wierzbicki AS, Naoumova RP, Boroujerdi MA, Sonksen PH, and Russell-Jones DL. Effects of growth hormone (GH) replacement therapy on very low density lipoprotein apolipoprotein B100 kinetics in patients with adult GH deficiency: a stable isotope study. J Clin Endocrinol Metab 84: 307-316, 1999[Abstract/Free Full Text].

4.   Cummings, MH, Watts GF, Umpleby AM, Hennessy TR, Naoumova R, Slavin BM, Thompson GR, and Sonksen PH. Increased hepatic secretion of very-low-density lipoprotein apolipoprotein B-100 in NIDDM. Diabetologia 38: 959-967, 1995[ISI][Medline].

5.   Donkin, SS, McNall AD, Swencki BS, Peters JL, and Etherton TD. The growth hormone-dependent decrease in hepatic fatty acid synthase mRNA is the result of a decrease in gene transcription. J Mol Endocrinol 16: 151-158, 1996[Abstract].

6.   Edén, S. Age- and sex-related differences in episodic growth hormone secretion in the rat. Endocrinology 105: 555-560, 1979[ISI][Medline].

7.   Elam, MB, Simkevich CP, Solomon SS, Wilcox HG, and Heimberg M. Stimulation of in vitro triglyceride synthesis in the rat hepatocyte by growth hormone treatment in vivo. Endocrinology 122: 1397-1402, 1988[Abstract].

8.   Elam, MB, Wilcox HG, Cagen LM, Deng X, Raghow R, Kumar P, Heimberg M, and Russell JC. Increased hepatic VLDL secretion, lipogenesis, and SREBP-1 expression in the corpulent JCR:LA-cp rat. J Lipid Res 42: 2039-2048, 2001[Abstract/Free Full Text].

9.   Elam, MB, von Wronski MA, Cagen L, Thorngate F, Kumar P, Heimberg M, and Wilcox HG. Apolipoprotein B mRNA editing and apolipoprotein gene expression in the liver of hyperinsulinemic fatty Zucker rats: relationship to very low density lipoprotein composition. Lipids 34: 809-816, 1999[ISI][Medline].

10.   Elam, MB, Wilcox HG, Solomon SS, and Heimberg M. In vivo growth hormone treatment stimulates secretion of very low density lipoprotein by the isolated perfused rat liver. Endocrinology 131: 2717-2722, 1992[Abstract].

11.   Etherton, TD. The biology of somatotropin in adipose tissue growth and nutrient partitioning. J Nutr 130: 2623-2625, 2000[Abstract/Free Full Text].

12.   Flores-Morales, A, Ståhlberg N, Tollet-Egnell P, Lundeberg J, Malek RL, Quackenbush J, Lee NH, and Norstedt G. Microarray analysis of the in vivo effects of hypophysectomy and growth hormone treatment on gene expression in the rat. Endocrinology 142: 3163-3176, 2001[Abstract/Free Full Text].

13.   Foretz, M, Guichard C, Ferre P, and Foufelle F. Sterol regulatory element binding protein-1c is a major mediator of insulin action on the hepatic expression of glucokinase and lipogenesis-related genes. Proc Natl Acad Sci USA 96: 12737-12742, 1999[Abstract/Free Full Text].

14.   Frick, F, Bohlooly YM, Lindén D, Olsson B, Törnell J, Edén S, and Oscarsson J. Long-term growth hormone excess induces marked alterations in lipoprotein metabolism in mice. Am J Physiol Endocrinol Metab 281: E1230-E1239, 2001[Abstract/Free Full Text].

15.   Frick, F, Oscarsson J, Vikman-Adolfsson K, Ottosson M, Yoshida N, and Edén S. Different effects of IGF-I on insulin-stimulated glucose uptake in adipose tissue and skeletal muscle. Am J Physiol Endocrinol Metab 278: E729-E737, 2000[Abstract/Free Full Text].

16.   Frohman, LA, and Bernardis LL. Growth hormone secretion in the rat: metabolic clearance and secretion rates. Endocrinology 86: 305-312, 1970[Medline].

17.   Gause, I, Isaksson O, Lindahl A, and Edén S. Effect of insulin treatment of hypophysectomized rats on adipose tissue responsiveness to insulin and growth hormone. Endocrinology 116: 945-951, 1985[Abstract].

18.   Gibbons, GF. Assembly and secretion of hepatic very-low-density lipoprotein. Biochem J 268: 1-13, 1990[ISI][Medline].

19.   Hansen, LH, Madsen B, Teisner B, Nielsen JH, and Billestrup N. Characterization of the inhibitory effect of growth hormone on primary preadipocyte differentiation. Mol Endocrinol 12: 1140-1149, 1998[Abstract/Free Full Text].

20.   Horton, JD, and Shimomura I. Sterol regulatory element-binding proteins: activators of cholesterol and fatty acid biosynthesis. Curr Opin Lipidol 10: 143-150, 1999[ISI][Medline].

21.   Jansson, JO, Ekberg S, Isaksson O, Mode A, and Gustafsson JÅ. Imprinting of growth hormone secretion, body growth, and hepatic steroid metabolism by neonatal testosterone. Endocrinology 117: 1881-1889, 1985[Abstract].

22.   Koopmans, SJ, Kushwaha RS, and DeFronzo RA. Chronic physiologic hyperinsulinemia impairs suppression of plasma free fatty acids and increases de novo lipogenesis but does not cause dyslipidemia in conscious normal rats. Metabolism 48: 330-337, 1999[ISI][Medline].

23.   Lewis, GF. Fatty acid regulation of very low density lipoprotein production. Curr Opin Lipidol 8: 146-153, 1997[ISI][Medline].

24.   Li, X, Catalina F, Grundy SM, and Patel S. Method to measure apolipoprotein B-48 and B-100 secretion rates in an individual mouse: evidence for a very rapid turnover of VLDL and preferential removal of B-48- relative to B-100-containing lipoproteins. J Lipid Res 37: 210-220, 1996[Abstract].

25.   Lindén, D, Sjöberg A, Asp L, Carlsson L, and Oscarsson J. Direct effects of growth hormone on production and secretion of apolipoprotein B from rat hepatocytes. Am J Physiol Endocrinol Metab 279: E1335-E1346, 2000[Abstract/Free Full Text].

26.   Malaisse, WJ, Malaisse-Lagae F, King S, and Wright PH. Effect of growth hormone on insulin secretion. Am J Physiol 215: 423-428, 1968[Free Full Text].

27.   Nielsen, JH. Effects of growth hormone, prolactin, and placental lactogen on insulin content and release, and deoxyribonucleic acid synthesis in cultured pancreatic islets. Endocrinology 110: 600-606, 1982[Abstract].

28.   Ntambi, JM. Regulation of stearoyl-CoA desaturase by polyunsaturated fatty acids and cholesterol. J Lipid Res 40: 1549-1558, 1999[Abstract/Free Full Text].

29.   Oscarsson, J, Olofsson SO, Bondjers G, and Edén S. Differential effects of continuous versus intermittent administration of growth hormone to hypophysectomized female rats on serum lipoproteins and their apoproteins. Endocrinology 125: 1638-1649, 1989[Abstract].

30.   Oscarsson, J, Ottosson M, and Edén S. Effects of growth hormone on lipoprotein lipase and hepatic lipase. J Endocrinol Invest 22: 2-9, 1999[Medline].

31.   Oscarsson, J, Ottosson M, Vikman-Adolfsson K, Frick F, Enerbäck S, Lithell H, and Edén S. GH but not IGF-I or insulin increases lipoprotein lipase activity in muscle tissues of hypophysectomised rats. J Endocrinol 160: 247-255, 1999[Abstract/Free Full Text].

32.   Pierluissi, J, Pierluissi R, and Ashcroft SJ. Effects of growth hormone on insulin release in the rat. Diabetologia 19: 391-396, 1980[ISI][Medline].

33.   Pittner, RA, Bracken P, Fears R, and Brindley DN. Insulin antagonises the growth hormone-mediated increase in the activity of phosphatidate phosphohydrolase in isolated rat hepatocytes. FEBS Lett 202: 133-136, 1986[ISI][Medline].

34.   Rizza, RA, Mandarino LJ, Genest J, Baker BA, and Gerich JE. Production of insulin resistance by hyperinsulinaemia in man. Diabetologia 28: 70-75, 1985[ISI][Medline].

35.   Rizza, RA, Mandarino LJ, and Gerich JE. Effects of growth hormone on insulin action in man. Mechanisms of insulin resistance, impaired suppression of glucose production, and impaired stimulation of glucose utilization. Diabetes 31: 663-669, 1982[Abstract].

36.   Roberts, TJ, Azain MJ, Hausman GJ, and Martin RJ. Interaction of insulin and somatotropin on body weight gain, feed intake, and body composition in rats. Am J Physiol Endocrinol Metab 267: E293-E299, 1994[Abstract/Free Full Text].

37.   Salomon, F, Cuneo RC, Hesp R, and Sonksen PH. The effects of treatment with recombinant human growth hormone on body composition and metabolism in adults with growth hormone deficiency. N Engl J Med 321: 1797-1803, 1989[Abstract].

38.   Semenkovich, CF. Regulation of fatty acid synthase (FAS). Prog Lipid Res 36: 43-53, 1997[ISI][Medline].

39.   Shimano, H. Sterol regulatory element-binding proteins (SREBPs): transcriptional regulators of lipid synthetic genes. Prog Lipid Res 40: 439-452, 2001[ISI][Medline].

40.   Shimomura, I, Bashmakov Y, Ikemoto S, Horton JD, Brown MS, and Goldstein JL. Insulin selectively increases SREBP-1c mRNA in the livers of rats with streptozotocin-induced diabetes. Proc Natl Acad Sci USA 96: 13656-13661, 1999[Abstract/Free Full Text].

41.   Shimomura, I, Matsuda M, Hammer RE, Bashmakov Y, Brown MS, and Goldstein JL. Decreased IRS-2 and increased SREBP-1c lead to mixed insulin resistance and sensitivity in livers of lipodystrophic and ob/ob mice. Mol Cell 6: 77-86, 2000[ISI][Medline].

42.   Sjöberg, A, Oscarsson J, Borén J, Edén S, and Olofsson SO. Mode of growth hormone administration influences triacylglycerol synthesis and assembly of apolipoprotein B-containing lipoproteins in cultured rat hepatocytes. J Lipid Res 37: 275-289, 1996[Abstract].

43.   Sjöberg, A, Oscarsson J, Boström K, Innerarity TL, Edén S, and Olofsson SO. Effects of growth hormone on apolipoprotein-B (apoB) messenger ribonucleic acid editing, and apoB 48 and apoB 100 synthesis and secretion in the rat liver. Endocrinology 130: 3356-3364, 1992[Abstract].

44.   Sjöberg, A, Oscarsson J, Olofsson SO, and Edén S. Insulin-like growth factor-I and growth hormone have different effects on serum lipoproteins and secretion of lipoproteins from cultured rat hepatocytes. Endocrinology 135: 1415-1421, 1994[Abstract].

45.   Sonksen, PH, Greenwood FC, Ellis JP, Lowy C, Rutherford A, and Nabarro JD. Changes of carbohydrate tolerance in acromegaly with progress of the disease and in response to treatment. J Clin Endocrinol Metab 27: 1418-1430, 1967[ISI][Medline].

46.   Sparks, JD, and Sparks CE. Insulin regulation of triacylglycerol-rich lipoprotein synthesis and secretion. Biochim Biophys Acta 1215: 9-32, 1994[ISI][Medline].

47.   Thorngate, FE, Raghow R, Wilcox HG, Werner CS, Heimberg M, and Elam MB. Insulin promotes the biosynthesis and secretion of apolipoprotein B-48 by altering apolipoprotein B mRNA editing. Proc Natl Acad Sci USA 91: 5392-5396, 1994[Abstract].

48.   Tollet-Egnell, P, Flores-Morales A, Ståhlberg N, Malek RL, Lee N, and Norstedt G. Gene expression profile of the aging process in rat liver: normalizing effects of growth hormone replacement. Mol Endocrinol 15: 308-318, 2001[Abstract/Free Full Text].

49.   Zammit, VA, Waterman IJ, Topping D, and McKay G. Insulin stimulation of hepatic triacylglycerol secretion and the etiology of insulin resistance. J Nutr 131: 2074-2077, 2001[Abstract/Free Full Text].

50.   Zechner, R. The tissue-specific expression of lipoprotein lipase: implications for energy and lipoprotein metabolism. Curr Opin Lipidol 8: 77-88, 1997[ISI][Medline].


Am J Physiol Endocrinol Metab 283(5):E1023-E1031
0193-1849/02 $5.00 Copyright © 2002 the American Physiological Society