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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1999;19:832-839.)
© 1999 American Heart Association, Inc.

Original Contributions

Cholesterol and Lipoprotein Metabolism in Aging

Reversal of Hypercholesterolemia by Growth Hormone Treatment in Old Rats

Paolo Parini; Bo Angelin; Mats Rudling

From the Metabolism Unit, Center for Metabolism and Endocrinology, Department of Medicine, and the Molecular Nutrition Unit, Center for Nutrition and Toxicology, Novum, Karolinska Institute at Huddinge University Hospital, Huddinge, Sweden.

Correspondence to Mats Rudling, MD, PhD, CME, M63, Huddinge University Hospital, S-141 86 Huddinge, Sweden. E-mail mats.rudling{at}cnt.ki.se

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Abstract—Plasma cholesterol levels increase with age, as does the incidence of coronary heart disease. The mechanisms responsible for the age-related hypercholesterolemia are not well understood. An interesting hypothesis suggests that the relative deficiency in growth hormone (GH), which occurs with aging, contributes to the development of the age-related hypercholesterolemia, because GH has beneficial effects on cholesterol metabolism. In the present work, we tested this hypothesis by the administration of GH to normal rats of varying ages. Plasma lipids and hepatic cholesterol metabolism were characterized in 2-, 12-, and 18-month-old male Sprague-Dawley rats. In 2-month-old rats, GH specifically stimulated the hepatic low density lipoprotein (LDL) receptor expression in a dose-dependent way, both at the protein level and at the mRNA level. Concomitantly, plasma cholesterol increased by 30% within the large high density lipoprotein and LDL fractions. In 12-month-old animals, cholesterol 7-hydroxylase (C7OH) activity was reduced, whereas hepatic LDL receptors and plasma total cholesterol were unchanged. GH treatment (1 mg · kg^-1 · d^-1) normalized the activity of C7OH and had effects on plasma cholesterol and LDL receptors similar to those seen in 2-month-old animals. In 18-month-old rats, plasma cholesterol was increased 2-fold, whereas hepatic LDL receptor expression and C7OH activity were similar to those of the 12-month-old animals. Infusion of GH to 18-month-old rats had similar effects on hepatic C7OH and LDL receptors as seen in 12-month-old rats. However, GH treatment strongly reduced the hypercholesterolemia in 18-month-old animals. We conclude that the age-dependent increase of plasma cholesterol in rats can be reversed by the administration of GH, presumably through the pleiotropic effects of this hormone on lipoprotein metabolism.

Key Words: aging • cholesterol • cholesterol 7-hydroxylase • growth hormone • LDL receptor

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Elevated levels of plasma cholesterol, particularly LDL cholesterol, are known to be associated with an enhanced risk for atherosclerosis and coronary heart disease.^{1 2} Total and LDL cholesterol levels increase with age,^{3 4} as does the incidence of cardiovascular disease.⁵ The mechanisms behind this age-related increase in plasma cholesterol are still incompletely characterized. Of particular interest is the finding of a gradual decline in the fractional clearance of LDL from the circulation with age^{6 7 8 9} and evidence of the reduced expression of hepatic LDL receptors (LDLRs) with increasing age in some species.^{10 11} The capacity for body cholesterol removal through the conversion of cholesterol to bile acids is also progressively reduced with age,¹² and a decrease in the activity of the rate-limiting enzyme in bile acid biosynthesis, cholesterol 7-hydroxylase (C7OH), has been demonstrated in the aging rat.^{13 14} In addition, there is some evidence that the synthesis of apolipoprotein (apo) B-100 in VLDL may be increased with age.⁹

A number of explanations to these findings have been discussed, including both dietary and hormonal factors. An interesting hypothesis¹⁵ states that the critical changes in cholesterol and lipoprotein metabolism depend on the progressive decrease in growth hormone (GH) secretion, which occurs with normal aging.^{16 17} In previous studies, mainly based on experiments in hypophysectomized rats, we established that GH has an important role in cholesterol homeostasis,¹⁵ both by modulating the expression of hepatic LDLRs^{18 19 20 21} and by controlling the activity of C7OH.²² In addition, GH stimulates hepatic VLDL²³ and apoE²⁴ secretion and may influence the ratio between apoB-100 and apoB-48 through stimulation of apoB mRNA editing in the rat.^{20 25}

To further characterize the role of GH in the physiological regulation of cholesterol and lipoprotein metabolism and particularly to evaluate the possible relevance of reduced GH secretion for the development of hypercholesterolemia with age, we performed studies in normal rats of varying ages, both with and without infusion of GH. We were able to show that when plasma cholesterol was significantly increased in old rats, the hyperlipidemic pattern could be reversed by infusion with GH. These effects may be partially mediated via stimulation of LDLR activity and bile acid synthesis but probably also reflect the pleiotropic effects of GH on lipoprotein metabolism.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Materials
Osmotic minipumps (model 2001, delivering 1 µL solvent per hour) were from Alza Corp. Recombinant human GH (Genotropin) and recombinant human insulin-like growth factor I (IGF-I) were kindly provided by AB Pharmacia & Upjohn, Stockholm, Sweden. All other materials were obtained from previously described sources.^{19 20 26 27 28 29}

Animals and Experimental Procedures
Altogether, 75 male Sprague-Dawley rats (ALAB, Sollentuna, Sweden) were used in 4 separate experiments. The animals were adapted to the environment at least 1 week before the experiments. Young rats were purchased at 7 weeks of age, whereas old animals were obtained at 6 months of age and kept at the animal facilities until 12 or 18 months of age. Rats were housed under standardized conditions, with free access to water and chow; the light cycle hours were between 6 AM and 6 PM The studies were approved by the Institutional Animal Care and Use Committee. Osmotic minipumps were implanted subcutaneously in the dorsal region. A small (5- to 10-mm) incision was made, and a subcutaneous tunnel was created with a pair of forceps. Minipumps were put in place and the skin was sutured. Controls were sham-operated with the same surgical procedure. The presence of minipumps only did not alter plasma lipoproteins; this was verified in a separate control experiment (not shown). When the experiments were terminated, the rats were anesthetized with ether between 10 and 11 AM, blood was drawn by cardiac puncture, and the animals were killed by cervical dislocation. The livers were immediately removed, and 1 piece of fresh liver (1 g) was taken for preparation of microsomes for subsequent assay of enzyme activities as described below. The remaining liver was immediately frozen in LN₂.

Plasma IGF-I was determined in serum by radioimmunoassay after separation of IGFs from IGF-binding proteins by acid-ethanol extraction and cryoprecipitation. To minimize interference from the remaining IGF-binding proteins, des1-3–IGF-I was used as the radioligand.³⁰

Size Fractionation of Lipoproteins by Fast Protein Liquid Chromatography (FPLC)
Equal volumes of plasma from every rat were pooled (5 mL) for each group, and the density was adjusted to 1.21 g/mL with KBr. After ultracentrifugation at 100x10³g for 48 hours, the supernatant was removed and adjusted to 2 mL with 0.15 mol/L NaCl, 0.01% EDTA, and 0.02% NaN₃, pH 7.3. One milliliter of this solution (corresponding to 2.5 mL of plasma) was injected onto a 540x18-mm Superose 6B column after filtration through a Millipore 0.45-µm HA filter; 2-mL fractions were collected at a flow rate of 1 mL/min.²⁹ In the experiment illustrated in Figure 1, the column was 470 mm long.

View larger version (36K): [in this window] [in a new window]   Figure 1. Effects of increasing GH dose to normal rats. Eight-week-old animals (5 per group) were infused subcutaneously with the indicated amounts of GH for 6 days. A, Plasma total cholesterol in all animals; results shown are mean±SEM. B, Plasma lipoprotein patterns after separation by FPLC. Pooled plasma from each group was separated on a Superose column after ultracentrifugation. Two-milliliter fractions were collected and assayed for cholesterol. , Control group; , 1 mg · kg-1 · d-1; , 2 mg · kg-1 · d-1; and , 3.5 mg · kg-1 · d-1 of GH. C, LDLR expression assayed by ligand blotting, using rabbit 125I–ß-VLDL. Hepatic membranes were prepared from pooled liver samples from each group and separated by SDS-PAGE (6% polyacrylamide). D, Hepatic LDLR mRNA levels assayed by solution hybridization in total RNA extracts. Data represent mean±SEM. *P<0.05, **P<0.005 vs untreated animals.

SDS–Polyacrylamide Gel Electrophoresis (PAGE) Separation of Apolipoproteins
Pooled plasma from each group was separated in 2-mL fractions by FPLC. Equal volumes from fractions 22 to 24 (VLDL), 25 to 27 (IDL), 30 to 32 (LDL), and 35 to 37 (HDL) were pooled, and 1.1 mL was precipitated with trichloroacetic acid (15% final concentration), delipidated by washing twice with acetone, and solubilized in 200 µL of loading buffer as described elsewhere.²⁹ After boiling the samples for 5 minutes in the presence of 5% (vol/vol) 2-mercaptoethanol, 100 µL was loaded onto 4% to 20% gradient SDS-polyacrylamide gels and separated for 4 hours at 45 mA. Gels were stained with Coomassie blue. For reference, wide-range molecular-mass standards (Bio-Rad Laboratories), as well as human LDL and HDL, were used.

Lipid Extracts
Liver homogenates and hepatic microsomes were prepared as previously described.^{27 28} The lipid fraction was obtained by extraction with chloroform/methanol (2:1, vol/vol), dried under a stream of N₂, and dissolved in 200 µL methanol.

Cholesterol Assay
Cholesterol in plasma, FPLC fractions, liver extracts, and hepatic microsome extracts was assayed with Boehringer Mannheim free or total cholesterol assay kits, with the use of a 5.2 mmol/L cholesterol standard (Merck, catalog No. 141 64).

Ligand Blot Assay of LDLR
Hepatic membranes were prepared as described previously.¹⁸ Membranes from pooled liver samples were separated by 6% SDS-PAGE under nonreduced conditions. Size markers were reduced with 2-mercaptoethanol and boiled. Filters were incubated with ¹²⁵I-labeled rabbit ß-migrating VLDL (ß-VLDL), as described.¹⁸ Filters were exposed on Dupont Cronex film. The LDLR expression was quantified from the 120-kDa bands by using a Fujix Bio-imaging analyzer (BAS 2000, Fuji Photo Film Co). Background was obtained by counting irrelevant filter parts of the same area. The data are presented as photostimulated luminescence per microgram of protein loaded.

Activities of C7OH and HMG-CoA Reductase
Microsomes were prepared by differential ultracentrifugation of individual liver homogenates in the absence of fluoride as described previously.^{12 26 27} The activity of C7OH was determined as the formation of 7-hydroxycholesterol (pmol · mg protein^-1 · min^-1) from endogenous microsomal cholesterol by using isotope dilution mass spectrometry.²⁷ Microsomal 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity was assayed by determining the conversion of HMG-CoA to mevalonate and expressed as picomoles formed per milligram protein per minute.²⁶ The enzyme assays were carried out in duplicate.

Total Nucleic Acid Preparation
Total nucleic acid (TNA) extracts were prepared as described.³¹ The concentration of TNA in the samples was measured by absorbance at 260 nm and assuming that 1 optical density unit was equivalent to 40 µg TNA per mL.

Quantification of mRNA
The mRNA levels for the LDLR and C7OH were quantified by a solution hybridization titration assay with mouse -³⁵S–UTP-cRNA-probes corresponding to nucleotides 1247 to 1308 in the human LDLR cDNA and to nucleotides 646 to 813 in the rat C7OH genomic DNA.³² Editing of apoB mRNA was determined by primer extension analysis with the use of total RNA.^{20 33}

Statistics
Data are presented as mean±SEM. The significance of differences between groups was tested by 1-way ANOVA followed by a planned comparison or by post hoc comparisons of group means according to least significant difference or honestly significantly difference methods (Statistica software, Stat Soft). To stabilize the variances, data were logarithmically transformed when the assumption for no correlation between means and variances was violated.³⁴

	Results

Top Abstract Introduction Methods Results Discussion References

 
Previous information on the role of GH in lipoprotein metabolism has mainly been gained from studies in hypophysectomized rats,^{18 19 20 23 24 25} and thus, we first wanted to assess the effects of GH administration in normal young rats. Eight-week-old male rats were given a subcutaneous infusion of recombinant human GH at increasing doses 1, 2, and 3.5 mg GH · kg^-1 · d^-1. After 6 days of infusion, the animals were killed and plasma and livers were collected. GH infusions increased plasma total cholesterol slightly, although this rise was not statistically significant (Figure 1A), and FPLC separation of plasma lipoproteins showed a slight increase, predominantly within the large HDL particles (Figure 1B). Determination of the hepatic LDLR expression showed a dose-dependent stimulatory effect after the infusion of GH (Figure 1C), and this effect was evident also at the mRNA level (Figure 1D).

Many of the actions of GH are mediated by the GH-dependent production of IGF-I.¹⁶ To determine whether the effects on lipoprotein metabolism observed in normal young rats given GH could be reproduced by the administration of IGF-I to normal rats, we repeated the experiment by using recombinant human IGF-I. After 6 days of infusion of 2 and 4 mg · kg^-1 · d^-1 of IGF-I, respectively, hepatic LDLR expression was not altered (Figure 2A). Plasma total cholesterol was unchanged (Figure 2B), but separation of plasma lipoproteins by FPLC indicated a slight increase within LDL particles (Figure 2C). Thus, in consonance with our previous findings in hypophysectomized rats,²⁰ IGF-I could not reproduce the effects of GH on lipoprotein metabolism in normal rats.

View larger version (41K): [in this window] [in a new window]   Figure 2. Effects of IGF-I in normal rats. Eight-week-old animals (5 per group) were treated by subcutaneous infusion for 6 days with the indicated amounts of IGF-I. A, LDLR expression as assayed by ligand blotting, using rabbit 125I–ß-VLDL. Membranes from pooled livers were separated by SDS-PAGE (6% polyacrylamide) and subsequently transferred onto a nitrocellulose filter. B, Total plasma cholesterol in all animals (mean±SEM). C, Plasma lipoprotein patterns after separation by FPLC. Two-milliliter fractions were collected, after separation on a Superose column, and cholesterol content was determined. , Control group; , 2 mg · kg-1 · d-1; and , 4 mg · kg-1 · d-1 of IGF-I.

To explore the possible relationship between GH secretion and hepatic lipoprotein metabolism, we then investigated young (8 weeks) and 12-month-old rats that were given a GH infusion of 1 mg · kg^-1 · d^-1 for 6 days in comparison with untreated animals. The plasma levels of IGF-I were 38% lower in the old untreated controls (Table 1), indicative of a reduced secretion of GH in these rats.¹⁷ The infusion of GH did not alter this difference between young and old animals. Analysis of plasma total cholesterol revealed a modest increase of cholesterol in old untreated rats (Table 1), which was due to an increase within LDL and large HDL particles (Figure 3A). GH infusion significantly increased plasma total cholesterol in both young and old animals (Table 1). This treatment did not reverse the increases in LDL and HDL cholesterol in the old rats, and both groups of rats responded with an elevation of cholesterol within large HDL particles (Figure 3A). Determination of the triglyceride content in FPLC fractions showed no difference between untreated young and old rats (Figure 3B), whereas the infusion of GH increased triglycerides slightly (30%) in the VLDL of old animals.

View this table: [in this window] [in a new window]   Table 1. Effects of 6 Days of GH Infusion in 8-Week-Old and 12-Month-Old Male Rats

View larger version (31K): [in this window] [in a new window]   Figure 3. Effect of GH in 8-week-old and 12-month-old rats. Animals (5 per group) were infused subcutaneously with 1 mg · kg-1 · d-1 of GH for 6 days. Untreated rats of corresponding ages served as controls. Pooled plasma from each group was separated on a Superose column, after ultracentrifugation. Two-milliliter fractions were collected and assayed for cholesterol (A) or triglyceride (B) content. , 8-week-old controls; , 8-week-old rats infused with GH; , 12-month-old controls; and , 12-month-old rats infused with GH. C, LDLR expression assayed by ligand blotting, using rabbit 125I–ß-VLDL. Hepatic membranes were prepared from pooled liver samples from each group and separated by SDS-PAGE (6% polyacrylamide). Data are presented as photostimulated luminescence (PSL) per microgram protein loaded. D, Enzymatic activity of C7OH. The activity was determined after incubation of freshly prepared liver microsomes. Results show the mean±SEM. *P<0.001, **P<0.05 vs 8-week-old controls. #P<0.001 vs 12-month-old controls. W indicates weeks; M, months.

Quantification of the hepatic LDLR expression showed a similar level of expression in 8-week- and 12-month-old rats, and the infusion of GH increased hepatic LDLR expression slightly (by 25% to 50%) in both groups (Figure 3C). The hepatic LDLR mRNA levels were similar in young and old rats (Table 1), and there was no alteration by GH treatment. Analysis of the C7OH enzymatic activity in hepatic microsomes revealed a 60% lower level (P<0.001) in the 12-month-old rats compared with the young rats (Figure 3D). In the old animals, GH infusion increased the activity of C7OH (to 130%, P<0.001) to a level similar to that observed in untreated young controls. A response to GH treatment was also seen in the young animals, the C7OH activity being increased by 40% (P<0.05, Figure 3D). Despite the differences found in C7OH activity, no differences were found between young and old rats when the hepatic C7OH mRNA abundance was determined by solution hybridization (Table 1). GH infusion doubled the C7OH mRNA abundance in the young rats (P<0.05), whereas only a slight increase was observed in the old animals. There were no significant differences in hepatic total cholesterol between the groups, whereas GH treatment significantly increased hepatic microsomal free cholesterol in the young rats (P<0.05, Table 1).

Thus, there was a clear reduction of C7OH activity in the 12-month-old rats, and this situation could be normalized by GH treatment. However, the modest increase of plasma total cholesterol in these animals was not statistically significant compared with the corresponding values in young controls. In the rat, a doubling of plasma cholesterol has been reported between 12 and 18 months of age.³⁵ To further explore whether treatment with GH could improve age-induced changes in lipid metabolism, we therefore repeated the experiment in 18-month-old animals. GH was infused at a rate of 1 mg · kg^-1 · d^-1 for 6 days, and 8-week-old rats served as the reference. Determination of plasma IGF-I showed a 34% reduction in the untreated 18-month-old rats (Table 2). Again, GH treatment did not alter the difference in plasma IGF-I levels between young and old rats.

View this table: [in this window] [in a new window]   Table 2. Effects of 6 Days of GH Infusion in 8-Week-Old and 18-Month-Old Male Rats

Analysis of plasma total cholesterol in this experiment revealed a 2-fold increase in the 18-month-old rats compared with the young ones (Table 2). In agreement with our previous results, GH treatment tended to increase plasma total cholesterol in young rats. In contrast, GH infusion dramatically reduced plasma total cholesterol in the 18-month-old animals (P<0.001), to a level that was similar to that of young GH-treated rats (Table 2). Lipoprotein profile analysis by FPLC showed that the age-induced plasma cholesterol in 18-month-old rats was within all lipoprotein classes (Figure 4A). The infusion of GH to these animals clearly reduced cholesterol in all fractions, so that the lipoprotein pattern of GH-treated, 18-month-old rats resembled that of GH-treated young rats. In young GH-infused rats, plasma cholesterol again increased mainly within large HDL and LDL. Analysis of triglycerides in FPLC fractions showed higher concentrations in VLDL particles of untreated 18-month-old rats (Figure 4B). GH infusion reduced VLDL triglycerides in old animals, whereas VLDL triglycerides increased in 8-week-old rats. SDS-PAGE separation of the apolipoproteins in FPLC fractions revealed changes in consonance with the alterations of the lipid profiles (Figure 4C). Particularly, there was an increase in LDL apoB-100 in 18-month-old rats; this abnormality was reversed by the infusion of GH. Thus, it was evident that GH had contrasting effects on the lipoprotein pattern in young and 18-month-old animals.

View larger version (41K): [in this window] [in a new window]   Figure 4. Plasma lipoprotein profiles in 8-week- and 18-month-old rats after 1 mg · kg-1 · d-1 GH infusion. Animals (5 per group) were infused subcutaneously for 6 days. Untreated rats of corresponding ages served as controls. Pooled plasma from each group was separated by FPLC after ultracentrifugation. Two-milliliter fractions were collected and assayed for cholesterol (A) or triglycerides (B). , 8-week-old controls; , 8-week-old rats infused with GH; , 18-month-old controls; and , 18-month-old rats infused with GH. C, Separation of apolipoproteins in FPLC fractions. Pooled plasma from each group was separated by FPLC, and apolipoproteins were further separated by SDS-PAGE as described in Methods. W indicates weeks; M, months.

Despite the dramatic differences in plasma cholesterol and lipoprotein profiles, the hepatic LDLR expression in the 18-month-old rats was also similar to that of young animals (Figure 5A). GH infusion did not alter hepatic LDLR expression in either old or young animals. It was thus clear that there was no major change in hepatic LDLR expression with aging.

View larger version (45K): [in this window] [in a new window]   Figure 5. Effects of GH infusion [1 (mg/kg)d] in 8-week- and 18-month-old rats. Each group consisted of 5 animals, and untreated rats of corresponding ages served as controls. A, LDLR expression in hepatic membranes assayed by ligand blot, using rabbit 125I–ß-VLDL. Data are presented as photostimulated luminescence (PSL) per microgram protein loaded. B, Enzymatic activity of C7OH in freshly isolated liver microsomes. Results represent mean±SEM. #P<0.05, *P<0.001 vs 8-week-old controls. C, Enzymatic activity of HMG-CoA reductase in freshly isolated liver microsomes. Results are mean±SEM. *P<0.05 vs 8-week-old controls. P<0.05 vs 18-month-old controls. W indicates weeks; M, months.

Microsomal activity of C7OH in the 18-month-old animals was 35% lower (P<0.05) than that in the young ones (Figure 5B). In young rats, GH treatment stimulated C7OH activity, inducing a 70% increase. Also in 18-month-rats, a stimulation of C7OH activity was seen, though of lesser magnitude (Figure 5B). Similar results were obtained when the mRNA abundance for C7OH was measured in hepatic TNA extracts (Table 2). As in the previous experiment, no differences were detected in hepatic microsomal free cholesterol content between young and old rats. GH infusion again increased hepatic microsomal free cholesterol in young rats. Hepatic total cholesterol was similar in young and old animals, and GH infusion significantly reduced its concentration in young animals only.

The drastic effect of GH treatment on plasma lipoproteins in the 18-month-old animals occurred in contrast to more moderate changes in LDLR and C7OH activities. This prompted us to search for other changes induced by GH. Assay of the activity of liver microsomal HMG-CoA reductase, the rate-limiting enzymatic step in cholesterol biosynthesis, did not show any difference between young and 18-month-old animals, however (Figure 5C). This finding suggests that the hypercholesterolemia is not explained by an elevated hepatic synthesis of cholesterol. GH treatment stimulated HMG-CoA reductase activity in both groups of animals. Finally, we determined the apoB mRNA editing by primer extension analysis of total hepatic RNA. No differences were found in the degree of mRNA editing between the 18-month-old rats compared with young animals.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The results of the present study strongly support the concept that GH has an important role in the physiological regulation of cholesterol and lipoprotein metabolism. The reversal of the disturbed lipoprotein pattern in 18-month-old male rats by a moderate dose of GH implies that, although other mechanisms may be involved, a relative GH deficiency contributes to the development of age-related hypercholesterolemia in this species. However, the present data do not permit any definite identification of a single mechanism by which GH exerts its effect in this situation. Instead, the data rather indicate that the pleiotropic effects of GH on lipoprotein metabolism result in compensatory adjustments of age-related changes at a number of regulatory steps.

The present data confirm and extend previous studies of cholesterol metabolism in the aging rat.^{13 14 35 36 37 38 39} Thus, a significant hypercholesterolemia was present in 18-month-old rats, whereas only a minor increase was detected in 12-month-old animals. The age-induced increase in plasma cholesterol was within all lipoprotein classes and was associated with an increased apoB-100 protein in LDL. Triglycerides were also elevated, particularly in VLDL. The hepatic content of total and microsomal cholesterol was unaltered even in the 18-month-old rats. Ståhlberg et al¹³ reported unchanged levels of free cholesterol in hepatic microsomes of 1-, 6-, and 24-month-old rats and observed an increased total cholesterol only in livers from 24-month-old animals, indicating that hepatic cholesterol may accumulate in rats of very old age. Our findings of an essentially unaltered hepatic LDLR expression are in agreement with previous reports by Stange and Dietschy³⁹ and Nanjee et al.³⁷ The activity of HMG-CoA reductase was unchanged in old rats despite their hypercholesterolemia, again in agreement with Ståhlberg et al,¹³ who found no change in HMG-CoA reductase activity when comparing 6- and 24-month-old animals. From the literature, it appears that HMG-CoA reductase activity is higher in rats up to 4 to 5 weeks of age,^{13 14} which may indicate that a decrease occurs during sexual maturation and not later in life.

Of particular interest is the finding of a clear reduction of C7OH activity already in 12-month-old rats. This observation supports the data of Ståhlberg et al,¹³ who found a progressive reduction of C7OH activity when comparing animals at the ages of 1 month, 6 months, and 24 months. Thus, a reduced C7OH activity, and—since HMG-CoA reductase activity is maintained—a diminished relative conversion of cholesterol to bile acids occurs relatively early in the aging process. It is important to note that no change in microsomal free cholesterol, the substrate pool for the C7OH enzyme, was observed, and that the changes in enzyme activity were not paralleled by any change in the C7OH mRNA levels. The latter observation would indicate that nontranscriptional mechanisms of regulation are involved in the age-induced reduction of enzyme activity. In most situations, hepatic C7OH activity is strictly regulated at the transcriptional level in the rat.^{40 41 42} However, evidence of posttranscriptional regulation has actually been observed in hypophysectomized (GH-deficient) rats, wherein enzyme activity is considerably reduced despite increased mRNA levels.²²

Despite a pronounced reduction of C7OH activity, there was only a minor increase in plasma cholesterol in 12-month-old rats. C7OH has previously been regarded as an important determinant for the level of cholesterol in plasma because of its key role in the excretion of cholesterol from the body. Stimulation of bile acid production by interruption of the enterohepatic circulation of bile acids is well known to lower plasma total and LDL cholesterol, presumably through induction of the hepatic LDLR.^{43 44 45 46} Although it has been shown that plasma cholesterol may drop after the transient overexpression of hepatic C7OH,⁴⁷ the elimination of this enzyme by gene knockout does not result in altered plasma lipids.⁴⁸ Previous studies on hypophysectomized rats have also indicated that the link between plasma cholesterol and hepatic C7OH may at times be absent.²² Although it is still reasonable to assume that a decreased C7OH activity may amplify the effects of other age-related changes in lipoprotein metabolism, our data would indicate that the hypercholesterolemia appearing in 18-month-old rats is not solely dependent on a reduced activity of C7OH. In preliminary experiments, we did not find any change in the editing of apoB mRNA in old rats, an observation that would speak against the possibility that a relative predominance of apoB-100 in VLDL secreted from the liver explains a slower lipoprotein turnover in the older animals.⁴⁹ In conclusion, the clear age-induced hypercholesterolemia in 18-month-old rats cannot simply be explained by a single alteration of hepatic cholesterol synthesis, bile acid production, LDLR expression, or apoB mRNA editing. However, combined discrete effects on all of these processes may very well be of great importance. Such effects are indeed difficult to demonstrate reliably with available assays.

The administration of GH to young rats was associated with a dose-dependent increase of hepatic LDLR expression, both at the protein and the mRNA level. Plasma cholesterol increased slightly, predominantly within large HDL and LDL particles. The mechanisms responsible for the latter change were not explored further. Thus, GH also exerts clear effects on lipoprotein metabolism in normal rats when the doses are adequately high. Furthermore, these effects are specific for GH, in that they could not be reproduced by the infusion of IGF-I. In consonance with our present results, we have not been able to obtain similar effects on LDLR or apoB mRNA editing in hypophysectomized rats when GH was substituted with IGF-I.²⁰ Also, in normal middle-aged humans, GH and IGF-I exert different effects on plasma LDL cholesterol levels.⁵⁰

To evaluate the possible contribution of a relative GH deficiency to the age-related hypercholesterolemia in rats, we chose to substitute old animals (12-month- and 18-month-old) with a dose of GH (1 mg · kg^-1 · d^-1) that did not influence cholesterol metabolism to a major degree in young rats. This treatment did not significantly change plasma IGF-I levels, and the dose may thus be considered relatively low. Nevertheless, the infusion of GH to hypercholesterolemic 18-month-old rats was associated with a very clear reversal of the changes in plasma lipoproteins. It was more difficult to exactly define a specific point of action responsible for this overall effect. GH affects a number of regulatory steps in lipid and lipoprotein metabolism.^{15 16 51} The fact that the lipoprotein pattern was reversed despite an increased HMG-CoA reductase activity would indicate that the turnover of lipoproteins was stimulated. The increase in hepatic LDLR expression was relatively modest, as was the change in C7OH activity. The fact that more pronounced effects were observed in response to GH treatment in the 12-month-old animals may suggest that the oldest rats were relatively resistant to infusion with GH. The 12-month-old rats displayed a response that was similar to that obtained in the young animals, both in C7OH and lipoprotein pattern. It is of interest to note that plasma IGF-I levels were not changed in these groups of rats, either. The possible contribution of a GH-induced stimulation of the hepatic secretion of apoE or of an increased peripheral clearance of lipoproteins cannot be evaluated from the present study. It seems reasonable to assume that the pleiotropic effects of GH on lipoprotein metabolism are responsible for the favorable changes in plasma lipoprotein patterns in the 18-month-old rats.

In conclusion, our studies have demonstrated the reversal of age-related hypercholesterolemia in the rat by infusion of GH. Our findings further support the concept¹⁵ that GH has an important role in modulating lipoprotein metabolism under physiological conditions.

	Acknowledgments

 
This study was supported by grants from the Swedish Medical Research Council (03X-7137); the Swedish Society for Medical Research; the Swedish Foundation for Strategic Research; the Ax:son Johnson, Jeansson, Widengren, and Ruth and Richard Julin Foundations; the Swedish Heart-Lung Foundation; "Förenade Liv" Mutual Group Life Insurance Co; the foundation of Female Old Servants; and the Karolinska Institute. We thank Ingela Arvidsson, Ewa Ellis, and Lilian Larsson for technical assistance.

Received March 24, 1998; accepted July 28, 1998.

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