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Endocrinology Vol. 148, No. 6 2845-2853
Copyright © 2007 by The Endocrine Society

Adipocytokines and the Regulation of Lipid Metabolism in Growth Hormone Transgenic and Calorie-Restricted Mice

Zhihui Wang1, Michal M. Masternak1, Khalid A. Al-Regaiey and Andrzej Bartke

Geriatrics Research (Z.W., M.M.M., A.B.), Department of Physiology and Internal Medicine, School of Medicine, Southern Illinois University, Springfield, Illinois 62794-9628; Division of Diabetes, Endocrinology, and Metabolism (Z.W.), School of Medicine, Vanderbilt University, Nashville, Tennessee 37232-6303; and Department of Physiology (K.A.A.-R.), College of Medicine, King Saud University, Riyadh, Saudi Arabia 11461

Address all correspondence and requests for reprints to: Zhihui Wang, M.D., Ph.D., Division of Diabetes, Endocrinology, and Metabolism, School of Medicine, Vanderbilt University, 712 PRB, 2220 Pierce Avenue, Nashville, Tennessee 37232-6303. E-mail: zhihui.wang{at}vanderbilt.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Chronic elevation of GH induces resistance to insulin and hyperinsulinemia in both humans and animals, whereas calorie restriction (CR) improves peripheral insulin sensitivity in many species. To investigate the mechanisms that lead to insulin resistance in animals with high levels of GH as well as the mechanisms that might improve insulin sensitivity, we fed GH-overexpressing transgenic mice ad libitum or subjected them to 30% CR. We then assayed the plasma adipocytokines levels related to insulin sensitivity, plasma lipid levels, and tissue triglycerides accumulation and examined adipocyte morphology. Furthermore, we evaluated mRNA expression and protein levels of enzymes or regulators involved in regulating hepatic lipid metabolism. Our results suggest that decreased plasma adiponectin, increased plasma resistin and cholesterol, and elevated levels of TNF-{alpha} and IL-6 in adipocytes may all contribute to the insulin resistance observed in GH-Tg mice. Increased accumulation of triglycerides and impaired adipocytes differentiation in GH-transgenic mice provide plausible mechanisms for the alterations of adipocytokines. Hepatic and muscle insulin resistance in these mice is probably related to excessive accumulation of fatty acids and their metabolites. An increase in plasma adiponectin and decrease in plasma IL-6, triglycerides, and cholesterol levels in response to CR may improve insulin sensitivity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ADIPOSE TISSUE PERFORMS its important functions in energy storage and is well documented as an active endocrine organ. Several signaling molecules secreted by adipocytes, such as adiponectin, leptin, resistin, TNF-{alpha}, IL-6, and fatty acids, influence insulin sensitivity, glucose homeostasis, and lipid metabolism in different ways (1). Adiponectin regulates insulin sensitivity and glucose homeostasis via the activation of AMP-activated protein kinase (AMPK) in the liver and muscle, which phosphorylates and inactivates acetyl-coenzyme A carboxylase (ACC), and then decreases the production of malonyl CoA, a substrate for fatty acid synthase (FAS) and a potent inhibitor of mitochondrial transportation of fatty acids (1). This process decreases fatty acid synthesis, promotes fatty acid oxidation, and reduces the accumulation of triglycerides in liver and muscle (1). Adiponectin also reduces the expression of enzymes involved in gluconeogenesis including glucose-6-phosphatase and phosphoenolpyruvate carboxykinase (PEPCK) in the liver (2) and directly stimulates glucose uptake in muscle and adipocytes by activating AMPK (3), thus regulating insulin sensitivity and energy homeostasis.

Leptin has been widely accepted as an adiposity signal controlling food intake and energy expenditure (4). By binding to its receptor, leptin initiates a phosphorylation cascade via Janus kinase/signal transducer and activator of transcription 3 pathway and then phosphorylates and activates AMPK and peroxisome proliferator-activated receptor (PPAR)-{gamma} coactivator (PGC)-1{alpha} (5). Thus, it down-regulates lipogenic enzymes such as ACC and FAS, up-regulates lipid oxidation-related enzymes such as carnitine palmitoyl transferase (CPT)-1 and acyl-CoA oxidase (ACO), and therefore regulates lipogenesis and fatty acid oxidation (5).

Resistin is recognized as an antiadipogenic factor and an inducer of insulin resistance in rodents (6), although its physiological role in humans remains to be elucidated (7). It inhibits adipocyte differentiation (8), impairs glucose tolerance, decreases insulin-mediated glucose uptake in cultured 3T3-L1 adipocytes, and increases hepatic glucose production in rats (9). TNF-{alpha} and IL-6, proinflammatory cytokines involved in chronic inflammation and malignancy, have been repeatedly shown to contribute to insulin resistance. TNF-{alpha} impairs the insulin signaling pathway by blocking tyrosine kinase activity of insulin receptors (IRs) and inducing serine phosphorylation of IR substrate (IRS) (10, 11). IL-6 inhibits IR signal transduction in the liver, which is mediated at least partly by the induction of suppressor of cytokine signaling-3 (12). It also may increase circulating free fatty acids (FFAs) and decrease adiponectin secretion (13, 14).

Abnormal lipid metabolism is one of the characteristics of insulin resistance and can affect gene expression and intracellular signaling pathways, thus leading to energy imbalance, impaired insulin action, and metabolic syndrome. Triglycerides, FFAs, and cholesterol have all been suggested as role players in the development of insulin resistance (15, 16, 17). The accumulation of triglycerides in nonadipose tissue was reported to interfere with insulin-stimulated phosphatidylinositol 3 kinase (PI3K) activation and subsequent glucose transporter-4 translocation to the plasma membrane, which leads to decreased glucose uptake. Elevated FFAs impair the ability of insulin to suppress hepatic glucose output and stimulate glucose uptake into skeletal muscle as well as inhibit insulin secretion from pancreatic ß-cells (18). Therefore, the enzymes involved in regulating lipid metabolism [lipoprotein lipase (LPL), hormone-sensitive lipase (HSL), and FAS] as well as key transcriptional factors regulating lipid metabolism [PPAR-{alpha}, PPAR-{gamma}, PGC-1{alpha}, and sterol regulatory element binding proteins (SREBPs)] are likely to be linked to insulin sensitivity.

Chronic elevation of GH induces resistance to insulin and hyperinsulinemia in both humans and animals (19). Transgenic (Tg) mice overexpressing bovine (b) GH produce high levels of circulating GH and consequently have increased plasma levels of IGF-I. It has been reported that the insulin signaling pathways in the liver (20) and skeletal muscle (21) were impaired, which may be associated with the insulin resistance exhibited by these animals (22, 23). These mice have an increase in the relative mass of internal organs, and although their body fat content (24) and body fat percent (25) are reduced, they display resistance to insulin.

Calorie restriction (CR) is the most effective intervention that delays aging and extends life span (26). It also improves insulin action in peripheral tissues of many species including mice, rats, rhesus and cynomolgus monkeys, and humans (27, 28, 29, 30, 31). The physiological mechanisms for the improvement of insulin sensitivity by CR may include decreases in circulating fatty acid concentrations and intramyocellular triglycerides, and the changes of adipocyte secretion of cytokines including adiponectin and leptin. Additional mechanisms remain to be fully elucidated.

The purpose of the present study was to investigate the relationship between adipocytokines, the regulation of lipid metabolism, and insulin resistance. In addition, we wanted to elucidate the mechanisms that contribute to insulin resistance observed in GH-Tg mice and address the possible mechanisms by which CR improves insulin sensitivity. Our findings suggest that the alterations of adipocytokines and lipid levels in GH-Tg mice might be due to the excess accumulation of triglyceride in adipocytes and the impaired adipocyte differentiation. Changes in the expression or protein levels of enzymes or transcriptional factors involved in lipid metabolism might lead to excess accumulation of fatty acids and their metabolites in the liver, thus impairing insulin signal conduction and consequent insulin sensitivity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and CR
Male GH-Tg mice and their normal male siblings were used. These animals were originally produced by microinjecting the bGH structural gene fused with the promoter of the rat PEPCK gene into the pronuclei of fertilized mouse eggs (32). The hemizygous Tg mice used in this study were produced by mating GH-Tg males with normal C57BL/6 x C3H F1 hybrid females. GH-Tg mice and their normal littermates were fed ad libitum (AL), housed at 22 ± 2 C and 12-h light, 12-h dark cycle. CR started at 5 months of age by receiving 90% of daily food consumption of AL control group for 1 wk, then 80% for the second week, and maintaining 70% for the rest of study. At the age of 7 months, animals were anesthetized by isoflurane between 0800 and 1000 h after overnight fasting, bled by cardiac puncture, and decapitated; epididymal white adipose tissues (WAT), liver, and hindlimb muscles were removed; frozen in dry ice; and stored at –70 C. Thus, we had four groups, eight to 10 animals per group: normal mice fed AL (N-AL), normal mice subjected to CR (N-CR), GH-Tg mice fed AL (Tg-AL), and GH-Tg mice subjected to CR (Tg-CR).

Plasma sample analyses
Plasma glucose levels were measured using a glucometer (LifeScan, Milpitas, CA), insulin levels were determined using Ultra Sensitive rat insulin ELISA kits (Crystal Chem Inc., Chicago, IL). Adiponectin and resistin levels were assayed using mouse adiponectin/resistin ELISA kits (Linco Research, St. Charles, MO). Leptin levels were evaluated using mouse leptin ELISA kit (Crystal Chem Inc.). TNF-{alpha} and IL-6 were measured using mouse TNF-{alpha}/IL-6 ELISA kits (Biosource, Camarillo, CA). Plasma triglycerides were determined using a triglyceride (liquid) reagent set (Pointe Scientific, Inc., Canton, MI). Plasma FFAs were assayed using optimized enzymatic colorimetric assay (Roche, Indianapolis, IN). Plasma cholesterol was measured using a cholesterol (liquid) reagent set (Pointe Scientific).

Total RNA extraction, cDNA transcription, and real-time PCR
Total hepatic and skeletal muscle RNA was extracted using the phenol-chloroform method (33). The RNA was reverse transcribed into cDNA using iScript cDNA synthesis kit (Bio-Rad, Hercules, CA). The relative expression of the genes was analyzed by real-time PCR according to the protocol described previously (34). The primers are listed in Table 1Go. After evaluation, ß2-microglobulin was used as a housekeeping gene and the equation of 2A – B/2C – D [A = cycle threshold (Ct) number of the gene of interest in the first control sample, B = Ct number of the gene of interest in each sample, C = Ct number of the housekeeping gene in the first control sample, D = Ct number of the housekeeping gene in each sample] was used as described previously (34). Thus, the relative expression of the first control sample was expressed as 1, and the relative expression of all other samples was calculated by this equation. The results from the N-AL group were averaged, and all other outputs were divided by this average to get the fold change of expression of the genes of interest, compared with this control group.


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  		TABLE 1. Sequence of primers for gene expression used in real-time PCRs

 
Total lipid extraction and tissue triglycerides assay
Muscle, liver, or WAT (35–50 mg) were homogenized in an organic extraction solution of 2:1 chloroform-methanol in a 20:1 volume to weight ratio and kept at 4 C overnight with gentle shaking; 0.6% NaCl was added to half volumes, an emulsification was formed by vortexing, and then samples were spun at 800 x g for 20 min at 4 C. After extracting the organic phase, samples were evaporated to dryness under nitrogen and reconstituted in 30–100 µl of PBS containing 1% Triton X-100. Triglyceride concentrations were measured using a triglyceride (liquid) reagent set (Pointe Scientific).

Histology
Epididymal WAT was fixed in 4% phosphate-buffered paraformaldehyde (pH 7.4), embedded in paraffin, sectioned, and stained with hematoxylin and eosin.

Western blotting
Each group included six samples. Briefly, the tissues were homogenized in an ice-cold lysis buffer containing 20 mM Tris, 150 mM NaCl, 1% Triton-X (pH 7.5), 1% phosphatase inhibitor cocktail I (Sigma, St. Louis, MO), 1% phosphatase inhibitor cocktail II (Sigma), and 1% protease inhibitor (Pierce, Rockford, IL). The protein concentration was determined using BCA protein assay kit (Pierce). The Western blot procedure was performed to analyze protein levels as described previously (35). The specific antibodies we used included anti-FAS antibody (BD Biosciences, Franklin Lakes, NJ), anti-ACC antibody (Cell Signaling, Beverly, MA), anti-phospho-AMPK-{alpha} antibody (Cell Signaling), anti-PGC-1 antibody (Chemicon, Temecula, CA), and anti-PPAR-{alpha} antibody (Sigma).

Statistical analyses
The results are reported as the mean ± SEM. Statistical analysis was performed using two-way ANOVA followed by Fisher’s protected least significant difference test. We also used Student’s t tests to evaluate the effects of diet within phenotypes and phenotype within diets. P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of phenotype and CR on body weight
As expected, the body weights of GH-Tg mice were dramatically increased, compared with their normal littermates (P < 0.0001, Fig. 1Go). After 30% CR, there was a consistent and significant reduction in the body weight of normal and Tg mice. The reduction of body weight by CR was 24% in normal mice (P = 0.0001) and 26% in Tg mice (P = 0.0002) by the end of study (Fig. 1Go).


Figure 1
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  		FIG. 1. Growth curves of normal (N) and bGH Tg mice fed AL or subjected to 30% CR. Each point represents the average body weights of nine to 10 animals per group.

 
Effects of phenotype and CR on the levels of adipocytokines
Although there were no significant differences in plasma glucose levels, Tg mice displayed increased plasma insulin levels, compared with their normal littermates (Table 2Go). CR did not affect plasma insulin levels in either normal or GH-Tg mice.


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  		TABLE 2. Plasma data from normal (N) and PEPCK-bGH Tg mice fed AL or subjected to 30% CR

 
Plasma adiponectin levels are usually negatively correlated to the body fat content (36). Interestingly, we found that plasma adiponectin levels were dramatically reduced in GH-Tg mice regardless of diet, compared with the levels measured in normal mice (Table 2Go), even though GH-Tg mice have reduced relative body fat content, compared with their normal littermates. CR significantly increased plasma adiponectin levels in both normal and Tg mice.

Consistent with the lower body fat, GH-Tg mice displayed a trend of reduced plasma leptin levels. However, it was not statistically significant (Table 2Go). CR decreased leptin levels in GH-Tg but not normal mice.

Resistin was recognized as an antiadipogenic factor and an inducer of insulin resistance in rodents (6). Consistent with this view, plasma resistin levels were elevated in GH-Tg mice; CR increased its levels in normal but not GH-Tg mice.

Plasma TNF-{alpha} levels were below the detectability limits of the assays used. GH-Tg genotype did not affect plasma IL-6 levels (Table 2Go) but increased TNF-{alpha} and IL-6 protein levels in the epididymal fat (Fig. 2Go). CR decreased plasma IL-6 levels in GH-Tg mice and appeared to lead to a change in the same direction in normal mice.


Figure 2
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  		FIG. 2. TNF-{alpha} (A) and IL-6 (B) protein levels in epididymal WAT from normal and PEPCK-bGH Tg mice fed AL, as assayed by ELISA (n = 8 per group), and each sample was measured in duplicate. Group values are expressed as the mean ± SE. In each group, values marked with a different superscript (a and b) are significantly different (P < 0.05).

 
Effects of phenotype and CR on plasma lipid levels and tissue triglycerides accumulation
The plasma triglyceride levels were reduced by 40% in GH-Tg mice (P < 0.005). CR suppressed its levels in normal mice but produced no detectable change in GH-Tg mice. Although GH-Tg mice had lower body fat and decreased plasma triglycerides, their plasma cholesterol levels were substantially elevated, compared with their normal siblings. CR did not alter plasma cholesterol in either normal or GH-Tg mice (Table 2Go).

Results from tissue triglyceride assay showed that GH-Tg mice have increased WAT triglyceride content but decreased liver triglyceride content. Skeletal muscle triglyceride content did not differ between genotypes (Fig. 3AGo).


Figure 3
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  		FIG. 3. A, Tissue triglycerides from normal and PEPCK-bGH Tg mice fed AL. Data are means ± SE (n = 9 per group). *, P < 0.05; **, P < 0.005. B, Epididymal adipocytes from N (left) and PEPCK-bGH Tg (right) mouse (10 times) fed AL.

 
Additionally, histological examination was performed. We observed that there were various adipocytes in heterogenous differentiation phases; the percentage of preadipocytes that are presumably unable to produce insulin-sensitizing hormone was increased dramatically (Fig. 3BGo) (1).

Effects of phenotype on the regulation of liver fatty acid synthesis, lipid mobilization, and fatty acid oxidation
The hepatic mRNA expression of lipogenic factors such as LPL, ACC-ß, and PPAR-{gamma} was up-regulated, whereas SREBP-1 mRNA expression was down-regulated in GH-Tg, compared with normal mice. At the same time, the hepatic expression of key enzymes or regulators that are responsible for triglyceride mobilization, mitochondria biogenesis, and fatty acid oxidation, including HSL, PGC-1{alpha}, AMPK-{alpha}2, CPT-1{alpha}, and ACO-1{alpha}, was substantially decreased in Tg mice (Table 3Go).


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  		TABLE 3. Relative mRNA expression in liver from normal (N) and PEPCK-bGH Tg mice subjected to 30% CR

 
Western blotting results from liver generally supported these findings. Although hepatic FAS protein levels were reduced in GH-Tg mice, the levels of ACC and PPAR-{alpha} proteins were elevated (Fig. 4Go), whereas the levels of phospho-AMPK-{alpha} (Thr172) protein, which is the activated form of AMPK, were reduced (Fig. 5Go).


Figure 4
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  		FIG. 4. Protein levels of enzymes involved in fatty acid synthesis in liver from normal and GH Tg mice subjected to 30% CR. Representative Western blots (A and C) and summary (B and D) of FAS (A and B) and ACC (C and D) protein levels. Relative protein levels are presented as fold change (mean ± SE) relative to N-AL group. Different superscripts denote significant difference at P < 0.05. Six animals per group were analyzed.

 

Figure 5
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  		FIG. 5. Protein levels of enzymes or transcriptional factors involved in fatty acid oxidation in liver from normal and GH Tg mice subjected to 30% CR. Representative Western blots (A, C, and E) and summary (B, D, and F) of PGC-1 (A and B), PPAR-{alpha} (C and D), and phospho-AMPK-{alpha} (Thr172) (E and F) protein levels. Relative protein levels are presented as fold change (mean ± SE) relative to N-AL group. Different superscripts denote significant difference at P < 0.05. Six animals per group were analyzed.

 
CR accelerated the turnover and degradation of fatty acid in liver from normal mice
Food deficiency had significant effect on the enzymes or transcriptional factors involved in the regulation of lipid metabolism in normal mice. Compared with N-AL mice, CR increased the mRNA expression of SREBP-1 and consequently up-regulated the expression of ACC-ß (Table 3Go). However, hepatic ACC protein levels did not differ between N-AL and N-CR mice (Fig. 4Go). CR also enhanced the expression of liver PPAR-{gamma}. Importantly, CR increased the expression of HSL, PGC-1{alpha}, AMPK, AMPK-{alpha} 2, CPT-1{alpha}, and ACO-1{alpha} (Table 3Go), which are related to the hydrolysis of triglycerides and the transport of fatty acids into mitochondria as well as mitochondrial biogenesis and oxidation. In addition, Western blotting results showed that CR reduced hepatic FAS and elevated PGC-1 protein levels in normal mice (Figs. 4Go and 5Go), providing further support for these findings.

In Tg mice, CR decreased the expression of LPL and increased the expression of PGC-1{alpha}. It also reduced the ACC and PPAR-{alpha} protein levels (Fig. 4Go) without altering SREBP-1, PPAR-{gamma}, HSL, AMPK, CPT-1{alpha}, or ACO-1{alpha}.

Effects of phenotype and CR on the regulation of skeletal muscle fatty acid synthesis, lipid mobilization, and fatty acid oxidation
In skeletal muscle, the mRNA expression of some lipogenic factors including LPL and ACC-ß were down-regulated in GH-Tg mice, but most of these factors including ACC-{alpha}, FAS, SREBP-1, and PPAR-{gamma} did not change, compared with their normal littermates (Table 4Go). More strikingly, the expression of key enzymes or regulators involved in triglycerides mobilization, mitochondria biogenesis, and fatty acid oxidation, such as PPAR-{alpha}, HSL, PGC-1{alpha}, AMPK, AMPK-{alpha}2, CPT-1{alpha}, and ACO-1{alpha}, was markedly decreased in GH-Tg mice (Table 4Go). CR increased the expression of ACC-{alpha} in normal mice but did not have much effect on the expression of other factors involved in lipid metabolism.


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  		TABLE 4. Relative mRNA expression in skeletal muscle from normal (N) and PEPCK-bGH Tg mice subjected to 30% CR

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
It is well documented that an increase in plasma GH induces resistance to the action of insulin in both human and animals (19). Excessive production of endogenous GH in acromegalic patients or administration of exogenous GH to animals leads to insulin resistance in different insulin target tissues (37, 38, 39, 40). However, the potential mechanisms by which elevated GH levels induce insulin resistance or reduce insulin sensitivity await further study. In the present study, we investigated the release of insulin sensitivity-related adipocytokines, the alteration of plasma and tissue lipid levels, and the relationship between these actions and insulin sensitivity. We also evaluated the effect of CR in normal and Tg mice overexpressing GH in an attempt to elucidate the mechanisms by which CR improves insulin sensitivity. GH transgenic mice exhibit chronically elevated circulating GH and IGF-I, marked stimulation of somatic growth, and reduced body fat content (24) and body fat percent (25). They are normoglycemic but highly hyperinsulinemic (23) and show slightly impaired or normal glucose tolerance as well as impaired insulin tolerance (Panici, J., and A. Bartke, unpublished data) when fed normal chow.

GH-Tg mice used in this study displayed the expected stimulation of somatic growth (Fig. 1Go). Glucose levels were normal but insulin levels were elevated. Two months of CR did not alter plasma glucose levels in either GH-Tg mice or their normal littermates. However, it numerically decreased insulin levels in normal mice (Table 2Go).

Our results indicate that insulin resistance in GH-Tg mice has a close relationship with the alterations of adipocytokine profile and lipid metabolism. Adiponectin, a plasma protein exclusively secreted by adipose tissue, is closely linked to insulin sensitivity, glucose homeostasis, and lipid metabolism (1, 2, 3). Epidemiological studies in humans and animals showed that circulating adiponectin levels are positively associated with insulin sensitivity and negatively associated with degree of adiposity. Our recent reports on insulin-sensitive Ames dwarf mice (41) provided further support for this association. Interestingly, we found that although GH-Tg mice have lower body fat content (24), they have decreased plasma adiponectin levels, which may be due to the elevated GH or insulin levels in these mice. GH was reported to reduce adiponectin secretion in human adipose tissue in vitro and in mice in vivo (42).

In cultured 3T3-L1 adipocytes, adiponectin mRNA levels were decreased by insulin or IGF-I (43). Consistent with these reports, GH-deficient (41) or GH receptor-deficient mice (35, 42) that have reduced circulating insulin and IGF-I levels were found to have increased plasma adiponectin levels. In the present study, CR increased adiponectin levels in both GH-Tg mice and their normal siblings.

These findings correlate with impaired insulin sensitivity in GH-Tg mice and improvement of insulin sensitivity by CR. Similar observations have been reported in acromegalic patients (43) and animals (42). The decrease of plasma leptin by CR in Tg mice may represent a compensatory response to reduced energy intake, thus increasing appetite and reducing energy expenditure. As expected, plasma resistin, the hormone closely associated with insulin resistance in rodents (6), was elevated in GH-Tg mice. In addition, two factors leading to insulin resistance, the WAT levels of TNF-{alpha} and IL-6 proteins, were also increased in Tg mice. The Tg animals showed reduced plasma triglyceride levels, whereas plasma cholesterol levels were elevated, compared with normal mice. CR decreased plasma triglycerides in normal but not GH-Tg mice; and, in contrast, it decreased plasma cholesterol levels in GH-Tg but not in normal mice. Unexpectedly, however, we found increased triglyceride accumulation in the WAT of GH-Tg mice. Morphological examination showed that the number of preadipocytes was dramatically increased. Unlike mature adipocytes, those preadipocytes are unable to produce insulin-sensitizing hormones (1) such as adiponectin. This observation provides a reasonable explanation for the detected alterations in plasma adipocytokine levels (Table 2Go). We postulate that the reduced plasma adiponectin levels and the elevated levels of plasma resistin and cholesterol as well as WAT, TNF-{alpha}, and IL-6 protein all play a role in inducing insulin resistance in Tg mice. These alterations of adipocytokines and lipid levels might be partly due to the excess accumulation of triglycerides in WAT and impaired adipocytes differentiation. CR might improve insulin sensitivity via increasing plasma adiponectin levels, whereas decreasing IL-6, triglycerides, and cholesterol levels.

Our results also suggest that insulin resistance in GH-Tg mice is closely linked to the alteration of hepatic lipid metabolism. It has been proposed that reduced insulin sensitivity is affected by the accumulation of intracellular lipid metabolites of triglycerides in peripheral tissues (44). Most studies to date in both humans and animals have found a strong relationship between hepatic triglyceride content and hepatic insulin sensitivity (45, 46, 47, 48). GH-Tg mice used in the present study showed less sensitivity to the action of insulin, indicated by the fact that they need more plasma insulin to maintain normal glucose levels. Furthermore, their insulin resistance was also supported by results of previous studies in which it was shown that in the liver, IR and IRS-1 phosphorylation, IRS-1/PI 3 kinase association, and PI 3 kinase activity have been highly activated under basal conditions, thus making the liver less sensitive to further stimulation by exogenous insulin in vivo (20). Furthermore, it has been suggested that in another line of GH-Tg mice, the overabundance of the p85{alpha} monomer competes with the p85-p110 heterodimer for binding to IRS-1 and activation of PI 3 kinase, thus causing insulin resistance (49). In the present study, GH transgenic mice had reduced hepatic triglyceride accumulation, which might be the result of increased triglyceride lipolysis or decreased triglyceride synthesis.

LPL, the key enzyme hydrolyzing circulating triglycerides from very low-density lipoprotein to FFAs, is rate limiting for uptake of triglyceride-derived fatty acid into tissue. ACC is a key regulatory enzyme for catalyzing the carboxylation of acetyl-CoA to form malonyl-CoA, thus providing a substrate for FAS. SREBP-1 regulates the expression of ACC and FAS. PPAR-{gamma} acts as a critical regulator of adipocyte differentiation, adipogenesis, and lipid metabolism. We found that the expression of LPL in the liver of Tg mice was enhanced substantially, which might be consequent to the elevated expression of PPAR-{gamma}, and it may increase the uptake of triglyceride-derived fatty acid into liver. More interestingly, we observed that both AMPK{alpha}2 mRNA expression and the phosphorylated AMPK{alpha} (Thr172) protein levels were reduced in GH-Tg mice. This is most likely caused by the reduced plasma adiponectin and increased plasma resistin levels because it was previously shown that plasma adiponectin strongly up-regulates the mRNA expression and the activity of AMPK, whereas plasma resistin has opposite effects. Reduced levels of activated AMPK levels might result in up-regulation of ACC expression, as our results showed. Additionally, elevated plasma insulin levels in Tg mice may also partly contribute to the up-regulation of ACC expression, which most likely leads to an increase in the level of malonyl CoA, the substrate of FAS, thus promoting FAS. In the liver from Tg mice, the expression of CPT-1, the key regulatory enzyme in the transport of long-chain fatty acid into mitochondria, was down-regulated; so was ACO-1, which is responsible for acyl-CoA oxidation. Furthermore, the expression of the transcriptional factor controlling the generation of mitochondria and oxidative phosphorylation, PGC-1, was also reduced. These data suggest that hepatic mitochondria fatty acid oxidation might be reduced in GH-Tg mice.

Our results are consistent with the results we obtained previously, indicating that fatty acid uptake is increased in GH-overexpressing GH-Tg mice but that the intracellular fatty acids are preferentially directed toward lipogenesis rather than oxidation. Actually, liver triglyceride accumulation in GH-Tg mice was decreased rather than increased (Fig. 3AGo), which might result from reduced SREBP-1 expression and enhanced TNF-{alpha} expression. Taken together, this suggests that in GH-Tg mice overexpressing GH, the pathway of triglyceride synthesis from fatty acid might be partly blocked, resulting in excess fatty acid accumulation in liver. This, in turn, provides a plausible explanation for the compromised insulin signal transduction observed previously (20, 50).

Furthermore, the impaired insulin signaling pathway in skeletal muscle from GH-Tg mice reported earlier (21) might also be linked to the alteration of muscle lipid metabolism. We observed that the expression of genes involved in lipid mobilization and oxidation (PPAR-{alpha}, HSL, PGC-1{alpha}, AMPK, AMPK-{alpha}2, CPT-1{alpha}, and ACO-1{alpha}) was significantly down-regulated in the skeletal muscle from GH-Tg mice, which might lead to an increase in triglyceride accumulation in this tissue (Table 4Go). Unexpectedly, we did not find any difference in muscle triglyceride content. These results suggest the synthesis of triglycerides from fatty acid might be at least partly impaired and that excess fatty acid accumulation rather than triglycerides may play a pivotal role in leading to muscle insulin resistance in GH-Tg mice. The specific mechanism is a target for future studies.

In conclusion, we found that GH-Tg mice overexpressing GH had reduced plasma adiponectin, elevated plasma resistin, and cholesterol and increased levels of TNF-{alpha} and IL-6 in adipocytes, which might contribute to the insulin resistance in these animals. Alterations of adipocytokine and lipid levels might be due to the excessive accumulation of triglycerides in adipocytes and impaired adipocytes differentiation. Changes in the expression or protein levels of enzymes or transcriptional factors involved in lipid metabolism might lead to excess accumulation of fatty acids and their metabolites in the liver and skeletal muscle, thus impairing insulin signal conduction and consequent insulin sensitivity. The present findings also suggest that CR might improve insulin sensitivity by enhancing the plasma adiponectin levels and reducing plasma IL-6, triglyceride, and cholesterol levels. Furthermore, CR may induce the hydrolysis of triglycerides and promote mitochondrial biogenesis and oxidation. This reduces triglyceride and fatty acid accumulation in the liver and results in improved insulin sensitivity.


   Acknowledgments
 
We thank Steve Sandstrom for his help in editing this manuscript.


   Footnotes
 
First Published Online March 8, 2007

1 Z.W. and M.M.M. contributed equally to this study. Back

Abbreviations: ACC, Acetyl-coenzyme A carboxylase; ACO, acyl-CoA oxidase; AL, ad libitum; AMPK, adenosine monophosphate-activated protein kinase; b, bovine; CPT, carnitine palmitoyl transferase; CR, calorie restriction; Ct, cycle threshold; FAS, fatty acid synthase; FFA, free fatty acid; HSL, hormone-sensitive lipase; IR, insulin receptor; IRS, IR substrate; LPL, lipoprotein lipase; PEPCK, phosphoenolpyruvate carboxykinase; PGC, PPAR-{gamma} coactivator; PI 3 kinase, phosphatidylinositol 3 kinase; PPAR, peroxisome proliferator-activated receptor; SREBP, sterol regulatory element binding protein; Tg, transgenic; WAT, white adipose tissue.

This work was supported by grants from the National Institute on Aging (AG-198899 and U19 AG023122) and the Ellison Medical Foundation.

Disclosure Statement: The authors have nothing to disclose.

Received September 25, 2006.

Accepted for publication February 28, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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