Adenovirus-Mediated High Expression of Resistin Causes Dyslipidemia in Mice
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内分泌学杂志 2005年第1期
Department of Medicine and Bioregulatory Science, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan
Address all correspondence and requests for reprints to: Kunihisa Kobayashi, Department of Medicine and Bioregulatory Science, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. E-mail: nihisak@intmed3.med.kyushu-u.ac.jp.
Abstract
The adipocyte-derived hormone resistin has been proposed as a possible link between obesity and insulin resistance in murine models. Many recent studies have reported physiological roles for resistin in glucose homeostasis, one of which is enhancement of glucose production from the liver by up-regulating gluconeogenic enzymes such as glucose-6-phosphatase and phosphoenolpyruvate carboxykinase. However, its in vivo roles in lipid metabolism still remain to be clarified. In this study, we investigated the effects of resistin overexpression on insulin action and lipid metabolism in C57BL/6 mice using an adenoviral gene transfer technique. Elevated plasma resistin levels in mice treated with the resistin adenovirus (AdmRes) were confirmed by Western blotting analysis and RIAs. Fasting plasma glucose levels did not differ between AdmRes-treated mice and controls, but the basal insulin concentration was significantly elevated in AdmRes-treated mice. In AdmRes-treated mice, the glucose-lowering effect of insulin was impaired, as evaluated by insulin tolerance tests. Furthermore, total cholesterol and triglyceride concentrations were significantly higher, whereas the high-density lipoprotein cholesterol level was significantly lower. Lipoprotein analysis revealed that low-density lipoprotein was markedly increased in AdmRes-treated mice, compared with controls. In addition, in vivo Triton WR-1339 studies showed evidence of enhanced very low-density lipoprotein production in AdmRes-treated mice. The expressions of genes involved in lipoprotein metabolism, such as low-density lipoprotein receptor and apolipoprotein AI in the liver, were decreased. These results suggest that resistin overexpression induces dyslipidemia in mice, which is commonly seen in the insulin-resistant state, partially through enhanced secretion of lipoproteins.
Introduction
OBESITY IS AN epidemic health hazard in industrialized countries and is strongly associated with increased prevalence of type 2 diabetes, hypertension, dyslipidemia, and atherosclerosis (1, 2). Although many epidemiological studies have suggested that increased adiposity predisposes to insulin resistance, the molecular mechanisms underlying this connection still remain unknown.
Many adipose tissue-derived factors have been reported, some of which influence glucose and lipid metabolism, such as free fatty acids (3, 4), leptin (5), TNF (6, 7), adiponectin (8, 9), and resistin (10).
Resistin, the expression of which is down-regulated by thiazolidinedione and up-regulated in diet-induced obesity as well as in genetic models of obesity and insulin resistance, impaired insulin action and glucose tolerance in normal mice (11). Furthermore, resistin antagonized the insulin effect on glucose uptake in differentiated 3T3-L1 adipocytes (11) and skeletal muscle L6 cells (12), suggesting that resistin directly links obesity to diabetes.
However, some studies reported the opposite observations, namely that resistin expression was significantly decreased in the white adipose tissue of several different models of obesity, compared with their lean counterparts (13), and its expression level was increased by thiazolidinedione (13, 14) and therefore cast doubt on the in vivo roles of resistin. More recently, however, it was reported that, whereas resistin mRNA expression was indeed suppressed in obese mice, plasma resistin levels were elevated in these mice (15), suggesting that posttranscriptional processes are more important for regulating the plasma resistin level.
Another group reported that resistin enhanced glucose production from the liver by reducing insulin-mediated suppression of gluconeogenesis and increasing glycogenolysis in vivo, suggesting that it blunts insulin action in the liver (16). Recently Banerjee et al. (17) reported the phenotype of mice lacking resistin. In these mice, the fasting blood glucose level was lower than that in wild-type controls due to reduced hepatic glucose production, suggesting a physiological role for resistin in maintaining glucose homeostasis in mice. In addition, they reported that phosphorylation of AMP-activated protein kinase was abrogated in the liver of these mice, implying that this kinase may be an important regulator of resistin signaling as well as the signaling of other adipocytokines such as adiponectin (18) and leptin (19).
These observations showing resistin’s prodiabetogenic properties in rodents may not readily be applied to humans. Some studies have reported a positive link of resistin to obesity and insulin resistance in humans (20, 21, 22), but the role of resistin in human insulin resistance still remains controversial, partly because human resistin is abundantly expressed in circulating mononuclear cells rather than adipocytes (23).
Many studies have suggested that obesity may be a factor in causing dyslipidemia, which is partially mediated by insulin resistance (24). The insulin-resistant state is often associated with abnormal lipoprotein metabolism, including hypertriglyceridemia, high levels of very low-density lipoprotein (VLDL), low levels of high-density lipoprotein (HDL) cholesterol, and small dense low-density lipoprotein (LDL). Lipid turnover studies in humans and animal models of insulin resistance and obesity have shown that the dyslipidemia associated with these conditions is predominantly due to elevated VLDL production (25, 26). However, the molecular mechanisms linking obesity and insulin resistance to VLDL hypersecretion still remain to be clarified. TNF, which is an adipocyte-secreted hormone that impairs insulin action similarly to resistin (7), has been shown to affect lipid metabolism by stimulating hepatic lipogenesis in rats in vivo (27). As previously mentioned, resistin specially attenuates insulin action in the liver, and therefore it is predicted that resistin would also affect lipoprotein metabolism independently of obesity. To understand the role of resistin in lipoprotein metabolism in mice, we induced resistin protein overexpression in C57BL/6 mice using an adenovirus-mediated gene delivery system (28) and investigated its in vivo effects on the lipid profiles and underlying mechanisms by which it impairs lipid metabolism.
Materials and Methods
Seven-week-old male C57BL/6 mice were purchased from SLC (Fukuoka, Japan) and housed in cages under controlled lighting conditions in a natural dark-light cycle (1800–0600 h). The mice were allowed free access to a regular chow diet (KBT Oriental, Saga, Japan) and sterile water.
Cloning of mouse resistin cDNA
Total RNA was prepared from differentiated 3T3-L1 cells using Isogen (Nippon Gene, Tokyo, Japan) according to the manufacturer’s instructions. Random primer-primed cDNA (1 μg) from the total RNA was used as a template for PCR cloning. The primers used were based on the mouse resistin nucleotide sequence (GenBank accession no. AF323080) as follows: 5'-CGGAATTCGGGATGAAGAACCTTTCATTTCCC-3' and 5'-CGGGATCCTCAGGAAGCGACCTGCAGCCTT-3'. After digestion with EcoRI and BamHI, the PCR fragment was subcloned into pBluescript KS (Stratagene, La Jolla, CA). A 6 x histidine tag was incorporated into the 3'-end of the resistin cDNA using fragment integration. The nucleotide sequences of the cloned cDNAs were determined by the dideoxy chain termination method using an ABI PRISM 377 DNA sequencer (Applied Biosystems Japan, Tokyo, Japan).
Preparation and administration of the resistin adenovirus
An adenovirus containing the full-length mouse resistin cDNA with the C-terminal 6 x histidine tag (AdmRes) was prepared using a commercial kit (Clontech, Palo Alto, CA). AdmRes and a control adenovirus containing ?-galactosidase (AdLacZ) were propagated in human embryonic kidney 293 cells, purified by CsCl gradient centrifugation, and stored at –80 C until use.
Sample preparation and Western blotting analysis
The 1 x 109 plaque-forming units/mouse of AdmRes and AdLacZ were injected into 8-wk-old mice via the internal jugular vein after anesthetization by an ip sodium pentobarbital injection as described previously (29).
Five days after the AdmRes or AdLacZ injection, blood was collected into tubes containing EDTA by puncturing the retroorbital plexus of anesthetized mice. After centrifugation, plasma samples were supplemented with NaN3 and phenylmethylsulfonyl fluoride and stored at 4 or –20 C.
Plasma samples (1 μl) of each mouse were boiled for 5 min in a sample buffer supplemented with 2% 2-mercaptoethanol. Samples were separated in 10 or 10–20% SDS-PAGE gels (Bio-Rad Laboratories Japan, Tokyo, Japan). The proteins were transferred to 0.2 μm nitrocellulose (Trans-Blot; Bio-Rad) or 0.2 μm polyvinyl difluoride membranes (Immun-Blot; Bio-Rad) in Tris/glycine buffer for 60 min at 60 mA, and the membranes were probed with primary antibodies diluted in appropriate buffers. Two kinds of IgG purified from a rabbit polyclonal antiserum against resistin (kindly provided by Dr. Tomoichiro Asano, University of Tokyo, and Linco Research, St. Charles, MO) were each used at a dilution of 1:1000. The secondary antibody (mouse antirabbit IgG-horseradish peroxidase; Amersham Biosciences, Piscataway, NJ) was used at a dilution of 1:5000. The signal was detected by a chemiluminescent reaction (ECL Plus; Amersham Biosciences), and the intensity was determined using a densitometer. All membranes were stained with Ponceau S to verify the quality of transfer and equivalent protein loading.
Measurements
Plasma total cholesterol, triglyceride, HDL cholesterol, and free fatty acid (FFA) concentrations were determined enzymatically. Plasma glucose levels were determined using the glucose oxidase method (glucose B test; Wako, Osaka, Japan). Immunoreactive insulin concentrations were determined using an ELISA kit (Shibayagi, Gunma, Japan). Plasma levels of resistin and adiponectin were measured using a RIA (Linco Research) and ELISA kit (Otsuka Pharmaceutical Co, Tokyo, Japan), respectively.
Lipoprotein analysis
Equal amounts of plasma samples (200 μl) were pooled from each mouse of PBS-treated, AdLacZ-treated, and AdmRes-treated group on d 5 (n = 7, respectively). Pooled samples (1000 μl) of each group were fractionated by sequential ultracentrifugation (30) at densities of less than 1.006 (VLDL), 1.006–1.019 (intermediate-density lipoprotein), 1.019–1.063 (LDL), and 1.063–1.21 g/ml (HDL). The lipoprotein fractions were then dialyzed and used for lipid analyses.
Insulin tolerance test
Insulin tolerance tests using 1 U/kg body weight of human regular insulin were conducted on AdmRes- or AdLacZ-treated mice after at least 6 h of fasting on d 5. At the indicated times after the injection, blood was drawn into tubes containing EDTA.
Triglyceride secretion rate (TGSR)
The TGSR was determined on d 5 using Triton WR-1339 (Tyloxapol; AMEND, Irvington, NJ) as described by Hirano et al. (31). Blood samples were collected at 0, 30, 60, and 90 min after iv injection of WR-1339 into mice. After checking the linearity (r > 0.98) of the plasma triglyceride increase in each mouse, the TGSR was calculated as the triglyceride increase per minute standardized by the mouse plasma volume.
RNA extraction and quantitative real-time PCR
Total RNA was extracted from approximately 30 mg tissue using Isogen and stored at –80 C for later analysis. The extracted RNA (5 μg) was converted to single-stranded cDNAs by a reverse transcriptase procedure with Superscript II (Invitrogen, Carlsbad, CA). The mRNA levels were quantified by real-time PCR using a LightCycler (Roche Diagnostics GmbH, Mannheim, Germany). Briefly, 1 μl of the cDNAs was placed in a 20-μl reaction volume containing 1 μl of each primer and 2 μl LightCycler-FastStartDNA Master SYBR Green I (Roche Diagnostics GmbH). Nucleotides, Taq DNA polymerase, and buffer were already included in the LightCycler-FastStartDNA Master SYBR Green I. The thermal cycling conditions comprised an initial denaturation step at 95 C (10 min), followed by 40 cycles of 95 C (0 sec), 60 C (15 sec) for ?-actin, apolipoprotein (Apo)E, ApoB, and ApoAI; 5 sec for lipoprotein lipase (LPL), microsomal triglyceride transfer protein (MTP), and LDL receptor); and 72 C (36 sec for ?-actin, ApoE, ApoB, and ApoAI; 15 sec for LPL, MTP, and LDL receptor).
The sense/antisense primers used were: LDL receptor (GenBank accession no. NM_010700), 5'-GAAGTCGACACTGTACTGACCACC-3' (nucleotide position 2143–2167)/5'-CTCCTCATTCCCTCTGAAAGCCAT-3' (nucleotide position 2329–2352) (32); LPL (GenBank accession no. NM_008509), 5'-AGTAGACTGGTTGTAT CGGG-3' (nucleotide position 529–548)/5'-AGCGTCATCAGGAGAAAGG-3' (nucleotide position 790–808); ?-actin (GenBank accession no. NM_007393), 5'-ACTGGGACGACATGGAGAAG-3' (nucleotide position 313–332)/5'-GGGGTGTT GAAGGTCTCAAA-3' (nucleotide position 450–469); MTP (GenBank accession no. NM_008642), 5'-TGAGCGGCTATACAAGCTCAC-3' (nucleotide position 114–134)/5'-CTGGAAGATGCTCTTCTCGC-3' (nucleotide position 314–333) (33); ApoB (GenBank accession no. XM_137955), 5'-GCCCATTGTGGACAAGTT GATC-3' (nucleotide position 2430–2451)/5'-CCAGGACTTGGAGGTCTTGGA-3' (nucleotide position 2531–2551) (34); ApoE (GenBank accession no. NM_009696), 5'-TGGGAGCAGGCCCTGAAACCGCTTC-3' (nucleotide position 151–174)/5'-G AGTCGGGCCTGTGCCGCCCTGCAC-3' (nucleotide position 364–387); and ApoAI (GenBank accession no. NM_012738), 5'-GGCAGAGACTATGTGTCCCAGTTT GA-3' (nucleotide position 188–213)/5'-GTCATCAGCGCGGGTTTGGCCTTCTC-3' (nucleotide position 707–733) (35). Threshold values were obtained when the fluorescent intensity was in the geometric phase of amplification, as determined by the LightCycler software (version 3.5). Products were verified by electrophoresis in 2% agarose gels.
Statistical analysis and ethical considerations
Values are presented as mean ± SEM unless otherwise indicated. Results were analyzed with Statview version 5 (SAS Institute Inc., Cary, NC) using unpaired Student’s t test or one-way ANOVA followed by comparisons using Bonferroni’s method. P < 0.05 denoted the presence of a statistically significant difference. The experimental protocol was approved by the Ethics Review Committee for Animal Experimentation of Kyushu University.
Results
High levels of resistin were present in plasma from AdmRes-treated mice
To confirm the protein expression of resistin, Western blotting analysis was performed under nonreducing and reducing conditions. Under nonreducing conditions, we detected a protein band of approximately 20,000 Mw in plasma samples from AdmRes-treated mice but not in those from AdLacZ-treated mice (Fig. 1A). This size was the same as that of a resistin dimer (36). Under reducing conditions, a protein band of approximately 10,000 Mw (monomer) was detected in the plasma samples from all groups (Fig. 1B). Quantification of the plasma resistin showed that the concentration was 45- to 50-fold higher in AdmRes-treated mice than in PBS- or AdLacZ-treated mice at d 5 (Table 1). The plasma resistin protein induced by AdmRes was detected at d 1, strongest at d 5, and then became weaker but could be still detected at d 9 (Fig. 1C).
FIG. 1. Western blotting analysis of plasma from PBS-, AdLacZ-, and AdmRes-treated mice at d 5 and time course of plasma resistin concentrations induced by AdmRes. Mice were iv injected with AdLacZ or AdmRes (1 x 109 plaque-forming units). Mouse plasma samples (1 μl) and recombinant resistin were separated in SDS-PAGE gels under nonreducing (A) and reducing (B) conditions and processed for immunoblotting analysis as described in Materials and Methods. C, Plasma samples were collected on the indicated day after AdmRes injection, and resistin concentrations were measured using a RIA kit (n = 4, respectively). The concentration at d 0 was 4.0 ± 0.2 ng/ml. *, P < 0.05 vs. d 0.
TABLE 1. Fasting plasma resistin and lipid levels in Mock-, AdLacZ-, and AdmRes-treated mice
We also investigated whether the secretion of other proteins from the liver was affected. The plasma levels of albumin did not differ between AdmRes-treated mice (2.4 ± 0.1 g/dl, n = 6) and AdLacZ-treated mice (2.5 ± 0.1 g/dl, n = 6). The plasma levels of insulin-sensitizing hormone, adiponectin, were the same for the two groups (AdmRes-treated mice, 18.72 ± 0.83 μg/ml, n = 4; AdLacZ-treated mice, 20.20 ± 0.82 μg/ml, n = 5).
Resistin caused attenuated insulin action
Plasma glucose (Fig. 2A) and insulin (Fig. 2B) levels were measured on d 5 after a 16-h fast. Fasting plasma glucose levels did not differ between AdmRes-treated and AdLacZ-treated mice, but the basal insulin concentration was significantly higher in AdmRes-treated mice than in controls. In insulin tolerance tests, the two groups showed a similar decrease in glucose until 30 min after the insulin injection. The glucose-lowering effect of insulin after 30 min was impaired in AdmRes-treated mice (Fig. 2C). The body weights were not significantly different between these two groups on d 5 (AdmRes-treated mice, 19.11 ± 0.30 g, n = 5; AdLacZ-treated mice, 19.14 ± 0.45 g, n = 5).
FIG. 2. Fasting plasma glucose and insulin concentrations and insulin tolerance tests in AdLacZ- and AdmRes-treated mice. Plasma glucose (A) and insulin (B) levels were measured on d 5 after a 16-h fast (n = 5–6). C, After ip injection of insulin (1 U/kg), blood samples were obtained at the indicated times from AdLacZ-treated (open circles, n = 5) and AdmRes-treated (closed circles, n = 5) mice. Initial absolute plasma glucose levels were not significantly different between AdmRes-treated mice (5.25 ± 0.62 mmol/liter) and AdLacZ-treated mice (6.74 ± 0.31 mmol/liter). Values are the mean ± SE of the percentage of the baseline glucose. *, P < 0.05.
Increased LDL in plasma from AdmRes-treated mice
As shown in Table 1, total cholesterol and triglyceride concentrations on d 5 were significantly higher in AdmRes-treated mice than controls. On the other hand, the HDL cholesterol level was significantly lower in AdmRes-treated mice. The non-HDL cholesterol (total cholesterol/HDL cholesterol) level was markedly higher in AdmRes-treated mice than controls. There were no significant differences in the plasma FFA levels among the three groups.
The cholesterol, triglyceride, and phospholipids in each lipoprotein fraction were determined using pooled plasma samples of each group (n = 7, respectively). LDL cholesterol was markedly increased in AdmRes-treated mice, compared with the other groups. The triglyceride and phospholipids in VLDL were slightly increased in AdmRes-treated mice (Fig. 3).
FIG. 3. Cholesterol (A), triglyceride (B), and phospholipid (C) contents of lipoproteins in PBS-, AdLacZ-, and AdmRes-treated mice. Each sample represents plasma pooled from mice treated with PBS (open columns), AdLacZ (hatched columns), or AdmRes (closed columns) and separated by sequential ultracentrifugation as described in Materials and Methods. IDL, Intermediate-density lipoprotein.
Plasma total cholesterol was increased in a time-dependent manner
The total cholesterol level was measured before and at 3, 5, and 8 d after the adenovirus injection. The cholesterol level in AdmRes-treated mice increased in a time-dependent manner. The cholesterol level was significantly higher in AdmRes-treated mice than the other groups after d 3 (Fig. 4).
FIG. 4. Plasma cholesterol changes in PBS-, AdLacZ-, and AdmRes-treated mice. Plasma cholesterol concentrations were measured in PBS-, AdLacZ-, and AdmRes-treated mice at various time points (days) before and after injection of PBS, AdLacZ, or AdmRes. Values are the mean ± SE (n = 6–8). *, P < 0.05.
Increased hepatic VLDL production rates in AdmRes-treated mice
To determine the mechanism of the dyslipidemia, we investigated the VLDL production rates in the livers of the mice. After an iv WR-1339 injection, AdmRes-treated mice showed a significantly higher TGSR than AdLacZ-treated mice (Fig. 5).
FIG. 5. Effect of resistin on the TGSR in AdLacZ- and AdmRes-treated mice. The TGSR was determined by measuring the increase in the plasma triglyceride concentration after an iv injection of Triton WR-1339. The TGSR is expressed as milligrams per minute. Data represent the mean + SE (n = 6).
Expressions of genes involved in lipoprotein metabolism were decreased in the muscle and liver of AdmRes-treated mice
The expressions of LDL receptor and ApoAI were significantly decreased in the liver of AdmRes-treated mice, compared with the those in the liver of AdLacZ-treated mice, and these may be causes of the high LDL and low HDL concentrations in the plasma of AdmRes-treated mice, respectively (Fig. 6). LPL expression was decreased in the muscle of AdmRes-treated mice, compared with that in the muscle of AdLacZ-treated mice, but the difference was not significant. The expressions of the genes encoding ApoB, ApoE, and MTP involved in VLDL assembly and secretion in the liver did not differ between the two groups.
FIG. 6. Relative gene expression levels of LDL receptor, ApoAI, ApoB, ApoE, and MTP in the liver and LPL expression in the muscle of AdLacZ- and AdmRes-treated mice at 5 d after the adenovirus injection. Total RNA was extracted from the liver and muscle, and single-stranded cDNAs were created as described in Materials and Methods. The mRNA levels were quantified by real-time PCR using a LightCycler. Gene levels are shown relative to the ?-actin expression level. Data are the mean + SE (n = 6).
Discussion
Resistin, also known as ADSF (37) and FIZZ3 (38), was originally reported as a possible link between obesity and diabetes mellitus (11). Recently it has been shown that resistin administration causes insulin resistance in the liver and glucose intolerance and that transgenic mice also have these characteristics (16, 40). Furthermore, ablation of resistin in mice impaired hepatic glucose production, suggesting a physiological function in maintaining glucose homeostasis in vivo (17). However, the in vivo effects of resistin on lipid metabolism still remain to be clarified. The aim of our study was to determine whether subchronic overexpression of resistin, using an adenovirus technique, impaired insulin action and subsequently affected lipoprotein metabolism in vivo.
The plasma glucose level in the fasted state was similar between AdmRes-treated mice and controls, whereas the insulin level was higher in AdmRes-treated mice than controls on d 5 after injection. In the insulin tolerance test, the glucose-lowering effect of insulin was impaired in AdmRes-treated mice. These results show subtle differences from previously reported transgenic models of resistin, which had similar fasting insulin levels and insulin tolerance to their controls (40). On the other hand, when recombinant resistin was administered to C57BL/6J mice, the mice showed an increased peak blood glucose level during glucose tolerance testing and a mildly attenuated insulin action during insulin tolerance testing, even though they showed no significant differences in the plasma glucose and insulin levels in the fasted state (11). The reason for these discrepant observations is probably differences in the degree, duration, or method of overexpression of this hormone.
The plasma cholesterol and triglyceride levels were significantly higher in AdmRes-treated mice than controls. Although most plasma cholesterol belongs to the HDL fraction in normal mice (41), AdmRes-treated mice showed a significantly lower HDL cholesterol level than the controls, and the calculated non-HDL cholesterol level was strikingly increased in AdmRes-treated mice. According to the plasma lipoprotein fractionation analysis, this increase in non-HDL cholesterol was mainly derived from elevated LDL cholesterol. These lipid profiles are consistent with those commonly observed in the insulin-resistant state in humans and diabetic mouse models such as C57BL/KsJ-db/db mice (42). These findings are particularly interesting because our experimental model was wild-type nonobese C57BL/6 mice fed on normal chow, suggesting that subchronic overexpression of resistin can affect lipid metabolism independently of obesity or a high fat diet.
Next, we investigated several factors that affect lipoprotein metabolism, such as VLDL secretion from the liver, LPL expression in the muscle, and LDL receptor and ApoAI expression in the liver. Previous kinetics studies suggested that the hypertriglyceridemia associated with insulin resistance was due to an increase in VLDL-triglyceride production by the liver (43, 44). The results of the WR-1339 study showed that the TGSR from the liver was significantly higher in AdmRes-treated mice than controls. It is possible that a moderate, but subchronic, increase in the TGSR could induce marked changes in the lipid profiles in AdmRes-treated mice. The time-dependent increase in cholesterol observed in AdmRes-treated mice may support this speculation. The expressions of genes involved in VLDL assembly and secretion, such as ApoB, ApoE, and MTP, did not differ between the two groups, consistent with the observations in a study on ob/ob mice (34). Insulin has been shown to acutely inhibit hepatic production of VLDL in both in vitro and in vivo studies (25, 45). Interestingly, however, chronically hyperinsulinemic and insulin-resistant obese human subjects and ob/ob mice were resistant to the inhibitory effects of insulin on VLDL production (34, 46). From the current results, it is possible that resistin is one of the factors that causes VLDL overproduction in obese subjects either by attenuating insulin action in the liver or by itself.
In the liver of AdmRes-treated mice, the expression of LDL receptor was decreased by 42%, compared with that in the liver of AdLacZ-treated mice, as estimated by quantitative real-time PCR analysis. Because the expression level of LDL receptors in the liver has been shown to be one of the major determinants of the plasma LDL level (47), this may also result in an increased level of plasma LDL. Similarly, the expression level of ApoAI in the liver of AdmRes-treated mice was decreased by 67%,compared with that in the liver of controls, which might have caused the low plasma HDL level in these mice.
We did not detect any significant differences in the FFA values among the three groups, whereas Pravenec et al. (39) reported that fat-specific resistin transgenic spontaneous hypertensive rats showed a higher serum FFA level. This difference may be due to differences in the dietary conditions (normal chow vs. a diet with 60% fructose), species, and/or genetic background. In addition, it should be noted that our model showed subchronic overexpression of plasma resistin, which is different from their transgenic model.
Obesity-related dyslipidemia is multifactorial, but our present results suggest that, independently of obesity, subchronic overexpression of resistin has in vivo effects that cause similar lipid profiles to those in the insulin-resistant state in humans and some diabetic mouse models. These effects are probably related to the increased VLDL production by the liver and the low removal rate of lipoproteins, which are probably brought about by the decreased expression of LDL receptors in the liver. Whether resistin itself or the secondary attenuation of insulin action gives rise to these phenomena remains to be clarified in future studies.
Acknowledgments
The authors thank Dr. Takahiko Oho (Department of Preventive Dentistry, Kyushu University Faculty of Dental Science) for helpful assistance in assays.
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Address all correspondence and requests for reprints to: Kunihisa Kobayashi, Department of Medicine and Bioregulatory Science, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. E-mail: nihisak@intmed3.med.kyushu-u.ac.jp.
Abstract
The adipocyte-derived hormone resistin has been proposed as a possible link between obesity and insulin resistance in murine models. Many recent studies have reported physiological roles for resistin in glucose homeostasis, one of which is enhancement of glucose production from the liver by up-regulating gluconeogenic enzymes such as glucose-6-phosphatase and phosphoenolpyruvate carboxykinase. However, its in vivo roles in lipid metabolism still remain to be clarified. In this study, we investigated the effects of resistin overexpression on insulin action and lipid metabolism in C57BL/6 mice using an adenoviral gene transfer technique. Elevated plasma resistin levels in mice treated with the resistin adenovirus (AdmRes) were confirmed by Western blotting analysis and RIAs. Fasting plasma glucose levels did not differ between AdmRes-treated mice and controls, but the basal insulin concentration was significantly elevated in AdmRes-treated mice. In AdmRes-treated mice, the glucose-lowering effect of insulin was impaired, as evaluated by insulin tolerance tests. Furthermore, total cholesterol and triglyceride concentrations were significantly higher, whereas the high-density lipoprotein cholesterol level was significantly lower. Lipoprotein analysis revealed that low-density lipoprotein was markedly increased in AdmRes-treated mice, compared with controls. In addition, in vivo Triton WR-1339 studies showed evidence of enhanced very low-density lipoprotein production in AdmRes-treated mice. The expressions of genes involved in lipoprotein metabolism, such as low-density lipoprotein receptor and apolipoprotein AI in the liver, were decreased. These results suggest that resistin overexpression induces dyslipidemia in mice, which is commonly seen in the insulin-resistant state, partially through enhanced secretion of lipoproteins.
Introduction
OBESITY IS AN epidemic health hazard in industrialized countries and is strongly associated with increased prevalence of type 2 diabetes, hypertension, dyslipidemia, and atherosclerosis (1, 2). Although many epidemiological studies have suggested that increased adiposity predisposes to insulin resistance, the molecular mechanisms underlying this connection still remain unknown.
Many adipose tissue-derived factors have been reported, some of which influence glucose and lipid metabolism, such as free fatty acids (3, 4), leptin (5), TNF (6, 7), adiponectin (8, 9), and resistin (10).
Resistin, the expression of which is down-regulated by thiazolidinedione and up-regulated in diet-induced obesity as well as in genetic models of obesity and insulin resistance, impaired insulin action and glucose tolerance in normal mice (11). Furthermore, resistin antagonized the insulin effect on glucose uptake in differentiated 3T3-L1 adipocytes (11) and skeletal muscle L6 cells (12), suggesting that resistin directly links obesity to diabetes.
However, some studies reported the opposite observations, namely that resistin expression was significantly decreased in the white adipose tissue of several different models of obesity, compared with their lean counterparts (13), and its expression level was increased by thiazolidinedione (13, 14) and therefore cast doubt on the in vivo roles of resistin. More recently, however, it was reported that, whereas resistin mRNA expression was indeed suppressed in obese mice, plasma resistin levels were elevated in these mice (15), suggesting that posttranscriptional processes are more important for regulating the plasma resistin level.
Another group reported that resistin enhanced glucose production from the liver by reducing insulin-mediated suppression of gluconeogenesis and increasing glycogenolysis in vivo, suggesting that it blunts insulin action in the liver (16). Recently Banerjee et al. (17) reported the phenotype of mice lacking resistin. In these mice, the fasting blood glucose level was lower than that in wild-type controls due to reduced hepatic glucose production, suggesting a physiological role for resistin in maintaining glucose homeostasis in mice. In addition, they reported that phosphorylation of AMP-activated protein kinase was abrogated in the liver of these mice, implying that this kinase may be an important regulator of resistin signaling as well as the signaling of other adipocytokines such as adiponectin (18) and leptin (19).
These observations showing resistin’s prodiabetogenic properties in rodents may not readily be applied to humans. Some studies have reported a positive link of resistin to obesity and insulin resistance in humans (20, 21, 22), but the role of resistin in human insulin resistance still remains controversial, partly because human resistin is abundantly expressed in circulating mononuclear cells rather than adipocytes (23).
Many studies have suggested that obesity may be a factor in causing dyslipidemia, which is partially mediated by insulin resistance (24). The insulin-resistant state is often associated with abnormal lipoprotein metabolism, including hypertriglyceridemia, high levels of very low-density lipoprotein (VLDL), low levels of high-density lipoprotein (HDL) cholesterol, and small dense low-density lipoprotein (LDL). Lipid turnover studies in humans and animal models of insulin resistance and obesity have shown that the dyslipidemia associated with these conditions is predominantly due to elevated VLDL production (25, 26). However, the molecular mechanisms linking obesity and insulin resistance to VLDL hypersecretion still remain to be clarified. TNF, which is an adipocyte-secreted hormone that impairs insulin action similarly to resistin (7), has been shown to affect lipid metabolism by stimulating hepatic lipogenesis in rats in vivo (27). As previously mentioned, resistin specially attenuates insulin action in the liver, and therefore it is predicted that resistin would also affect lipoprotein metabolism independently of obesity. To understand the role of resistin in lipoprotein metabolism in mice, we induced resistin protein overexpression in C57BL/6 mice using an adenovirus-mediated gene delivery system (28) and investigated its in vivo effects on the lipid profiles and underlying mechanisms by which it impairs lipid metabolism.
Materials and Methods
Seven-week-old male C57BL/6 mice were purchased from SLC (Fukuoka, Japan) and housed in cages under controlled lighting conditions in a natural dark-light cycle (1800–0600 h). The mice were allowed free access to a regular chow diet (KBT Oriental, Saga, Japan) and sterile water.
Cloning of mouse resistin cDNA
Total RNA was prepared from differentiated 3T3-L1 cells using Isogen (Nippon Gene, Tokyo, Japan) according to the manufacturer’s instructions. Random primer-primed cDNA (1 μg) from the total RNA was used as a template for PCR cloning. The primers used were based on the mouse resistin nucleotide sequence (GenBank accession no. AF323080) as follows: 5'-CGGAATTCGGGATGAAGAACCTTTCATTTCCC-3' and 5'-CGGGATCCTCAGGAAGCGACCTGCAGCCTT-3'. After digestion with EcoRI and BamHI, the PCR fragment was subcloned into pBluescript KS (Stratagene, La Jolla, CA). A 6 x histidine tag was incorporated into the 3'-end of the resistin cDNA using fragment integration. The nucleotide sequences of the cloned cDNAs were determined by the dideoxy chain termination method using an ABI PRISM 377 DNA sequencer (Applied Biosystems Japan, Tokyo, Japan).
Preparation and administration of the resistin adenovirus
An adenovirus containing the full-length mouse resistin cDNA with the C-terminal 6 x histidine tag (AdmRes) was prepared using a commercial kit (Clontech, Palo Alto, CA). AdmRes and a control adenovirus containing ?-galactosidase (AdLacZ) were propagated in human embryonic kidney 293 cells, purified by CsCl gradient centrifugation, and stored at –80 C until use.
Sample preparation and Western blotting analysis
The 1 x 109 plaque-forming units/mouse of AdmRes and AdLacZ were injected into 8-wk-old mice via the internal jugular vein after anesthetization by an ip sodium pentobarbital injection as described previously (29).
Five days after the AdmRes or AdLacZ injection, blood was collected into tubes containing EDTA by puncturing the retroorbital plexus of anesthetized mice. After centrifugation, plasma samples were supplemented with NaN3 and phenylmethylsulfonyl fluoride and stored at 4 or –20 C.
Plasma samples (1 μl) of each mouse were boiled for 5 min in a sample buffer supplemented with 2% 2-mercaptoethanol. Samples were separated in 10 or 10–20% SDS-PAGE gels (Bio-Rad Laboratories Japan, Tokyo, Japan). The proteins were transferred to 0.2 μm nitrocellulose (Trans-Blot; Bio-Rad) or 0.2 μm polyvinyl difluoride membranes (Immun-Blot; Bio-Rad) in Tris/glycine buffer for 60 min at 60 mA, and the membranes were probed with primary antibodies diluted in appropriate buffers. Two kinds of IgG purified from a rabbit polyclonal antiserum against resistin (kindly provided by Dr. Tomoichiro Asano, University of Tokyo, and Linco Research, St. Charles, MO) were each used at a dilution of 1:1000. The secondary antibody (mouse antirabbit IgG-horseradish peroxidase; Amersham Biosciences, Piscataway, NJ) was used at a dilution of 1:5000. The signal was detected by a chemiluminescent reaction (ECL Plus; Amersham Biosciences), and the intensity was determined using a densitometer. All membranes were stained with Ponceau S to verify the quality of transfer and equivalent protein loading.
Measurements
Plasma total cholesterol, triglyceride, HDL cholesterol, and free fatty acid (FFA) concentrations were determined enzymatically. Plasma glucose levels were determined using the glucose oxidase method (glucose B test; Wako, Osaka, Japan). Immunoreactive insulin concentrations were determined using an ELISA kit (Shibayagi, Gunma, Japan). Plasma levels of resistin and adiponectin were measured using a RIA (Linco Research) and ELISA kit (Otsuka Pharmaceutical Co, Tokyo, Japan), respectively.
Lipoprotein analysis
Equal amounts of plasma samples (200 μl) were pooled from each mouse of PBS-treated, AdLacZ-treated, and AdmRes-treated group on d 5 (n = 7, respectively). Pooled samples (1000 μl) of each group were fractionated by sequential ultracentrifugation (30) at densities of less than 1.006 (VLDL), 1.006–1.019 (intermediate-density lipoprotein), 1.019–1.063 (LDL), and 1.063–1.21 g/ml (HDL). The lipoprotein fractions were then dialyzed and used for lipid analyses.
Insulin tolerance test
Insulin tolerance tests using 1 U/kg body weight of human regular insulin were conducted on AdmRes- or AdLacZ-treated mice after at least 6 h of fasting on d 5. At the indicated times after the injection, blood was drawn into tubes containing EDTA.
Triglyceride secretion rate (TGSR)
The TGSR was determined on d 5 using Triton WR-1339 (Tyloxapol; AMEND, Irvington, NJ) as described by Hirano et al. (31). Blood samples were collected at 0, 30, 60, and 90 min after iv injection of WR-1339 into mice. After checking the linearity (r > 0.98) of the plasma triglyceride increase in each mouse, the TGSR was calculated as the triglyceride increase per minute standardized by the mouse plasma volume.
RNA extraction and quantitative real-time PCR
Total RNA was extracted from approximately 30 mg tissue using Isogen and stored at –80 C for later analysis. The extracted RNA (5 μg) was converted to single-stranded cDNAs by a reverse transcriptase procedure with Superscript II (Invitrogen, Carlsbad, CA). The mRNA levels were quantified by real-time PCR using a LightCycler (Roche Diagnostics GmbH, Mannheim, Germany). Briefly, 1 μl of the cDNAs was placed in a 20-μl reaction volume containing 1 μl of each primer and 2 μl LightCycler-FastStartDNA Master SYBR Green I (Roche Diagnostics GmbH). Nucleotides, Taq DNA polymerase, and buffer were already included in the LightCycler-FastStartDNA Master SYBR Green I. The thermal cycling conditions comprised an initial denaturation step at 95 C (10 min), followed by 40 cycles of 95 C (0 sec), 60 C (15 sec) for ?-actin, apolipoprotein (Apo)E, ApoB, and ApoAI; 5 sec for lipoprotein lipase (LPL), microsomal triglyceride transfer protein (MTP), and LDL receptor); and 72 C (36 sec for ?-actin, ApoE, ApoB, and ApoAI; 15 sec for LPL, MTP, and LDL receptor).
The sense/antisense primers used were: LDL receptor (GenBank accession no. NM_010700), 5'-GAAGTCGACACTGTACTGACCACC-3' (nucleotide position 2143–2167)/5'-CTCCTCATTCCCTCTGAAAGCCAT-3' (nucleotide position 2329–2352) (32); LPL (GenBank accession no. NM_008509), 5'-AGTAGACTGGTTGTAT CGGG-3' (nucleotide position 529–548)/5'-AGCGTCATCAGGAGAAAGG-3' (nucleotide position 790–808); ?-actin (GenBank accession no. NM_007393), 5'-ACTGGGACGACATGGAGAAG-3' (nucleotide position 313–332)/5'-GGGGTGTT GAAGGTCTCAAA-3' (nucleotide position 450–469); MTP (GenBank accession no. NM_008642), 5'-TGAGCGGCTATACAAGCTCAC-3' (nucleotide position 114–134)/5'-CTGGAAGATGCTCTTCTCGC-3' (nucleotide position 314–333) (33); ApoB (GenBank accession no. XM_137955), 5'-GCCCATTGTGGACAAGTT GATC-3' (nucleotide position 2430–2451)/5'-CCAGGACTTGGAGGTCTTGGA-3' (nucleotide position 2531–2551) (34); ApoE (GenBank accession no. NM_009696), 5'-TGGGAGCAGGCCCTGAAACCGCTTC-3' (nucleotide position 151–174)/5'-G AGTCGGGCCTGTGCCGCCCTGCAC-3' (nucleotide position 364–387); and ApoAI (GenBank accession no. NM_012738), 5'-GGCAGAGACTATGTGTCCCAGTTT GA-3' (nucleotide position 188–213)/5'-GTCATCAGCGCGGGTTTGGCCTTCTC-3' (nucleotide position 707–733) (35). Threshold values were obtained when the fluorescent intensity was in the geometric phase of amplification, as determined by the LightCycler software (version 3.5). Products were verified by electrophoresis in 2% agarose gels.
Statistical analysis and ethical considerations
Values are presented as mean ± SEM unless otherwise indicated. Results were analyzed with Statview version 5 (SAS Institute Inc., Cary, NC) using unpaired Student’s t test or one-way ANOVA followed by comparisons using Bonferroni’s method. P < 0.05 denoted the presence of a statistically significant difference. The experimental protocol was approved by the Ethics Review Committee for Animal Experimentation of Kyushu University.
Results
High levels of resistin were present in plasma from AdmRes-treated mice
To confirm the protein expression of resistin, Western blotting analysis was performed under nonreducing and reducing conditions. Under nonreducing conditions, we detected a protein band of approximately 20,000 Mw in plasma samples from AdmRes-treated mice but not in those from AdLacZ-treated mice (Fig. 1A). This size was the same as that of a resistin dimer (36). Under reducing conditions, a protein band of approximately 10,000 Mw (monomer) was detected in the plasma samples from all groups (Fig. 1B). Quantification of the plasma resistin showed that the concentration was 45- to 50-fold higher in AdmRes-treated mice than in PBS- or AdLacZ-treated mice at d 5 (Table 1). The plasma resistin protein induced by AdmRes was detected at d 1, strongest at d 5, and then became weaker but could be still detected at d 9 (Fig. 1C).
FIG. 1. Western blotting analysis of plasma from PBS-, AdLacZ-, and AdmRes-treated mice at d 5 and time course of plasma resistin concentrations induced by AdmRes. Mice were iv injected with AdLacZ or AdmRes (1 x 109 plaque-forming units). Mouse plasma samples (1 μl) and recombinant resistin were separated in SDS-PAGE gels under nonreducing (A) and reducing (B) conditions and processed for immunoblotting analysis as described in Materials and Methods. C, Plasma samples were collected on the indicated day after AdmRes injection, and resistin concentrations were measured using a RIA kit (n = 4, respectively). The concentration at d 0 was 4.0 ± 0.2 ng/ml. *, P < 0.05 vs. d 0.
TABLE 1. Fasting plasma resistin and lipid levels in Mock-, AdLacZ-, and AdmRes-treated mice
We also investigated whether the secretion of other proteins from the liver was affected. The plasma levels of albumin did not differ between AdmRes-treated mice (2.4 ± 0.1 g/dl, n = 6) and AdLacZ-treated mice (2.5 ± 0.1 g/dl, n = 6). The plasma levels of insulin-sensitizing hormone, adiponectin, were the same for the two groups (AdmRes-treated mice, 18.72 ± 0.83 μg/ml, n = 4; AdLacZ-treated mice, 20.20 ± 0.82 μg/ml, n = 5).
Resistin caused attenuated insulin action
Plasma glucose (Fig. 2A) and insulin (Fig. 2B) levels were measured on d 5 after a 16-h fast. Fasting plasma glucose levels did not differ between AdmRes-treated and AdLacZ-treated mice, but the basal insulin concentration was significantly higher in AdmRes-treated mice than in controls. In insulin tolerance tests, the two groups showed a similar decrease in glucose until 30 min after the insulin injection. The glucose-lowering effect of insulin after 30 min was impaired in AdmRes-treated mice (Fig. 2C). The body weights were not significantly different between these two groups on d 5 (AdmRes-treated mice, 19.11 ± 0.30 g, n = 5; AdLacZ-treated mice, 19.14 ± 0.45 g, n = 5).
FIG. 2. Fasting plasma glucose and insulin concentrations and insulin tolerance tests in AdLacZ- and AdmRes-treated mice. Plasma glucose (A) and insulin (B) levels were measured on d 5 after a 16-h fast (n = 5–6). C, After ip injection of insulin (1 U/kg), blood samples were obtained at the indicated times from AdLacZ-treated (open circles, n = 5) and AdmRes-treated (closed circles, n = 5) mice. Initial absolute plasma glucose levels were not significantly different between AdmRes-treated mice (5.25 ± 0.62 mmol/liter) and AdLacZ-treated mice (6.74 ± 0.31 mmol/liter). Values are the mean ± SE of the percentage of the baseline glucose. *, P < 0.05.
Increased LDL in plasma from AdmRes-treated mice
As shown in Table 1, total cholesterol and triglyceride concentrations on d 5 were significantly higher in AdmRes-treated mice than controls. On the other hand, the HDL cholesterol level was significantly lower in AdmRes-treated mice. The non-HDL cholesterol (total cholesterol/HDL cholesterol) level was markedly higher in AdmRes-treated mice than controls. There were no significant differences in the plasma FFA levels among the three groups.
The cholesterol, triglyceride, and phospholipids in each lipoprotein fraction were determined using pooled plasma samples of each group (n = 7, respectively). LDL cholesterol was markedly increased in AdmRes-treated mice, compared with the other groups. The triglyceride and phospholipids in VLDL were slightly increased in AdmRes-treated mice (Fig. 3).
FIG. 3. Cholesterol (A), triglyceride (B), and phospholipid (C) contents of lipoproteins in PBS-, AdLacZ-, and AdmRes-treated mice. Each sample represents plasma pooled from mice treated with PBS (open columns), AdLacZ (hatched columns), or AdmRes (closed columns) and separated by sequential ultracentrifugation as described in Materials and Methods. IDL, Intermediate-density lipoprotein.
Plasma total cholesterol was increased in a time-dependent manner
The total cholesterol level was measured before and at 3, 5, and 8 d after the adenovirus injection. The cholesterol level in AdmRes-treated mice increased in a time-dependent manner. The cholesterol level was significantly higher in AdmRes-treated mice than the other groups after d 3 (Fig. 4).
FIG. 4. Plasma cholesterol changes in PBS-, AdLacZ-, and AdmRes-treated mice. Plasma cholesterol concentrations were measured in PBS-, AdLacZ-, and AdmRes-treated mice at various time points (days) before and after injection of PBS, AdLacZ, or AdmRes. Values are the mean ± SE (n = 6–8). *, P < 0.05.
Increased hepatic VLDL production rates in AdmRes-treated mice
To determine the mechanism of the dyslipidemia, we investigated the VLDL production rates in the livers of the mice. After an iv WR-1339 injection, AdmRes-treated mice showed a significantly higher TGSR than AdLacZ-treated mice (Fig. 5).
FIG. 5. Effect of resistin on the TGSR in AdLacZ- and AdmRes-treated mice. The TGSR was determined by measuring the increase in the plasma triglyceride concentration after an iv injection of Triton WR-1339. The TGSR is expressed as milligrams per minute. Data represent the mean + SE (n = 6).
Expressions of genes involved in lipoprotein metabolism were decreased in the muscle and liver of AdmRes-treated mice
The expressions of LDL receptor and ApoAI were significantly decreased in the liver of AdmRes-treated mice, compared with the those in the liver of AdLacZ-treated mice, and these may be causes of the high LDL and low HDL concentrations in the plasma of AdmRes-treated mice, respectively (Fig. 6). LPL expression was decreased in the muscle of AdmRes-treated mice, compared with that in the muscle of AdLacZ-treated mice, but the difference was not significant. The expressions of the genes encoding ApoB, ApoE, and MTP involved in VLDL assembly and secretion in the liver did not differ between the two groups.
FIG. 6. Relative gene expression levels of LDL receptor, ApoAI, ApoB, ApoE, and MTP in the liver and LPL expression in the muscle of AdLacZ- and AdmRes-treated mice at 5 d after the adenovirus injection. Total RNA was extracted from the liver and muscle, and single-stranded cDNAs were created as described in Materials and Methods. The mRNA levels were quantified by real-time PCR using a LightCycler. Gene levels are shown relative to the ?-actin expression level. Data are the mean + SE (n = 6).
Discussion
Resistin, also known as ADSF (37) and FIZZ3 (38), was originally reported as a possible link between obesity and diabetes mellitus (11). Recently it has been shown that resistin administration causes insulin resistance in the liver and glucose intolerance and that transgenic mice also have these characteristics (16, 40). Furthermore, ablation of resistin in mice impaired hepatic glucose production, suggesting a physiological function in maintaining glucose homeostasis in vivo (17). However, the in vivo effects of resistin on lipid metabolism still remain to be clarified. The aim of our study was to determine whether subchronic overexpression of resistin, using an adenovirus technique, impaired insulin action and subsequently affected lipoprotein metabolism in vivo.
The plasma glucose level in the fasted state was similar between AdmRes-treated mice and controls, whereas the insulin level was higher in AdmRes-treated mice than controls on d 5 after injection. In the insulin tolerance test, the glucose-lowering effect of insulin was impaired in AdmRes-treated mice. These results show subtle differences from previously reported transgenic models of resistin, which had similar fasting insulin levels and insulin tolerance to their controls (40). On the other hand, when recombinant resistin was administered to C57BL/6J mice, the mice showed an increased peak blood glucose level during glucose tolerance testing and a mildly attenuated insulin action during insulin tolerance testing, even though they showed no significant differences in the plasma glucose and insulin levels in the fasted state (11). The reason for these discrepant observations is probably differences in the degree, duration, or method of overexpression of this hormone.
The plasma cholesterol and triglyceride levels were significantly higher in AdmRes-treated mice than controls. Although most plasma cholesterol belongs to the HDL fraction in normal mice (41), AdmRes-treated mice showed a significantly lower HDL cholesterol level than the controls, and the calculated non-HDL cholesterol level was strikingly increased in AdmRes-treated mice. According to the plasma lipoprotein fractionation analysis, this increase in non-HDL cholesterol was mainly derived from elevated LDL cholesterol. These lipid profiles are consistent with those commonly observed in the insulin-resistant state in humans and diabetic mouse models such as C57BL/KsJ-db/db mice (42). These findings are particularly interesting because our experimental model was wild-type nonobese C57BL/6 mice fed on normal chow, suggesting that subchronic overexpression of resistin can affect lipid metabolism independently of obesity or a high fat diet.
Next, we investigated several factors that affect lipoprotein metabolism, such as VLDL secretion from the liver, LPL expression in the muscle, and LDL receptor and ApoAI expression in the liver. Previous kinetics studies suggested that the hypertriglyceridemia associated with insulin resistance was due to an increase in VLDL-triglyceride production by the liver (43, 44). The results of the WR-1339 study showed that the TGSR from the liver was significantly higher in AdmRes-treated mice than controls. It is possible that a moderate, but subchronic, increase in the TGSR could induce marked changes in the lipid profiles in AdmRes-treated mice. The time-dependent increase in cholesterol observed in AdmRes-treated mice may support this speculation. The expressions of genes involved in VLDL assembly and secretion, such as ApoB, ApoE, and MTP, did not differ between the two groups, consistent with the observations in a study on ob/ob mice (34). Insulin has been shown to acutely inhibit hepatic production of VLDL in both in vitro and in vivo studies (25, 45). Interestingly, however, chronically hyperinsulinemic and insulin-resistant obese human subjects and ob/ob mice were resistant to the inhibitory effects of insulin on VLDL production (34, 46). From the current results, it is possible that resistin is one of the factors that causes VLDL overproduction in obese subjects either by attenuating insulin action in the liver or by itself.
In the liver of AdmRes-treated mice, the expression of LDL receptor was decreased by 42%, compared with that in the liver of AdLacZ-treated mice, as estimated by quantitative real-time PCR analysis. Because the expression level of LDL receptors in the liver has been shown to be one of the major determinants of the plasma LDL level (47), this may also result in an increased level of plasma LDL. Similarly, the expression level of ApoAI in the liver of AdmRes-treated mice was decreased by 67%,compared with that in the liver of controls, which might have caused the low plasma HDL level in these mice.
We did not detect any significant differences in the FFA values among the three groups, whereas Pravenec et al. (39) reported that fat-specific resistin transgenic spontaneous hypertensive rats showed a higher serum FFA level. This difference may be due to differences in the dietary conditions (normal chow vs. a diet with 60% fructose), species, and/or genetic background. In addition, it should be noted that our model showed subchronic overexpression of plasma resistin, which is different from their transgenic model.
Obesity-related dyslipidemia is multifactorial, but our present results suggest that, independently of obesity, subchronic overexpression of resistin has in vivo effects that cause similar lipid profiles to those in the insulin-resistant state in humans and some diabetic mouse models. These effects are probably related to the increased VLDL production by the liver and the low removal rate of lipoproteins, which are probably brought about by the decreased expression of LDL receptors in the liver. Whether resistin itself or the secondary attenuation of insulin action gives rise to these phenomena remains to be clarified in future studies.
Acknowledgments
The authors thank Dr. Takahiko Oho (Department of Preventive Dentistry, Kyushu University Faculty of Dental Science) for helpful assistance in assays.
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