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The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 61:323-331 (2006)
© 2006 The Gerontological Society of America

Adipocytokines and Lipid Levels in Ames Dwarf and Calorie-Restricted Mice

Zhihui Wang, Khalid A. Al-Regaiey, Michal M. Masternak and Andrzej Bartke

Geriatrics Research, Department of Physiology and Internal Medicine, School of Medicine, Southern Illinois University, Springfield.

Address correspondence to Zhihui Wang, MD, SIU School of Medicine, Department of Physiology and Internal Medicine, 801 N. Rutledge, Springfield, IL 62794-9628. E-mail: zwang2{at}siumed.edu


   Abstract
Top
 Abstract
Methods
 Results
 Discussion
 References
 
Ames dwarf mice are long-lived and insulin sensitive, and have a normal or reduced percentage of body fat. Calorie restriction (CR) is known to improve insulin sensitivity and reduce body fat. The purpose of this study was to evaluate the mechanism of improved insulin sensitivity in the Ames dwarfs and the effects of CR on adipose signaling and metabolism in normal and dwarf mice. Enhanced insulin sensitivity in dwarf mice may be partly due to increased release of adiponectin and the reduced release of tumor necrosis factor-{alpha} (TNF-{alpha}) and interleukin-6 (IL-6). Altered levels of adipocytokines might be consequent to the decreased lipid synthesis, plasma triglycerides, and free fatty acid levels. In normal mice, CR improves insulin sensitivity by affecting the release of adipocytokines, and decreasing circulating fatty acid and triglycerides concentrations as well as liver triglyceride accumulation. However, CR may reduce rather than enhance some of the insulin effects in the highly insulin-sensitive dwarf mice.

INSULIN resistance is one of the key factors in major health problems such as hyperglycemia, hypertension, hyperlipidemia, and cardiovascular diseases. Obesity plays an important role in the development of insulin resistance. As an active endocrine organ, adipose tissue secretes various peptide hormones that are involved in obesity, insulin resistance, and type II diabetes (1) including adiponectin, leptin, resistin, tumor necrosis factor-{alpha} (TNF-{alpha}), and interleukin-6 (IL-6). Adiponectin is a plasma protein secreted exclusively by adipose tissue (2). It regulates insulin sensitivity and glucose homeostasis via the activation of adenosine monophosphate (AMP)–activated protein kinase (AMPK) in the liver and muscle, which phosphorylates acetyl-coenzyme A carboxylase (ACC), and thus promotes fatty acid oxidation and decreases the accumulation of triglycerides in liver and muscle (1). Adiponectin also reduces the expression of enzymes involved in gluconeogenesis including glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK) in the liver (3), and directly stimulates glucose uptake in muscle and adipocytes by activating AMPK (4), thus regulating insulin sensitivity and energy homeostasis. Leptin has been well recognized as an adiposity signal. Leptin plasma levels are positively related to the fat mass; mice that are deficient in leptin (obese, ob/ob) or leptin receptor (diabetic, db/db) exhibit hyperphagia, obesity, and diabetes (5). Exogenous leptin administration to ob/ob mice reverses these abnormalities (6). Leptin activates neurons in the hypothalamic arcuate nucleus that express proopiomelanocortin (POMC) to release anorexic neuropeptide, {alpha}-melanocyte stimulating hormone (MSH), and inhibits neurons that express orexigenic neuropeptide Y (NPY) and agouti-related protein (AgRP) (7), and thus potently inhibits food intake. {alpha}-MSH binds to and acts on melanocortin 4 receptor (MC4R) located in the downstream neurons in paraventricular nuclei and the lateral hypothalamus, decreases food intake, and increases energy expenditure (7,8). {alpha}-MSH also increases the gene expression of uncoupling proteins (UCP) 1, UCP2, and UCP3 in adipose tissue, and thus promotes energy expenditure. Resistin is also involved in insulin resistance and obesity. Its plasma levels were reported to be elevated in the obese and insulin-resistant animals (9). Resistin administration to normal mice impaired glucose tolerance and action of insulin. Antidiabetic treatment with thiazolidinediones (TZDs) reduced resistin gene expression and protein levels (10,11). However, some reports showed different results: resistin messenger RNA (mRNA) expression in fat tissue was reduced in obese mice; plasma levels were decreased in insulin-resistant mice (12); and there was no correlation between resistin gene expression and plasma insulin levels or the insulin resistance, but there was a significant positive correlation between resistin gene expression and plasma glucose levels (13). TNF-{alpha} and IL-6 were first identified as proinflammatory cytokines involved in chronic inflammation and malignancy, and now are well documented as adipocytokines contributing 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) (14,15). TNF-{alpha} also down-regulates glucose transporter 4 (GLUT4) gene expression, induces the expression of suppressor of cytokine signaling (SOCS)-1 and SOCS-3, and suppresses the release of adiponectin. IL-6 inhibits IR signal transduction in liver, which is mediated at least partly by induction of SOCS-3 (16). It also may increase circulating free fatty acids (FFA) and decrease adiponectin secretion (17,18).

Lipid metabolism has a close relationship with insulin sensitivity and glucose homeostasis. The accumulation of triglycerides in nonadipose tissue was reported to interfere with insulin-stimulated phosphatidylinositol 3 kinase (PI3K) activation and subsequent GLUT4 translocation to the plasma membrane, which leads to decreased glucose uptake. Elevated FFA impairs the ability of insulin to suppress hepatic glucose output and to stimulate glucose uptake into skeletal muscle, as well as inhibits insulin secretion from pancreatic ß cells. Therefore, the enzymes involved in lipid metabolism such as lipoprotein lipase (LPL), hormone sensitive lipase (HSL), fatty acid synthase, as well as key transcriptional factors regulating lipid metabolism such as peroxisome proliferator-activated receptor-{gamma} (PPAR-{gamma}), PPAR-{gamma} coactivator 1 {alpha} (PGC-1 {alpha}), and sterol regulatory element binding proteins (SREBPs) are likely to be related to insulin sensitivity.

Calorie restriction (CR), the only effective intervention that delays aging and extends life span (19), also improves insulin action in peripheral tissues of many species including mice, rats, rhesus and cynomolgus monkeys, and humans (20–24). CR reduces plasma glucose and insulin levels, indicating an improvement of insulin sensitivity (25). However, CR did not affect the number or binding affinity of the IRs (26), the tyrosine kinase activity of the IR (27), the activation of PI3K (28), or the total GLUT4 or p85 subunit of PI3K in skeletal muscle (29). The physiological mechanisms for the improvement of insulin sensitivity by CR may include decreases in circulating fatty acid concentrations, intramyocellular triacylglycerol, and the changes of adipocyte secretion of cytokines including adiponectin and leptin. The mechanisms of improved insulin sensitivity induced by CR still remain to be fully elucidated.

The purpose of the present study was to investigate the relationship of adipocytokines, plasma and tissue lipid levels, and insulin sensitivity in Ames dwarf (df/df) mice and to evaluate the effects of CR on these relationships. Ames dwarf mice display primary hypopituitarism, diminutive body size, improved insulin sensitivity, and prolonged longevity (30). Though df/df mice are growth hormone (GH) deficient, young adult df/df mice are not obese and their relative body fat content at more advanced age is either similar to or lower than that of their normal littermates (31). We were interested in relating the levels of adipocytokines to the effects of CR and Ames dwarfism on insulin sensitivity and body composition. Whether there is a change in fat metabolism, which leads to a reduced percentage of body fat, and if these changes contribute to improved insulin sensitivity have not been previously investigated. Identification of these mechanisms should be helpful in understanding the role that adipocytokines and lipid metabolism play in regulating whole body insulin sensitivity.


   METHODS
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 Abstract
 Methods
Results
 Discussion
 References
 
Animals and CR
Male Ames dwarf mice and their normal littermates were fed ad libitum (AL) and housed at 22 ± 2°C with a 12-hour light/dark cycle. Animals were provided with a nutritionally balanced diet (Rodent Laboratory Chow 5001; not autoclaved; 23.4% protein, 4.5% fat, 5.8% crude fiber; LabDiet PMI Feeds, Inc., St. Louis, MO). CR started at 2 months of age by receiving 90% of daily food consumption of the AL control group for 1 week, then 80% for the second week, and maintaining 70% for the rest of study. At the age of 18 months, animals were anesthetized by isoflurane after overnight fasting between 8 AM and 10 AM, bled by cardiac puncture, and decapitated; epididymal white adipose tissues (WATs), liver, and hind-limb muscles were removed, frozen in dry ice, and stored at –70°C. Thus, we had four groups, 8–10 animals per group: normal mice fed AL (N-AL), normal mice subjected to CR (N-CR), Ames dwarf mice fed AL (df/df-AL), and Ames dwarf mice subjected to CR (df/df-CR).

Plasma Sample Analyses
Plasma glucose levels were measured using a glucose oxidase method (Sigma, St. Louis, MO), and insulin levels were determined using Ultra Sensitive Rat Insulin ELISA Kits (Crystal Chem Inc., Downers Grove, 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 Kits (Crystal Chem Inc., Downers Grove, IL). TNF-{alpha} and IL-6 were measured using Mouse TNF-{alpha}/IL-6 ELISA Kits (Biosource, Camarillo, CA). Plasma FFAs were assayed using optimized enzymatic colorimetric assays (Roche, Penzberg, Germany).

Total RNA Extraction, Complementary DNA Transcription, and Real-Time Polymerase Chain Reaction
For each sample, approximately 150 mg of tissue from individual animals was homogenized with 1 ml of cold 1x phosphate-buffered saline solution with 200 U RNAase inhibitor (Promega, Madison, WI). Homogenate (250 µl) was used for RNA extraction by phenol–chloroform solution (32). The RNA concentration was quantified using a spectrophotometer. Total mRNA (1 µg) was reverse-transcribed into complementary DNA using an iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA) according to the manufacturer's protocol. The amplification and quantification of specific genes was conducted by real-time polymerase chain reaction (RT–PCR) using iQ SYBR Green PCR Supermix (Bio-Rad) and specific forward and backward primers (Table 1). The program was designed as a denaturation step at 95°C for 2 minutes, followed by 45 cycles of 95°C denaturation for 15 seconds, 62°C (or 64°C) annealing for 30 seconds, and 72°C extension for 30 seconds. The fluorescence was read at the end of the 72°C extension. In addition, a melting curve was done for each reaction to evaluate the potential of nonspecific products. The data were analyzed and quantified using the Cepheid (Sunnyvale, CA) SmartCycler software. Ribosomal 18s was used as a housekeeping gene. Relative expression was calculated with the equation: 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, and D = Ct number of the housekeeping gene in each sample). Thus the relative expression of the first control sample was expressed as 1, and the relative expression of all other samples were calculated by using this equation. The results from 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 to this control group (33).


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  		Table 1. Sequence of Primers for Gene Expression Used in Real-Time Polymerase Chain Reactions.

 
Total Lipid Extraction, Plasma and Tissue Triglycerides Assay
Muscle, liver, or WAT (35–50 mg) was homogenized in an organic extraction solution of 2:1 chloroform/methanol in a 20:1 volume/weight ratio, and kept at 4°C overnight with gentle shaking; 0.6% NaCl was added to one-half volumes, an emulsification was formed by vortexing, then samples were spun at 800 g for 20 minutes at 4°C. After extracting the organic phase, samples were evaporated to dryness under nitrogen and dried samples were reconstituted in 30–100 µl of phosphate-buffered saline containing 1% Triton X-100. Triglyceride concentrations were measured using a Triglyceride (Liquid) Reagent Set (Pointe Scientific, Inc., Canton, MI).

Statistical Analyses
Results are presented as mean ± standard error of the mean (SEM). Statistical analysis was performed using a two-way analysis of variance (ANOVA) followed by Fisher's protected least significant difference test (PLSD). We also used t tests to evaluate the effects of diet within phenotypes and phenotype within diets. Values of p <.05 were considered significant.


   RESULTS
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 Methods
 Results
Discussion
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Effects of Genotype and CR on Body Weight
As expected, the body weights of Ames dwarf mice were reduced significantly compared to their normal littermates (Table 2). We have recently shown that the body growth of N-AL mice reached the maximum around 14 months of age, whereas the body growth of Ames df/df mice did not reach its maximum until 18 months in both AL and CR groups. CR decreased and delayed the body growth in normal and df/df mice but when these mice were killed there was no difference in the body weight between df/df-AL and df/df-CR mice (34).


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  		Table 2. Body Weight, Plasma Glucose, and Hormone and Adipocytokine Levels From Normal (N) and Ames df/df Mice Subjected to 30% Calorie Restriction (CR).

 
Effects of Genotype and CR on Insulin Sensitivity and Adipocytokines Expression
Plasma glucose levels were dramatically reduced in df/df mice compared with N-AL mice (p <.005; Table 2); CR did not affect the glucose level in normal mice, but increased it in df/df mice (p <.05). Plasma insulin was also greatly reduced in df/df mice compared with their normal littermates, regardless of the diet (p <.005; Table 2). CR did not alter the insulin levels in normal mice, whereas it elevated insulin levels in df/df mice (p <.05).

Neither df/df genotype nor CR changed the expression of adiponectin mRNA in WAT (Figure 1), but the plasma adiponectin levels were elevated significantly in both df/df-AL and df/df-CR mice compared with the levels measured in normal mice (p <.005 and.0005 respectively; Table 2). There was a trend for increased plasma adiponectin levels in df/df-CR mice compared with df/df-AL mice but it did not reach statistical significance (p =.0786; Table 2).


Figure 01
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  		Figure 1. Relative messenger RNA (mRNA) expression of adipocytokines in epididymal white adipose tissue (WAT) from normal (N) and df/df mice fed ad libitum (AL) or subjected to 30% calorie restriction (CR). Data from real-time polymerase chain reaction (PCR) were normalized by the housekeeping gene 18s rRNA. n = 7 in N-CR group, and n = 8 in other groups. Data are expressed as means ± standard error. In each group, values marked with a different superscript (a, b, c) are significantly different (p <.05). A, Relative mRNA expression of adiponectin. B, Relative mRNA expression of leptin. C, Relative mRNA expression of resistin. D, Relative mRNA expression of tumor necrosis factor (TNF)-{alpha}

 
Ames df/df genotype did not affect the leptin expression in WAT. CR reduced leptin expression in normal mice (p <.05) and appeared to lead to a change in the same direction in df/df mice (p =.08; Figure 1). However, plasma leptin levels were not affected by genotype or by CR in normal mice, and were increased by CR in df/df mice (p <.005; Table 2).

CR significantly increased the expression of resistin mRNA in normal mice (p <.05; Figure 1) as well as the plasma resistin levels in both normal and df/df mice (both p <.0001; Table 2). There were no differences between genotypes.

WAT TNF-{alpha} expression was reduced significantly in df/df mice (p <.05). IL-6 expression was measurable in normal animals but too low to be detected in df/df mice. CR numerically inhibited the TNF-{alpha} expression (p =.12) in normal mice, but had no effect in df/df mice (Figure 1). Plasma TNF-{alpha} and IL-6 levels were below the detectability limits of the assays used (Table 2). Because plasma TNF-{alpha} and IL-6 levels are mainly derived from WAT, we measured the TNF-{alpha} and IL-6 protein levels in epididymal white fat pads. We found that their levels were decreased by CR in df/df mice (p =.05 and.003, respectively) but not altered in normal animals (Figure 2).


Figure 02
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  		Figure 2. Tumor necrosis factor (TNF)-alpha (A) and interleukin-6 (IL-6) (B) protein levels in epididymal white adipose tissue (WAT) from normal (N) and Ames dwarf (df/df) mice fed ad libitum (AL) or subjected to 30% calorie restriction (CR), as assayed by enzyme-linked immunosorbent assay (ELISA). n = 6 per group, and each sample was measured in duplicate. Group values are expressed to the mean ± standard error. In each group, values marked with a different superscript (a, b) are significantly different (p <.05)

 
Effects of Genotype and CR on Lipid Metabolism-Related Enzymes and Related Transcription Factors in WAT
LPL is a key enzyme responsible for the transportation of fatty acid from plasma very low-density lipoprotein (VLDL) to adipocytes. The expression of LPL mRNA was numerically, but not significantly, reduced in df/df mice (p =.13). mRNA expression of fatty acid synthase (FAS), which is a major enzyme involved in triglyceride synthesis in adipocytes, was significantly reduced in df/df mice as compared to normal mice (p <.005, Figure 3), whereas CR greatly elevated its expression in df/df mice (p <.001) and numerically elevated its expression in normal mice (p =.1). Neither df/df genotype nor diet affected the expression of hormone-sensitive lipase mRNA in WAT.


Figure 03
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  		Figure 3. Relative messenger RNA (mRNA) expressions of lipid metabolism-related enzymes and transcription factors in epididymal white adipose tissue (WAT) from normal (N) and df/df mice fed ad libitum (AL) or subjected to 30% calorie restriction (CR). Data from real-time polymerase chain reaction (PCR) were normalized by the housekeeping gene 18s rRNA. n = 7 in the N-CR group, and n = 8 in other groups. Data are expressed as means ± standard error. In each group, values marked with a different superscript (a, b, c) are significantly different (p <.05). A, Relative mRNA expression of lipoprotein lipase (LPL). B, Relative mRNA expression of fatty acid synthase (FAS). C, Relative mRNA expression of hormone-sensitive lipase (HSL). D, Relative mRNA expression of peroxisome proliferator-activated receptor-{gamma} (PPAR-{gamma}). E, Relative mRNA expression of sterol regulatory element binding protein-1 (SREBP-1)

 
The mRNA expression of PPAR-{gamma}, the key transcription factor regulating lipogenesis in WAT, was numerically reduced in df/df mice (p =.11). The expression of SREBP-1, another important lipogenic transcription factor, was also numerically reduced by df/df genotype (p =.16, Figure 3). Expression of PPAR-{gamma} and SREBP-1 was not affected by CR in either genotype.

Effects of Genotype and CR on Plasma FFA and Tissue Triglyceride Levels
The plasma FFA levels were greatly reduced in df/df mice regardless of the diet (p <.05); CR reduced the FFA in normal mice but had no effect in df/df mice (p <.01; Table 2). Moreover, df/df mice had much lower plasma triglyceride levels compared with their normal siblings (p <.0005; Table 3). CR reduced plasma triglyceride in normal mice (p <.05), but elevated its levels in df/df mice (p <.05). Consistent with reduced plasma triglyceride levels, df/df mice also had a reduced WAT triglyceride concentration (p <.05; Table 3). WAT triglyceride level was elevated by CR in df/df mice (p <.05), but not in normal animals. In contrast to the results from plasma and WAT, we found that the liver triglycerides were significantly elevated in df/df mice (p <.001) and that CR dramatically reduced their levels in both normal and df/df mice (p <.05 and.0005, respectively; Table 3). Neither df/df genotype nor CR altered the muscle triglyceride levels.


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  		Table 3. Plasma and Tissue Triglycerides in Normal (N) and Ames df/df Mice Subjected to Calorie Restriction (CR).

 
Effects of Genotype and CR on Expression of Genes Related to Thermogenesis and Glucose Transport in WAT
UCP2 is the main protein involved in uncoupling mitochondria substrate oxidation and ATP production in white fat pads, and thus regulates the energy expenditure. UCP2 mRNA expression in WAT was reduced in df/df mice (p <.01; Figure 4), and CR numerically (but not significantly) reduced its expression in both normal and df/df mice. GLUT4 is the key transporter responsible for the glucose uptake by muscle and fat tissues. GLUT4 mRNA expression in WAT was dramatically elevated by CR in normal mice (p <.0001; Figure 4), but was not affected by genotype.


Figure 04
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  		Figure 4. Relative messenger RNA (mRNA) expression of thermogenic factors and glucose transporter in epididymal white adipose tissue (WAT) from normal (N) and df/df mice fed ad libitum (AL) or subjected to 30% calorie restriction (CR). The data from real-time polymerase chain reaction (PCR) were normalized by the housekeeping gene 18s rRNA. n = 7 in the N-CR group, and n = 8 in other groups. Data are expressed as means ± standard error. In each group, values marked with a different superscript (a, b, c) are significantly different (p <.05). A, Relative mRNA expression of uncoupling protein 2 (UCP2). B, Relative mRNA expression of glucose transporter4 (GLUT4)

 

   DISCUSSION
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 Methods
 Results
 Discussion
References
 
Ames dwarf mice (35) are homozygous for a mutation of the Prop 1 gene (36). They display primary deficiency of GH, prolactin (PRL), and thyroid stimulating hormone (TSH), improved insulin sensitivity, diminutive body size, and prolonged longevity (37). CR, which delays aging, extends longevity, and improves insulin sensitivity, further extends the life span of Ames df/df mice (38). The purpose of this study was to investigate the possible mechanisms of improved insulin sensitivity in Ames df/df and CR mice and to evaluate the effects of df/df genotype and CR on the adipocytokines and parameters related to lipid metabolism.

As expected, df/df mice displayed the reduced body weight and delayed growth that are consistent with the primary GH deficiency and the consequent lack of circulating insulin-like growth factor 1 (IGF-1) (39). The 30% CR significantly reduced body weight and delayed growth in normal and df/df mice, but when those mice were killed, CR had no significant effect on the body weight of df/df mice (34).

Glucose and insulin levels were reduced in dwarf mice by 23% and 82%, respectively, compared to the normal controls. This confirms our previous findings (39) and indicates that the insulin sensitivity was greater in dwarf mice. There was no difference in glucose levels between N-AL and N-CR mice. Plasma insulin levels were numerically reduced in N-CR mice compared to N-AL mice. This finding suggests that insulin sensitivity may have been improved by CR in normal animals. Dwarf mice subjected to CR had increased glucose and insulin compared to dwarf mice subjected to AL, perhaps due to those animals being fasted before death, whereas the CR animals were already adapted to lack of food during most of the night. Increased insulin levels provide a plausible explanation for the previously reported reduction in the IR protein levels in the liver from the same df/df CR mice (34). These findings suggest that, under conditions of this experiment, CR might reduce rather than enhance the responsiveness to insulin in these highly insulin-sensitive mice. This reduction might serve as a protective mechanism preventing hypoglycemia under conditions of limited availability of nutrients.

Our results indicate that the improved insulin sensitivity in dwarf mice compared to normal mice has a close relationship with the alterations of adipocytokine profile and lipid metabolism. Adiponectin, the plasma protein that plays a key role in the control of insulin sensitivity, was significantly elevated in dwarf mice as compared to normal mice. The expression of TNF-{alpha}, a hormone involved in insulin resistance, was dramatically decreased in WAT from dwarf mice. Moreover, the expression of LPL and FAS in WAT was reduced in dwarf mice. LPL, which is synthesized in adipocytes, is an enzyme involved in hydrolyzing the plasma triglycerides from chylomicrons or lipoproteins to fatty acid and glycerol. The fatty acids are transported to adipose tissue by fatty acid transport proteins (FATP), and then with the action of FAS, the FFAs are re-esterified to triglycerides in adipose tissue (40). PPAR-{gamma} and SREBP, two lipogenic transcription factors that regulate the expression of LPL and FAS, were also numerically reduced in dwarf mice. These results suggest that lipogenesis is reduced in dwarf adipose tissue. Young adult male df/df mice have a lower percentage of body fat compared with their normal littermates (31). We are tempted to speculate that the down-regulated lipid synthesis in df/df mice may prevent the accumulation of fat in these animals. This might be one of the adaptive responses to the effects of GH deficiency and hypothyroidism on fat accumulation. The reduced accumulation of triglycerides in dwarf adipose tissue found in this study supports this speculation and might contribute to the smaller size of adipocytes in dwarf mice (W. Zhihui, unpublished observations, 2004). Compared to those of normal animals, adipocytes of Ames dwarfs are presumably more sensitive to the action of insulin and release more insulin-sensitizing adipocytokines. In addition, the reduced plasma triglycerides and FFA may contribute to the improved insulin sensitivity in dwarf mice. Increases in circulating triglycerides and FFA are known to lead to both peripheral and hepatic insulin resistance by impairing insulin signaling pathways (41).

High accumulation of triglycerides in nonadipose tissue and especially in skeletal muscle is usually linked to insulin resistance. In the present study, liver triglyceride in dwarf mice was found significantly elevated, consistent with our findings of increased mRNA expression and protein levels of lipogenic PPAR-{gamma} in the liver (34), but we did not find any differences in muscle triglyceride levels. These results contrast with the improved insulin signaling in dwarf mice liver, suggested by the increased IR protein content, insulin-stimulated phosphorylation of IR and IRS, and the association of P85 regulatory subunit of PI3K and IRS-1 (42). We found that CR reduced liver triglycerides significantly in dwarf mice and numerically in normal mice (Table 3), consistent with the well-documented ability of CR to promote insulin sensitivity. This finding suggests that the elevated liver triglycerides in dwarf mice may represent energy storage to prevent the hypoglycemia when nutrients are in short supply. Further studies are needed to completely elucidate the role of liver triglyceride accumulation and metabolism in the control of insulin signaling pathway in these animals.

Our results suggest that the effects of CR on normal mice are associated with the expected improvement in insulin sensitivity. CR tended to decrease the expression of TNF-{alpha} in adipose tissue and it greatly reduced the plasma triglycerides and FFA levels as well as the liver triglycerides accumulation, most likely due to the decreased triglyceride synthesis. In this way CR would reduce the inhibitory effect of the high triglycerides and FFA on insulin signaling pathway. Reports concerning the effects of resistin are controversial. Resistin has been considered by some investigators as a hormone that induces insulin resistance. However, recent reports showed that resistin expression is severely suppressed in WAT in several types of obese, insulin-resistant animals such as ob/ob, db/db, tub/tub, KKAy, and diet-induced obese mice, and is stimulated by PPAR-{gamma} agonists in two standard rodent models of type 2 diabetes (12). This has cast some doubt on the hypothesis linking resistin with insulin resistance. In the present study, we found that the levels of resistin were not altered in dwarf as compared to normal mice, but CR dramatically elevated plasma resistin levels in both genotypes. The role of resistin in obesity and type 2 diabetes is still controversial and remains to be elucidated.

The effects of CR on dwarf mice are difficult to interpret. We found that CR elevated plasma adiponectin and resistin levels, reduced the TNF-{alpha} and IL-6 protein levels in adipose tissue, and decreased liver triglyceride content. Those results suggest that CR might improve insulin sensitivity in dwarf mice. However, the plasma triglyceride levels and triglyceride accumulation in WAT were elevated by CR in dwarf mice. This elevation might be due to the increased plasma insulin levels and elevated FAS expression in adipose tissue of df/df-CR animals observed in the present study. Insulin has been shown to decrease fatty acid oxidation and increase triglyceride synthesis. In addition, CR tended to decrease the expression of UCP2 in dwarfs and consequently may have caused decreased thermogenesis, which in turn may have contributed to the elevated triglycerides. These alterations in triglyceride metabolism might dampen the responsiveness of highly insulin-sensitive dwarf mice to the action of insulin, thus preventing hypoglycemia under conditions of CR.

Summary
The present study indicates that the improved insulin sensitivity in Ames dwarf mice may be due to the increased release of adiponectin and reduced release of TNF-{alpha} and IL-6. Altered secretion of these adipocytokines might be consequent to the decreased lipid synthesis and reduced plasma triglycerides and FFA levels. Our results also suggest that CR may differentially affect insulin sensitivity of normal and dwarf mice. In normal mice, CR improves the insulin sensitivity by affecting the release of adipocytokines and decreasing circulating fatty acid concentration, triglyceride concentration, and liver triglyceride accumulation. However, in highly insulin-sensitive dwarf mice, CR may reduce rather than enhance some aspects of the responsiveness to insulin. Further studies will be necessary to determine how these responses are related to extension of life span by CR in both normal and Ames dwarf mice.


   Acknowledgments
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 Results
 Discussion
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This study was supported by grants from the National Institute on Aging (AG-198899 and U19 AG023122), as well as by the Ellison Medical Foundation.

We thank Marty Wilson, Michael Bonkowski, and Jacob Panici for expert technical assistance.

Khalid A. Al-Regaiey is now with the Department of Physiology, College of Medicine, King Saud University, Riyadh, Saudi Arabia.


   Footnotes
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Decision Editor: James R. Smith, PhD

Received July 1, 2005

Accepted September 19, 2005


   References
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